AN ABSTRACT OF THE THESIS OF
Gabino Garcia de los Santos for the degree of Doctor of Philosophy in Crop
Science presented on August 18, 1997. Title: Crossing Behavior, RAPD
Analysis and Chlorophyll Fluorescence in Relationship to the Geographic
Adaptation of Birdsfoot Trefoil (Lotus corniculatus L.)
Abstract
Redacted for Privacy
approved:
ey J. Steiner
Birdsfoot trefoil (Lotus corniculatus L.) is a popular perennial, nonbloating forage legume used for pasture, hay and silage throughout the
temperate regions of Europe, Asia Minor, North Africa and North and South
America. It is regarded as the most morphologically and biochemically variable
species in the genus. Research investigating the relationships of
morphological, ecological and genetic characteristics describing birdsfoot
trefoil germplasm has not been done. This research was conducted to
investigate if the geographic and ecological origins of birdsfoot trefoil
genotypes are related to differences in: (i) crossing compatibility among diverse
genotypes, (ii) morphological traits, (iii) PCR-RAPD banding patterns, and (iv)
temperature response of chlorophyll Photosystem II variable fluorescence. The
28 genotypes were classified by morphological characteristics, 130
polymorphic random amplified polymorphic DNA bands, and eight ecological
characteristics of the original collection sites. The ease of introgressing 27
exotic genotypes into other germplasm backgrounds was determined by using
bidirectional crosses with a domestic and exotic genotype tester. The
chlorophyll fluorescence transients ratios (FTR) were determined from eight
genotypes that were selected by their ecological diversity with measurements
made from 10 to 40° C in 5° C increments for 33 minutes from the time of initial
dark adaptation in 3 minute increments.
Morphological similarities among genotypes were related to the general
geographic proximities of their collection sites and their genetic similarity based
on RAPD markers. Utilizing genetic, morphological and ecological descriptions
revealed combinations of variation among genotypes that would not be
observed with single measurements. Incompatibility among crosses was
expressed as either an inability of plants to set pods or F1 progeny resulting
from crosses producing inviable pollen. Reproductive barriers were
environmentally neutral and randomly distributed through the among the
genotypes. Intermediate crosses could be identified to bridge any combination
of genotypes that were incompatible. The eight genotypes differed in their FTR
responses and were grouped into two classes. However, no associations were
found between genotype similarities by FTR with genetic or ecologic
similarities.
0 Copyright by Gabino Garcia de los Santos
August 18, 1997
All Rights Reserved
Crossing Behavior, RAPD Analysis and Chlorophyll Fluorescence in
Relationship to the Geographic Adaptation of Birdsfoot Trefoil (Lotus
comiculatus L.)
By
Gabino Garcia de los Santos
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented August 18, 1997
Commencement June 1998
Doctor of Philosophy thesis of Gabino Garcia de los Santos presented on
August 18, 1997
APPROVED:
Redacted for Privacy
Major Pr 9fe sor
pi
fe ting Crop Science
\./
Redacted for Privacy
Chair of Department of Crop and Soil Science
Redacted for Privacy
Dean of Graduate /School
I understand that my thesis will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of my
thesis to any reader upon request.
Redacted for Privacy
Gabino Garcia de los Santos, Author
ACKNOWLEDGMENTS
Thanks must be given to my major professor Dr. Jeffrey J. Steiner for his
support, guidance, friendship and understanding. I would also like to thank the
rest of my committee members Dr. Stephen M. Griffith, Dr. Aaron Liston, Dr.
Patrick M. Hayes, and Dr. Russell S. Karow, for their suggestions and thorough
reviews of my manuscripts, and Dr. Thomas J. Wolpert for serving as my
graduate representative.
Thanks and appreciation are also extended to Chris Poklemba, for his
help and constant advice for the laboratory analyses and data collection.
I would like to give special thanks to Consejo Nacional de Ciencia y
Tecnologia (CONACYT), my Mexican sponsoring institution who supported me
during all my academic program, Colegio de Postgraduados for its constant
support, and also Oregon State University for its partial support of my
scholarship expenses.
To the staff of the USDA-ARS, National Forage Seed Production
Research Center, for their constant friendship.
Finally, thanks to my beloved wife and daughters who always agreed my
decisions and shared all the difficult times with me without conditions.
TABLE OF CONTENTS
Page
CHAPTER 1. GENERAL INTRODUCTION
1
CHAPTER 2. LITERATURE REVIEW
6
Origin and Importance of Birdsfoot Trefoil
Ecological Adaptation
Plant Morphology
Root
Stem and Leaf
Flower and Pod
Germplasm Diversity
Germplasm Management
Introduction
Germplasm Characterization
Plant Descriptors
Morphological Descriptors
Cytologic and Genetic Analysis
Molecular Descriptors
Physiological Descriptors
Geographic Descriptors
Matrix Correlation and Trait Relationship
21
CHAPTER 3. ADAPTIVE ECOLOGY OF Lotus corniculatus L.
GENOTYPES: I. PLANT MORPHOLOGY AND RAPD
CHARACTERISTICS
24
Abstract
Introduction
Materials and Methods
Genetic Materials and Collection Site Ecological
Descriptions
Genotype Descriptions
Morphological Characters
Molecular Characters
DNA Extraction and Quantification
RAPD Analysis
Statistical Analysis
Identification of Genetically Diverse Groups
6
6
7
7
7
8
9
10
10
11
12
14
15
17
18
19
24
26
29
29
33
33
36
36
37
39
39
TABLE OF CONTENTS (cont'd)
Page
Results and Discussion
Morphological Classification
Genetic Classification
Classification Method Comparisons
Conclusions
References
CHAPTER 4. ADAPTIVE ECOLOGY OF Lotus corniculatus L.
GENOTYPES: II. REPRODUCTIVE COMPATIBILITY AND
INTROGRESSION ABILITY
Abstract
Introduction
Materials and Methods
Genetic Materials
Measures of Reproductive Success
Ease of Introgression with USA and SWI Testers
Cross-compatibility Among Exotic Genotypes
Statistical Analysis
Results and Discussion
Characterization of Parental Genotypes
Ease of Exotic Genotype Introgression with USA and
SWI Testers
Cross-compatibility Among Diverse Genotypes
Conclusions
References
41
41
53
55
56
58
62
62
63
65
65
67
67
68
68
70
70
71
76
79
80
CHAPTER 5. ADAPTIVE ECOLOGY OF Lotus corniculatus L.
GENOTYPES:III. CHLOROPHYLL FLUORESCENCE AND
ADAPTATION
82
Abstract
Introduction
Materials and Methods
Selection of Genotypes
Fluorescence Reappearance Measurement
Data Analysis
Results and Discussion
Fluorescence Reappearance Curves
82
83
85
85
85
87
89
89
TABLE OF CONTENTS (cont'd)
Paae
Association of Chlorophyll Fluorescence Response with
other Classification Characteristics
Conclusions
References
89
97
98
CHAPTER 6. COMMON CONCLUSION
101
BIBLIOGRAPHY
104
APPENDICES
120
Appendix 1 Intra-accession variation assessment of the eight
birdsfoot trefoil genotypes.
121
Appendix 2 Binary data matrix of the 18 morphological traits
and the 71 characters states
122
Appendix 2 (cont'd)
123
LIST OF FIGURES
Figure
EAU
3.1
Ward's cluster analysis classification of 28 exotic old world birdsfoot
trefoil genotypes based on 18 morphological traits described by 71
binary characters states using Euclidian distance
46
3.2
Classification of 28 exotic old world birdsfoot trefoil genotypes
based on 130 RAPD product bands using Euclidian distance and
Ward's cluster analysis
52
4.1
Female (F) and male (M) cross compatibility between NC-83
and PI 234811 with 27 exotic genotypes measured as: A,
percentage of pod set; B, pod length; C, seeds per pod; and D, F1
pollen viability. Accession names at the top and bottom of A and D
indicate extremes combination values for pod set and pollen
viability, respectively. Box plots labeled with different letters
indicate mean comparison between male and female parents are
different at P s 0.05 according to LSD test
72
4.2
Female (F) and male (M) diallele cross compatibility among seven
exotic and one North american adapted genotypes measured as:
A, percentage of pod set; B, pod length; C, seeds per pod; D,
percentage of F1 pollen viability. Accessions used are: MOR, PI
31276; SWI, PI 234811; FRA2, PI 235525; ETH, PI 260268; NOR2,
P 1319823; RUS2, PI 325369; TUR, PI 464682; and USA, 'NC-83'.
Accession names at the bottom of D indicate poor combination
for pollen viability. Box plots labeled with different letters have
means significant different at P s 0.05 according to LSD test.
78
5.1
Fluorescence transient ratio (Fv/Fo) temperature response curves
for Morocco (MOR) and Switzerland (SWI) birdsfoot trefoil
genotypes at seven temperatures. Each point represents the
mean of five replications ± standard error of the mean
90
5.2
Fluorescence transient ratio (Fv/Fo) temperature response curves
for France (FRA2) and Ethiopia (ETH) birdsfoot trefoil
genotypes at seven temperatures. Each point represents the
mean of five replications ± standard error of the mean
91
LIST OF FIGURES (cont'd)
Figure
Elg2
5.3
Fluorescence transient ratio (Fv/Fo) temperature response curves
for Norway (NOR2) and Russia (RUS2) birdsfoot trefoil genotypes
at seven temperatures. Each point represents the mean of five
replications ± standard error of the mean
92
5.4
Fluorescence transient ratio (Fv/Fo) temperature response curves
for Turkey (TUR) and USA birdsfoot trefoil genotypes at seven
temperatures. Each point represents the mean of five replications
± standard error of the mean
93
5.5
Optimum temperature for fluorescent transient ratios (FTRoptImum)
for eight birdsfoot trefoil genotypes. Each point represents the
mean of the 12 observations per temperature taken every three
minutes
5.6
94
Classification of eight birdsfoot trefoil genotypes based
on all the chlorophyll fluorescence transient ratio measurements
made from temperatures ranging from 10 to 40° C using 12 times
(0-33 min) recorded at 3 min intervals with 10s excitation and using
Euclidian distance and Ward's cluster analysis
95
LIST OF TABLES
Table
Page
3.1
Geographic origin of the 28 birdsfoot trefoil accession
30
3.2
Ecoclimatic range matrix description for the 28 accessions of
birdsfoot trefoil
31
Plant morphological characters used for the birdsfoot trefoil
accession descriptions
34
List of primers used, oligonucleotide sequences, number of
bands RAPD product bands, and range of RAPD product size for
28 birdsfoot trefoil genotypes
38
Description of the 28 birdsfoot trefoil genotypes by 12
morphological qualitative traits.
42
Description of the 28 birdsfoot trefoil genotypes by six
quantitative traits.
44
Normalized Mantel statistic (Z) from comparisons of different
proximity matrices using RAPD, combinations of morphological,
ecological characters, and geographic distances
47
3.3
3.4
3.5
3.6
3.7
3.8
Association among genetic, morphological, and geographic
classifications with nine ecological descriptors for 28 birdsfoot trefoil
genotypes collection sites
49
3.9
Pearson's correlation coefficients (r) between four quantitative
morphological traits and nine ecological descriptors
50
3.10
Analysis of variance relationships for ten qualitative morphological
traits with nine ecological descriptors.
51
4.1
Pollen viability and self compatibility percentages of the 29
birdsfoot trefoil genotypes.
66
LIST OF TABLES (cont'd)
Table
4.2
4.3
5.1
Page
Female (F) and male (M) cross-compatibility between NC-83
and SWl with the 27 exotic genotypes measured as pod set and
pollen viability percentages
74
Means of female parent diallele cross-compatibility averages
among eight birdsfoot trefoil genotypes.
77
Ecological descriptions of eight birdsfoot trefoil genotype collection
86
sites.
5.2
Normalized Mantel statistic (Z) and probability values (F) from
comparisons of different Euclidian distance matrix for the eight
birdsfoot trefoil genotypes using chlorophyll fluorescence transient
ratios.
96
CROSSING BEHAVIOR, RAPD ANALYSIS AND CHLOROPHYLL
FLUORESCENCE IN RELATIONSHIP TO THE GEOGRAPHIC
ADAPTATION OF BIRDSFOOT TREFOIL (Lotus corniculatus L.)
CHAPTER 1
GENERAL INTRODUCTION
Birdsfoot trefoil (Lotus corniculatus L. ; Fabaceae; 2n = 4x = 24) is one of
the 100 Lotus species distributed throughout the temperate regions of Europe,
Asia Minor, North Africa and North and South America (Zandstra and Grant
1968; Gunn, 1983 and 1992).The L. corniculatus group encompasses as many
as 12 diploid and tetraploid species (Ball and Chrtkova-Zertova, 1968), which
are closely related and constitute complexes grouped by similar characters that
make them difficult to recognize (Arambarri, 1994). Birdsfoot trefoil is a popular
perennial, nonbloating forage legume used for pasture, hay, and silage. It is
regarded as the most morphologically (Larsen and Zertova, 1963; Forde and
De Latour, 1978; Steiner and Poklemba, 1994) and biochemically (Ross and
Jones, 1985) variable species in the genus.
The division of the species complex comprising L. corniculatus has
been conducted in different ways by various researchers who have primarily
relied on morphological trait differences. It is believed that because of its
morphological variability, it has been able to move into a large number of
different habitats and so today is distributed over a very wide area (ChrtkovaZertova, 1973).
Currently, the National Plant Germplasm System (NPGS) Lotus
collection contains 737 accessions collected or donated from 52 countries. The
collection is represented by 36 species, with an emphasis on Old World
accessions. Fifty-five percent of the accessions are birdsfoot trefoil (Green and
McFerson, 1994) and the most-represented countries in the collection are the
United States (44), Turkey (36) and Italy (33).
2
The role of germplasm in the improvement of cultivated plants has been
well recognized (Frankel & Hawkes, 1975; Hawkes, 1983; Holden & Williams,
1984). Plant germplasm resources serve as important sources of genes for
resistance to pests, diseases, and environmental stresses, and help to ensure
that potentially useful genetic variation is preserved (Astley, 1987). However, in
many cases, the available information about accessions may be so scant that it
is of little interest to potential users of germplasm collections. Because of this,
efficient conservation and utilization of the genetic potential held within
germplasm collections demands detailed knowledge about intraspecific
genetic variation (Beuselinck and Steiner, 1992). A prerequisite for efficient
conservation of crop genetic resources is to maintain a manageable number of
accessions that represent the genetic diversity of a taxon in ex situ germplasm
collections (Kresovich and McFerson, 1992).
Clearly the goal of germplasm management is to provide the user
community (plant breeders, other users of plant genetic resources, and
scientists in other disciplines) with genetic resources for crop improvement. In
this sense, the identification and utilization of valuable traits in collections can
be very helpful to meet future demands for improved cultivars (Strauss et al.,
1988).
Diverse accessions in collections such as that of the NPGS birdsfoot
trefoil collections have been described by the ecological region from which they
were collected (Steiner and Poklemba, 1994). Such information can be used to
determine if similar ecological regions correspond to specific genotype
adaptations. It is believed that apart from general geographic descriptions, little
is known about the significance of collection site ecology on genetic adaptation
of birdsfoot trefoil.
Classifying plant populations by ecological origin has proved to be an
effective method to select planting stocks for reforestation (Conk le and Westfall,
1984). However, such knowledge has not been widely utilized to determine
whether specific accessions or traits from distinct environments can be used to
develop improved cultivars adapted to geographically distant but ecologically
similar environments (Steiner, 1994).
3
Geographic diversity can be used to develop representative core
collections from a defined region (Charmet, 1993), and to describe functional
and physiological responses of accessions for particular species, including
birdsfoot trefoil (McGraw et al., 1989). However, it has been observed that
accessions may vary more due to the local ecological factors in which they are
found than by geographic distance, and geographic isolation may have a more
significant influence on trait expression than climate (Steiner and Poklemba,
1994).
In addition to the improvement of birdsfoot trefoil as a forage species,
one of the main objectives is to understand the origin of birdsfoot trefoil (Grant,
1991). Extensive inter-and intra-specific variation has been reported within the
L. corniculatus complex. However, many cultivars of birdsfoot trefoil have been
developed using a narrow germplasm base (Steiner and Poklemba, 1996).
Therefore, it would be useful to know the different levels of reproductive
compatibility of the accessions in order to identify possible routes to introgress
important traits from exotic germplasm that has not been previously utilized in
breeding programs.
Biochemical markers have been proposed to replace conventional
descriptors of accessions in plant collections (Marshall and Brown, 1975).
Biochemical markers used in conjunction with other kinds of descriptors, such
as morphological traits, may best describe, genetic and evolutionary
relationships and estimate which environments genotypes are best adapted
than any other single trait (Avise, 1994; Stuessy, 1990). Moreover, a broader
range of descriptors may better characterize the range of genetic variation in
collections than a limited number of descriptors (Beuselink and Steiner, 1992;
Millar and Westfall, 1992). Combination of characters may also provide a better
resolution to the genetic variation among accessions in germplasm collections.
Recently, a classification of birdsfoot trefoil based on seed polypeptides
4
(Steiner and Poklemba, 1994) only could be done after accession taxonomy
was first verified by cytology (Steiner, 1992).
Random amplified polymorphic DNA (RAPD) by the polymerase chain
reaction (PCR) has been proposed as a tool for several germplasm
applications (Williams et al., 1990). RAPD markers can be used to determine
taxonomic identities, inter-specific gene flow, assessments of kindship
relationships, and analyses of mixed genome samples (Hadrys et al., 1992).
RAPDs can also be used to estimate phenotypic diversity among plant
populations (Chalmers et al., 1992; Huff et al., 1993) and to detect genetic
diversity when selecting parental strains for new crosses and germplasm
preservation (Sobral and Honeycutt, 1994). RAPD analyses have been used to
determine hybridization along vertical zonations in natural populations
(Crowhurst et al., 1991) and to verify levels of fecundity in birdsfoot trefoil
crosses (Beuselinck and Steiner, 1996). There is a general consensus that
RAPDs are a quick and efficient screening tool for germplasm collection
management as well as a technique suitable for more extensive studies of
systematics in Lotus species.
Furthermore, numerous research efforts have been made to identify
factors that correlate whole-plant physiological functions to adaptation.
Correlations between temperature traits and leaflet shape have been shown,
however, climatic and morphological characters show poor correlation
relationships (Kramina, 1994).
Methods identifying and describing the characteristic temperature optima
in species using the rate and magnitude of the reappearance of photosystem II
(PSII) have been described (Ferguson and Burke, 1991). PSII variable
fluorescence (Fv) can be easily monitored in plant leaves that have been
previously dark-adapted (Moffat et al., 1990; Peeler and Naylor, 1988). Recent
investigations report that the optimal temperatures for Fv dark recovery after
illumination are related to cool and warm-season species adaptation,
suggesting that indirect temperature dependent chlorophyll fluorescence
measurements might be used to determine plant temperature optima (Burke,
5
1990). The technique has been proved to be effective in soybean, potato, and
tobacco (Ferguson and Burke, 1991)
In birdsfoot trefoil cv. Leo, previous observations indicate that faster rates
of stem elongation and biomass production are related to high (25 C) rather
than low (10°C) temperatures (Carter and Morris, 1994). These results agree
with preliminary PSII Fv observations made with the same cultivar by Steiner
and Burke (1994, unpublished data). However, broad germplasm screening
has not been done with this technique and may prove be very helpful for
characterizing accessions in germplasm collections to determine their optimal
adaption.
The objectives of this study are to investigate if the geographic and
ecological origins of birdsfoot trefoil genotypes are related to differences in: (i)
crossing compatibility among diverse genotypes, (ii) morphological traits, (iii)
PCR-RAPD banding patterns, and (iv) chlorophyll Photosystem II variable
fluorescence temperature response.
6
CHAPTER 2
LITERATURE REVIEW
Origin and Importance of Birdsfoot Trefoil
The genus Lotus belongs to the Fabaceae family, tribe Lotoideae and is
a large polymorphic group that is comprised of approximately 100 annual and
perennial species that are widely distributed throughout the world (Grant,
1965). Its greatest diversity occurs in the Mediterranean basin which is
considered the regional center of origin.
Birdsfoot trefoil (Lotus corniculatus L.) is a widespread and variable
herbaceous species containing tetraploid (2n = 4x = 24) perennial races that
have utility for high quality livestock feed, is non bloating, and can be used for
pasture, hay, or silage purposes. The quality of birdsfoot trefoil compares
favorably with alfalfa and white clover for nutritive value. Birdsfoot trefoil is
native to Europe and has become naturalized in South and North America
where it is an important forage species (Seaney and Henson, 1970; Beuselinck
and Grant, 1995).
Ecological Adaptation
Birdsfoot trefoil is adapted to temperate northern hemisphere climates
and also grows in cooler regions of the southern hemisphere tropics and
subtropics (Duke, 1981). Suitable to varied types of soils from clays to sandy
loams, it even grows on droughty, infertile, acidic, or mildly alkaline soils, as
well as on mine spoils and under saline and waterlogged conditions. This
species is considered better adapted to wet or waterlogged soil conditions than
alfalfa (Medicago sativa L.) because it is not susceptible to Phytophthora
megasperma Drechs. (Chi and Sabo, 1978). Birdsfoot trefoil is a long-day
7
plant, with most North American cultivars requiring a 16 to 18 hour photoperiod
for full flowering (Beuselinck and Grant, 1995). Shorter photoperiods may
restrict flowering and produce plants with a prostrate or rosette growth habit
(Seaney and Henson, 1970).
Plant Morphology
Root
Birdsfoot trefoil has a well-developed, long, branching taproot with
numerous lateral roots that reach a depth of 1 to 1.1 m (Duke, 1981). Lateral
roots extend outward at almost right angles to the main axis and form a dense
mat in the upper 30 to 60 cm of the soil (McDonald, 1946). The taproot does not
penetrate as deeply as alfalfa and the distribution of branches in the upper soil
is generally more extensive. Plants have the ability to live for about two to four
years, making reseeding necessary for persistence. Recently, birdsfoot trefoil
genotypes have been found that persist through the production of rhizomes and
plant breeders have transferred this trait into commercially accepted genetic
lines (Pedersen et al., 1986; Beuselinck et al., 1992; Beuselinck and Steiner,
1996).
Stem and Leaf
There is considerable variation in leaf and stem morphology within
birdsfoot trefoil. Size, shape, color and pubescence of stems vary greatly
among different genotypes (Chrtkova-Zertova, 1973). The stems vary from
prostrate to somewhat erect, and the overall length of the stems can vary
greatly also. In general, stems of the erect types are smaller in diameter and
less rigid than stems of alfalfa (Seaney and Henson, 1970). Mature plants have
several branched stems arising from a single crown. When it grows under
8
favorable conditions, the main stems can reach a length of 60 to 90 cm. Stem
length is generally shorter than alfalfa.
Each leaf is composed of five leaflets, three attached to the terminal end
of the petiole and two at the base. Leaves are attached alternately on opposite
sides of the stem. Leaflets are typically obovate, although shape may vary from
rounded to oblanceolate. Typically the leaflets close around the petiole and
stem during the night, similar to the night-closing of leaves of other forage
species such as white clover (McDonald, 1946). Because roots develop from
callous tissue of stem internodes, plants can be easily propagated by stem
cuttings.
Flower and Pod
The inflorescence is an umbel having four to eight florets attached to the
end of a relatively long peduncle. Each floret consists of a calyx with five united
sepals and a typical legume corolla with five petals. Two petals are fused
together to form the keel which is enclosed by two wings petals and the
standard. The color of the petals varies from a light to dark yellow and may be
tinted with pale orange or red stripes. Keel tips may be yellow, brown, or red
(Buzzel and Wilsie, 1963).
Reproductive parts of the flower are composed of ten stamens and a
single pistil. One stamen is attached individually to the base of the flower, while
the other nine form a tube surrounding the ovary. The fused stamens may have
different lengths (McDonald, 1946).
On average, five to six pods are borne at right angles to the tip of the
peduncle, giving the appearance of a bird's foot, and thus its name. Mature
pods are regularly brown to almost black. The cultivar Viking may have average
pod dimensions of 25 mm in length and 3 mm in diameter (Winch et al., 1985).
Each pod contains 15 to 20 seeds attached to the ventral suture and dehisce
along both sutures and twist spirally to release the seeds.
9
Germplasm Diversity
Plants vary greatly in growth habit size, shape, color and pubescence of
stems and leaves. Varieties within the birdsfoot trefoil group consist of two
distinct types commonly known as "European" and "Empire" types. The main
differences between these forms are their growth habit, maturity, and winter
hardiness (Seaney and Henson, 1970). In Europe, these types are found: the
sparsely and densely pubescent types with calyx teeth shorter than the tube are
found mainly in northern countries. Those with glabrous or sparse pubescence,
smaller leaflets, calyx teeth slightly longer than calyx tube, are found along
coastal western and northern Europe. Those with dense pubescence, calyx
teeth slightly longer than the calyx tube are found in central and southern
Europe (Chrtkova-Zertova, 1973).
The Empire-type (also known as New-York type) is a naturalized ecotype
from Albany, NY that has finer stems, a more prostrate growth habit, is 11 to 14
days later in flowering, and is more indeterminate than the European type.
Erect growing cultivars that are recommended for hay or rotational grazed
pastures include Cascade, Douglas, Granger, Leo, Maitland, Mansfield, Tana
and Viking. Semi-erect or spreading cultivars include Empire and Empire
strains and are recommended for permanent pastures and intense grazing
(Duke, 1981).
When grown under field conditions, it is relatively easy to distinguish
between Empire- and European-type cultivars. However, growth habit, date of
flowering, and the gross morphological characteristics of European-type
cultivars are quite similar, and the identification of specific cultivars becomes
extremely difficult (Seaney and Henson, 1970).
Birdsfoot trefoil is also polymorphic for cyanogenic glucosides, which
can be hydrolyzed by enzymatic action to produce free hydrocyanic acid (Grant
and Sidhu, 1967; Phillips, 1968). Most collections of birdsfoot trefoil contain
accessions with cyanogenic glucoside and the corresponding enzyme for
hydrolysis. It has been reported that cyanogenesis is controlled by a single
10
dominant gene. Plants that have the recessive gene lack the enzyme
necessary for hydrolysis of the cyanogenetic glucoside (Dawson, 1941). The
concentration of hydrocyanic acid in some Lotus species is poisonous to
animals (Gurney and White, 1941; Henry, 1938; Jones, 1978), but there have
been no authenticated reports of birdsfoot trefoil causing HCN poisoning when
grazed. There are examples of birdsfoot trefoil plants that are acyanogenic, and
some attempts have been made to isolate an acyanogenic strain of birdsfoot
trefoil by recurrent selection (Seaney and Henson, 1970).
Germplasm Management
Introduction
The International Board of Plant Genetic Resources (IBPGR) developed
the concept of a global network of gene banks; base collections support the
active collection, and are used for distribution and evaluation of germplasm.
The necessity for developing core collections that are representative of the
genetic diversity of a crop species and its wild relatives has been discussed
(Frankel, 1984, and Brown, 1989). The accessions not included in the core
collection are maintained as a reserve collection. The reserve collection
demands care and must remain an integral part of the overall active collection.
If difficult situations such as limited finances arise, the core collection must have
priority over the reserve collection. Taxonomic origin and known characteristics
of accession holdings are traditionally considered in gene bank management.
The ability to identify redundant accessions in collections is important.
Molecular techniques have proved useful to identify repetitive accessions in
collections of Musa spp. (Jarret, 1987) and Solanum spp. (Huaman, 1984). For
repositories with very large collections, molecular techniques represent an
important tool toward more detailed characterization of individual accessions
(Melchinger et al., 1991; Smith and Smith, 1991).
11
Germplasm Characterization
Germplasm characterization is a systematic evaluation of genetically
controlled traits for a collection of accessions (Frankel, 1987; Williams, 1989b).
Accession characterizations are useful for designating core collections and
should provide a standardized record of plant attributes together with passport
data (Frankel and Brown, 1984). Passport data includes the key information
about the collection site and environment from which an accession originated.
Data used in accession characterization should be linked to passport and
evaluation data (Steiner and Green, 1996). Evaluation is a prerequisite for the
germplasm collections (Frankel, 1989b). Information about germplasm
collection holdings makes choosing and utilizing appropriate accessions
easier. Without characterization, collections mostly remain as curiosities that
are not effectively utilized.
The primary level of collection characterization is done using
morphologic characters, either throughout plant development or at maturity,
and the primary user is the germplasm collection curator (I BPGR, 1985). Large-
scale systematic evaluations can be performed by a variety of scientists
interested in crop improvement and production research (Williams, 1989). The
initial phase of evaluation is usually performed by the germplasm manager
during initial accession seed increase. Observations are usually limited to a
small number of morphological features, adaptability at the site of seed
increase, and any obvious characteristics of potential economic value. These
preliminary observations enable the germplasm worker to forward the materials
to other researchers for complementary testing (Peeters and Williams, 1984).
However, this level of characterization is of limited value to plant breeders since
different traits are usually desired for cultivar development (Frankel, 1986).
Wide applicability of characterization data can be ensured by using
standardized descriptors (National Research Council, 1993). Characterization
data are also useful for validating accession identity when accessions are
12
regenerated. Characterizations at the time of regeneration are efficient because
additional plantings are not required for evaluation.
Plant Descriptors
The classification and organization of genotypes into systematic classes
can be done using a wide array of morphological, biochemical, and molecular
descriptions. Regardless of the type of characters chosen for analysis, all are
assumed to be genetically regulated. No characters can be viewed in a vacuum
and should be evaluated in reference to independent data sets (Crawford,
1990). There are three basic kinds of descriptors: (i) those that involve the
organism itself, such as morphology, cytology, genetics, and chemistry; (ii)
those that come from organism to organism interactions which include
cytogenetics and some reproductive biology traits (e. g., pollination, etc.); and
(iii) data that involve organism-environment interactions such as geographic
distribution and ecology (Stuessy, 1990).
The necessity of efficient methods to evaluate genetic diversity and
maintain germplasm collections has been expressed. Core collections can
minimize repetition of similar or duplicated accessions within the collection and
be used to represent the genetic diversity of a species (Holbrook et al., 1993),
but selection of a broad range of appropriate descriptors may be a more
effective way to characterize the range of genetic variation (Beuselinck and
Steiner, 1992; Millar and Westfall, 1992).
Access to comprehensive accession documentation is essential to
ensure conservation and to promote the use of genetic resources. Recent
efforts have focused on reviewing historic documents linked with collections
that include plant exploration and introduction inventories, original trip reports,
correspondences and evaluation notes. Passport information has been
organized and placed in the Germplasm Resources Information Network
(GRIN) to supplement existing data for some species (Green and McFerson,
1994).
13
With the development of molecular biology tools, the potential for
defining crop genetic pools is limitless and can expand the understanding of
the range of genetic variation accessible to plant breeders. In the case of
birdsfoot trefoil, it has been stated that most of the genes that code important
traits are completely uncharacterized and will take years to isolate (Grant and
Altosaar, 1994).
Currently, some characterization and evaluation data are available for
the Lotus collection and as are descriptor lists (Green and Bohening, 1993).
Numerous studies have looked at genetic relationships among and within
different Lotus taxa. These have included the use of cytology (Larsen and
Zertova, 1963; Grant, 1965; Zandstra and Grant, 1968; Small et al., 1984),
isozymes (De Lautour et al., 1978; Raelson and Grant, 1988; Raelson and
Grant, 1989), seed proteins (Sammour et al., 1993; Steiner and Poklemba,
1994; Steiner et al., 1994b), agronomic and herbage quality measurements for
plant decumbency and winter injury (McGraw, et al., 1989), and DNA (Steiner
et al., 1994b). While studies of birdsfoot trefoil and related species have been
published, comprehensive morphological, geographical, and genetic studies
are still needed (Grant and Small, 1996).
14
Morphological Descriptors
Several kinds of descriptors can be used to describe intraspecific
variation among accessions. However, to the collector in the field,
morphological traits and ecological adaptation may be the most relevant.
Morphological variation within an accession can help collectors keep records
and help them estimate the amount of diversity in the field while collecting.
Two types of approaches have been used to study intraspecific variation
(Hanelt, 1986). Formal classifications are based on a few, easily recognizable
characters that allow rapid assessment of the variation among collections
(Perrino and Hammer, 1983). Informal classifications on the other hand, are
less popular for describing crop variation as time demands are greater. These
include morphological traits such as pod, leaf, and bract characteristics
(Brandenburg, and Schneider, 1988).
Morphological characters have been the most used plant classification
descriptors. These kinds of data have the advantage of being easily observed
and therefore their description of variability (Davies and Heywood, 1963). Floral
traits have been more useful for systematic classification and have been used
more extensively than many vegetative characters (Schuyler, 1971). However,
vegetative descriptors such as leaf and pod shape tend to be more plastic and
variable than floral traits which makes them more complicated to use for
classification purposes (White, 1979). Both vegetative and floral characters
have been used for many taxonomic problems (Stuessy, 1990).
The evaluation of genetic diversity provides the breeder with the
appropriate genetic materials for crop improvement. Morphological, agronomic
and geographic characterization have been used to study the variability of
crops such as pearl millet (Ouendeba et al., 1995), and cotton (Gossypium
arboreoum L. and G. herbaceum) (Hutchinson, 1964). Morphological and
crossing data have been also used to classify Solanum species into sections
(Correll, 1962; Hawkes, 1990). In alfalfa, (Medicago sativa) morphological and
agronomic variation among 41 accessions from the Middle East was employed
to separate genotypes from different regions (Smith et al., 1995).
15
Birdsfoot trefoil is considered one of the most morphologically variable
species of the entire genus Lotus. Using specific morphological and anatomic
characteristics, the variability of the species and its taxonomic significance has
been evaluated in northern and central Europe (Chrtkova-Zertova, 1973). The
variability present in birdsfoot trefoil can form series of specific traits related to
geographic origin. Native plants in northern Europe are prostrate with short
internodes and small leaflets (Bonnemaison and Jones, 1986). In the French
Alpes, at around 200 m, birdsfoot trefoil and L. alpinus ansh. can be
discriminated by differences in calyx size and cauline hair length (Blaise et al.,
1991). Currently there is a need for a standardized morphology descriptor list
information for collected birdsfoot trefoil accessions.
Cytologic and Genetic Analysis
Chromosome data including number, size and shape, meiosis behavior,
and DNA characteristics have been used in taxonomy (Lewis, 1969).
Cytogenetic data provide the taxonomist hereditary bases for evolutionary
divergence (Giannasi, 1979). Few studies have been done to determine the
genetic basis of taxonomic characters through crossing studies. In addition,
cytogenetic descriptors can reveal relationships among taxa by chromosome
homologies through natural or artificial hybridization (Stuessy, 1990). One way
of establishing genetic relationships among taxa is by producing artificial
hybrids (Stuessy, 1990). Progeny of hybrids can be tested for fertility upon
maturation. Pollen viability of hybrids is a commonly used and quick method to
test fertility. Various stains (Hauser and Morrison, 1964) or agar methods can
be used to test pollen viability (Han, 1989).
It has been reported that the taxa L. tenuis ansh., L. cornicu/atus and L.
alpinus may be characterized by using ploidy-level descriptors since these
species have both diploid or tetraploid forms (Small et all., 1984). In assessing
Lotus relationships of species chromosome numbers need to be assessed for
reliable identification (Jones and Turkington, 1986). The autotetraploid origin of
16
birdsfoot trefoil has been postulated from chromosome description studies of L.
cornicu/atus and L. glaber (Wernsman, et al., 1964). These observations were
later complemented by crossing studies indicating that birdsfoot trefoil could
have been arisen as an autotetraploid from L. glaber (Negri and Veronesi,
1989).
Some crop germplasm advisory committees have recommended the use
of cytological techniques as a high-priority descriptor to verify and describe
accessions in germplasm collections (Roat, 1989).
In birdsfoot trefoil, many taxonomic discrepancies in the NPGS collection
have been rectified by using somatic chromosome numbers to differentiate
diploid and tetraploids before classifying accessions by other traits (Steiner and
Poklemba, 1994). It has been mentioned that results reporting cytological data
can help resolve placement of Lotus species and improve communication
between researchers (Steiner, 1994). Karyotype analysis, however does not
appear to be a suitable method to investigate the parentage of birdsfoot trefoil
because chromosome repatterning has occurred during the evolutionary
development of closely related diploid species. Approaches such as
chromosome banding, isozymes, and other molecular systematic methods
should be considered (Grant, 1991).
Cytologic studies of birdsfoot trefoil suggests that evolutionary changes
are still occurring (Angulo and Real, 1977; Fernandez, 1981). Recently,
comprehensive reviews have been published stating that there is still
insufficient cytogenetic and biochemical data available for birdsfoot trefoil to
draw definite conclusions about its origin (Grant and Small, 1996).
Molecular Descriptors
17
Descriptions of the morphology of vegetative and reproductive structures
and agronomic assessments have been the main methods to characterize
genetic resources. However, in recent years, new molecular techniques have
increasingly been used to describe plant germplasm collection holdings (Rao
and Riley, 1994). The advantages of DNA markers for germplasm
characterization are: 1) any part of the plant can be sampled 2) only small
amounts of DNA are required 3) generally the method of inheritance is simple,
and 4) conditions have no effect on expression (Lowe et al., 1996)
Molecular marker technologies are effective diagnostic tools to assess
accession identity, degree of relatedness within and among accessions of a
collection, and in germplasm conservation (Kresovich et al., 1993). In addition,
molecular markers can objectively estimate basic taxonomic relationships of
germplasm because they are not subject to environmental effects and therefore
are a reliable index of plant genotype (Bernatzky and Tanks ley, 1989). For
instance, a reasonable estimate of phenotypic diversity can be obtained with
molecular markers when specific selection criteria are not available (Burr et al.,
1983). Collections can be analyzed to determine where additional collections
should be made and may also reduce the need for maintaining genetically
similar samples.
Compared with other methods for genetic diversity analysis (e. g.,
isozymes or morphology), DNA marker are exceptionally sensitive,
reproducible and technically routine (Paterson et al., 1991). Evolving
technologies are also a potential tool to effectively and efficiently identify
genetic redundant germplasm collections based on genetic relatedness
(Kresovich et al., 1994). The application of molecular markers has the potential
to accelerate the accumulation of information in comparison to more traditional
morphologic evaluation methods (Sobral and Honeycutt, 1994)
A number of molecular DNA techniques are currently available (Weising
et al., 1995). A popular technique is the polymerase chain reaction (PCR) to
generate random amplified polymorphic DNA fragments (RAPDs) (Williams et
18
al., 1990). RAPDs have been proposed as a tool for several different
applications (Williams et al., 1990). This technique has proved to be useful for a
variety of genetic analysis including, genome mapping, fingerprinting, and
phylogeny reconstruction (Lamboy, 1994a, b), to estimate phenotypic diversity
among plant populations (Chalmers et al., 1992; Huff et al., 1993; Sweeney
and Danneberger, 1995), and as an effective and cost-efficient method for
purposes such as characterization of plant genetic resources (Transue et al.,
1994), detecting genetic variation (Hultqist et al., 1996), classification of
cultivars (Zheng et al., 1991), and to determine phylogenetic relationships
(Gunter et al., 1996). It is believed that some of the systematic discrepancies in
birdsfoot trefoil and Lotus corniculatus complex may be clarified by the use of
these molecular techniques (Campos et al., 1994). The limitations of RAPD
markers have been reviewed (Wolfe and Liston, in press).
Physiological Descriptors
The prediction of the suitable environments for agricultural plant growth
and the prediction of genotype responses to the stresses should provide
assessment procedures for germplasm collection management (Peacock,
1984; Crowson, 1970). Variation for physiological adaptation associated with
leaf photosynthesis has been reported in wild populations of various crops
(Lynch et al., 1992). Because of the utility of chlorophyll fluorescence
technique, it has been recommended for monitoring plant stress under various
environmental conditions such as chilling and drought stress (Larcher and
Neuner, 1988; Ogren, 1990), freezing stress (Somersalo and Krause, 1989),
and cold climatization and extreme temperatures (Boese and Huner, 1990;
Somersalo and Krause, 1989).
Changes in chlorophyll fluorescence have been used to characterize
plants with different tolerances to environmental stresses (Smillie and
Hetherington, 1983), to rank species in order of chilling sensitivity (Smillie,
1979; Peeler and Naylor, 1988) and frost sensitivity (Sundbom, Strand and
19
Hallgren, 1982). Other reports show the feasibility of using the chlorophyll
fluorescence technique for screening genotypes for high temperature tolerance
(Moffat et al., 1990). Recent methodological advances in measuring the rate
and magnitude of the reappearance of photosystem II (PSII) (Ferguson and
Burke, 1991) have shown the potential usefulness of classifying accessions in
germplasm collections based on association of whole-plant responses with
plant performance in diverse ecological environments. In birdsfoot trefoil cv.
Leo, three genotypes grown in growth chambers had faster stem elongation
rates and greater biomass production accumulation at 25 C than at 10 C
(Carter and Morris, 1994). These results are in agreement with preliminary PSII
Fv with the same cultivar (Burke and Steiner, 1994 unpublished data).
In general, studies where physiological characteristics have been
measured are very scarce (Gonzalez et al., 1995). There is little known about
physiological trait variation related to leaf photosynthesis or to variable
fluorescence response (Gonzalez et al., 1995), and broad germplasm
screening has not been done with this technique.
Geographic Descriptors
The largest portion of species genetic diversity is accounted for by
geographic range. However, apart from general geographic descriptions, little
is known about the significance of collection site ecology on ex situ collection
genetic structures (Hamrick and Godt, 1989). Important climatic and geographic
factors include total rainfall, humidity, soil moisture, seasonal and diurnal
temperatures, available light, latitude, longitude, and elevation. Latitude,
longitude and elevation can be used to facilitate mapping plant distributions
(Nairn, 1961). The geographic origin of an accession can be easily determined
using an electronical global positioning system at the time of collection or from
map coordinate data estimated after aquisition. Such information is commonly
20
included with the accession passport data and is often available from NPGS
data bases (Green and McFerson, 1994; Steiner and Poklemba, 1994).
The data elements required to classify accessions using ecological
descriptions can easily be recorded along with general site and accession
information (Steiner and Greene, 1996). Utilizing habitat data to describe the
ecology of collection sites may be another useful approach for examining
germplasm collection diversity and help estimate the ecologic adaptation of
different germplasm accessions. A list of ecological descriptors that would be
helpful for classifying accessions has been proposed (Steiner and Greene,
1996). Ecological descriptor data such as these may also help with the
planning of future germplasm collection trips and be used to determine where
accessions may be found that are less likely to be related to entries already in
germplasm collections.
Ecological data can be used at different levels of the taxonomic
hierarchy. Ecological data require less investment of time compared to other
kinds of data (Davis, 1968). Ecological data are unique in that they deal with
the organism itself. Hence, they can be very helpful for the assessment of
affinities, hypothesis of evolutionary processes, and biogeographical
considerations (Stuessy, 1990).
Geographic information has been used to develop core collections of
accessions from a defined region (Charmet, 1993) and to describe functional
physiological responses (McGraw et al., 1989). However, it has been found that
accessions may vary more based on the local ecological factors in which they
are found than by geographic distance (Steiner and Poklemba, 1994). For
some species, the ecogeographic origin has been used in the absence of
evaluation data to select core accessions (Spagnoletti, 1993).
Using individual birdsfoot trefoil traits, geographic distribution trends
have been observed. For instance, low growth, glaucousness, fleshy leaflets,
pale flower color, and dark keel color are more frequent as we go northward in
Europe, whereas the density of indumentum and the number of flowers per
inflorescence diminishes (Chrtkova-Zertova, 1973).
21
Ecology may be a key descriptor to better understand the dynamics of
evolution and gene exchange in the L. corniculatus complex. There have been
several studies of Lotus species variation in several geographic regions, but
there is still a need for better classification of species boundaries (Grant and
Small, 1996). Standardized and detailed ecological descriptor information for
collected accessions in germplasm collections is needed.
Matrix Correlation and Trait Relationships
Similarities among group of classified accessions need to be assessed.
For instance, in clustering techniques, a measure is needed to compare the
degree of correspondence between different solutions (Morey and Agresti,
1984). The two measures most frequently used by classification researchers
are the Kappa statistic (Cohen, 1960) and a statistic proposed by Rand (1971).
Both have been applied to describe the relative agreement between two
solutions of the same set of data.
Numerous indices have been suggested to measure the degree of
relationships among classifications using different sets of data. The matrix
correlation coefficient, which is simply the Pearson cross-product correlation
has been proposed to evaluate clustering results (Sokal and Rohlf, 1962). This
coefficient is equivalent to the standardized form of the Mantel statistic (Mantel,
1967). Since its introduction, this matrix correlation coefficient index has been
widely employed as standard method in numerical phenetic studies. It is a
measure of the degree of fit of a classification to a set of data and is a criteria for
evaluating the efficiency of various clustering techniques (Farris, 1969; Williams
and Clifford, 1971). Mantel's test statistic is a common method for measuring
the degree of correspondence between classifications (Tatineni et al., 1996).
This method has become popular and is being applied to a variety of research
situations. In birdsfoot trefoil, the mantel statistic has been used to test
relationships between geography and morphology, genetics, and chemical
constituents (Grant and Small, 1996).
22
Associations among genetic, environmental, and geographic variables
has been extensively reviewed. Understanding the relationships between
specific traits and environmental factors may be useful to understand the
adaptation of genetic resources to the environments according to their specific
uses (Hedrick, 1986; Beuselinck and Steiner, 1992).
Winter low temperature for instance, has been shown to be related to the
frequency of cyanogenic white clover plants in regions of Europe and North
America (Daday, 1954; Pederson et al., 1996). Lower frequencies of
cyanogenic white clover plants were observed at higher altitudes, and lower
temperatures (Daday, 1959; Foulds and Grime, 1971).
The integration of molecular data to whole-plant form and function has
been suggested as a way to characterize diversity (Smith and Smith, 1989a, b;
Smith et al., 1990). Correlations among molecular markers and whole plant
traits responses may be of value to predict performance. In crimson clover (T.
incarnatum L.) flowering time was found to be associated with specific RAPD
markers (Steiner et al., 1998).
Patterns of morphologic diversity have been examined in numerous
studies, and genetic diversity has been observed to be extremely large for most
traits in wild genotypes such as Lupinus angustifolius (Clements and Cowling,
1994). In red clover collections, correlation between spring recovery and winter
hardiness has suggested that evaluation of only one of these traits would be
sufficient for germplasm records (Kouarne and Quesenberry, 1993).
Associations have been found for plant winterhardiness and seed yield with
herbage yield (McGraw et al., 1989), and for flowering percentage and seed
chalcid resistance with herbage tannin content (Steiner and Beuselinck, 1998).
In Turkey, birdsfoot trefoil morphologic variation and chromosome number are
correlated with altitude (Small, 1984). However, no relationships between keel
color and environmental factors have been observed in a morphology study of
genotypes conducted in central and southern part of north Europe (ChrtkovaZertova, 1973).
Few studies have investigated associations of intraspecific genetic
variation with collection site variation to identify germplasm accessions that
23
contain useful traits (von Bothmer and Seberg, 1995; Green and McFerson,
1994). The use of simple correlation or multivariate analysis can reveal trait
association and unique combinations of traits not found in the majority of the
collection accessions. All species are affected by microclimatic factors and very
often high correlations exist, not always tested experimentally. Critical factors
may determine distribution of particular genotypes (Davis, 1968). More work is
needed to explore associations among different traits within germplasm
collections (Brown, 1989).
24
CHAPTER 3
ADAPTIVE ECOLOGY OF Lotus corniculatus L. GENOTYPES: I.
PLANT MORPHOLOGY AND RAPD CHARACTERISTICS
Abstract
)
Birdsfoot trefoil (Lotus corniculatus L.) is a widely distributed polymorphic
)
perennial forage legume species found in wild and naturalized populations
throughout temperate regions of Europe, Asia Minor, North Africa, and North
and South America. Efficient utilization of germplasm collections requires an
understanding about the range of variation present. Research investigating the
use of morphological, ecological, and genetic characteristics describing
birdsfoot trefoil germplasm has not been done. The objectives of this research
were to: (i) characterize and compare morphological and RAPD classifications
of 28 ecologically diverse genotypes from the NPGS birdsfoot trefoil collection,
and (ii) determine the relationship between RAPD and morphological
descriptors with ecological characteristics of the collection sites of the
genotypes. The genotypes were classified by 18 morphological characteristics,
130 polymorphic random amplified polymorphic DNA bands, and eight
ecological characteristics of the original collection sites. The 28 genotypes
were polymorphic and classified into five cluster groups. Morphological
similarities among genotypes were related to the general geographic
proximities of their collection sites and their genetic similarity based on RAPDs.
Genotype morphology was also associated with the general ecology of the
collection site habitat. However, ecological similarity of the genotypes is not
related to their genetic similarity. The lack of similarity between the genetic and
ecological classifications suggests that genotypes adapted to similar but
geographically distant habitats were derived from different progenitors but have
acquired similar phenotypes. Combining genetic, morphological, and
25
ecological descriptors reveals combinations of variation among the birdsfoot
trefoil genotypes that would not be apparent with any single measurement.
Introduction
26
The genus Lotus is a large polymorphic and widely distributed group
comprised of approximately 200 annual and perennial species (Grant, 1965).
Birdsfoot trefoil is the most important and widely distributed species of the
genus. It is generally known as tetraploid species with 2n = 4x = 24 somatic
chromosomes, but has been reported to have diploid populations (Grant, and
Small, 1996). The greatest genetic diversity for birdsfoot trefoil occurs in the
Mediterranean basin (Grant, 1991).
Birdsfoot trefoil is native to Europe and western Asia but also widely
naturalized throughout temperate regions of South and North America where it
has become an important forage species. It has a high nutritive value and is
non-bloating when grazed directly (Seaney and Henson, 1970; Beuselinck and
Grant, 1995). Birdsfoot trefoil is also tolerant of acid, infertile, and poorly
drained soils.
The role of germplasm in the improvement of cultivated plants has been
well recognized (Frankel and Hawkes, 1975; Hawkes, 1983; Holden and
Williams, 1984). Ex situ collections of wild germplasm have substantially
contributed to the development of improved cultivars. These collections contain
genes that code for resistance to pests, diseases, and environmental stresses,
and help to ensure that potentially useful genetic variation is preserved for
future needs (Astley, 1987).
The collection and preservation of wild populations is considered to be
vital for the future breeding and improvement of birdsfoot trefoil. Even though
the primary objective of breeding in North America has been to increase forage
yields, other specific breeding objectives have involved incorporating traits
such as seed pod shatter resistance, herbage tannin content, and increased
seed yield and seed vigor. There is a wide range of variability within the USDA­
ARS NPGS birdsfoot trefoil collection. However, this collection has not been
widely characterized. Birdsfoot trefoil accessions have been characterized by
seed globulin polypeptides (Steiner and Poklemba, 1994), random amplified
27
polymorphic DNA (RAPD) (Steiner and Beuselinck, 1998), and specific
agronomic traits (McGraw et al., 1989; Steiner and Beuselinck, 1998). More
thorough characterization of the range of genetic variation available for crop
improvement in this species will result in better utilization (Beuselinck and
Steiner, 1992).
Genetic resource collections are often poorly characterized because of
the lack of descriptive and pedigree information about the accessions that
comprise the collections. However, a definition of representative samples of
accessions from the larger working collections is required to better manage and
conserve these resources more efficiently (Frankel, 1986). Once the
representative samples of the collection have been defined, the accessions can
be comprehensively described using diverse kinds of descriptors (Beuselinck
and Steiner, 1992). Prioritizing descriptive characters in such a way would
serve as genetic indicators to represent the existing diversity of accessions
within a collection (Beuselinck and Steiner, 1992).
Core collections have been proposed as tools to study and utilize
genetic diversity represented by large germplasm collections (Brown, 1989).
Ideally, a core collection should represent most of the genetic diversity found in
a larger collection and thus allow extrapolation of information to the entire
collection. Understanding the genetic diversity within collections facilitates
resource utilization and has been cited as a reason for characterizing
collections holdings (Strauss et al., 1988; Beuselinck and Steiner, 1992). There
is a lack of information about the NPGS Lotus collection because only pieces of
information have been assembled from different research efforts. Hence, the
systematic evaluation of collection genetic diversity is needed to understand
the relationships among accessions and the corresponding environments from
which they were collected to efficiently utilize these resources (Steiner and
Greene, 1996).
Different types of descriptor data have been employed in the
classification of species. Traditionally, quantitative and qualitative traits that are
easily measured and interpreted, such as plant morphology, cytology, genetics
and chemistry, are used to characterize germplasm accessions (Stuessy,
28
1990). However, very often it is necessary to combine different kinds of plant
descriptors to obtain reliable classifications. Examples of germplasm
biochemical descriptors include isozymes (Doebley, 1989) and DNA markers
(Lamboy, 1994a; Paterson et al., 1991). Patterns of genetic variation needed for
efficient utilization of germplasm may be revealed by the combination of
qualitative or quantitative morphological traits, flavonoid composition, isozymes
and molecular techniques (Beer, et al., 1993). RAPD molecular markers are a
useful tool for characterizing germplasm (Tingey et al., 1993). Because data
can be efficiently and inexpensively generated, RAPDs can be routinely used to
answer specific germplasm questions (Skroch et al., 1992). RAPD markers
have been shown to detect intraspecific variation in birdsfoot trefoil and
differences among Lotus species (Campos et al., 1994). Because birdsfoot
trefoil is a highly variable species, the overall phenotypic resemblance among
the accessions makes it a very difficult species to characterize. Thus a
combination of descriptors are necessary to accurately distinguish accessions.
The objectives of this research were to: (i) characterize and compare
morphological and RAPD classifications of 28 ecologically diverse genotypes
from the NPGS birdsfoot trefoil collection, and (ii) determine the relationship
between RAPD and morphological descriptors with ecological characteristics of
the collection sites of the original accessions.
29
Materials and Methods
Genetic Materials and Collection Site Ecological Descriptions
Twenty-eight native birdsfoot trefoil genotypes from the NPGS birdsfoot
trefoil collection were selected based on the geographic and ecological
diversity of the sites from which they were originally collected (Table 3.1). The
environmental and ecological characteristics of the accession collection sites
(Table 3.2) were determined by original passport information or estimated by
retroclassification when actual collection site coordinates were unknown
(Steiner and Greene, 1996). The environmental and ecological
characterization data used for classification were acquired from the U.S.
Environmental Protection Agency and National Oceanic and Atmospheric
Administration (EPA / NOAA) Global Ecosystems Databases (Kineman and
Ohrenschall, 1992, 1994). The twenty-eight genotype collection sites were
described by: (i) ecoregions of the continents (Bailey, 1989); (ii) lowest (low)
and highest (high) monthly temperature, and annual average (average)
temperature, monthly and annual accumulated precipitation (precipitation)
monthly and annual accumulated snow depth (snow)
,
,
monthly and annual
percentage of sunshine hours (cloudiness) (Leemans and Cramer, 1992); and
(iii) modal elevation (elevation) (Fleet Numeric Oceanographic Center, 1992).
An estimate of geographic distances (Dgeog) among all genotype
collection sites was based on latitude and longitude coordinates using the
equation developed by A. Afonin, Vavilov Research Institute, St. Petersburg,
Russia (personal communication, 1996):
Dgeog = (( ABS (°Longitude of site 1
°Longitude of site 2) x ( 6378 / 2) x
(COS (°Latitude of site 1) + COS (°Latitude of site 2))2 +
(ABS (°Latitude of site 1
°Latitude of site 2) x 6378)2)
0.2
The ecological distance among genotype collection sites was
determined by calculating the Euclidean distance of the standardized values of
elevation, cloudiness, temperature (average, high, and low), and precipitation.
30
Table 3.1. Geographic origin of the 28 birdsfoot trefoil accessions.
Entry
Identification
Country
City
---- no. - -­
Location
° Iat
PI 31276
MOR
Morocco
PI 180171
CZE
PI 227512t
PI 234670
° long.
33.63 N
49.25 N
04.87 W
Czech Rep.
Sefrou
Tabor
IRA1
Iran
Komkun
29.00 N
53.00 E
FRA1
France
Montombam
48.05 N
01.41 E
PI 234811
SWI
Chur
FRA2
46.87 N
43.36 N
09.53 E
PI 235525t
PI 2511431'
MAC
ETH
PI 260692t
PI 267060
PI 290717t
PI 93-94
PI 315082t
PI 315454
ITA1
Italy
POL
Poland
DireDawa
Perugia
Warsaw
41.59 N
09.37N
21.26 E
PI 260268
Switzerland
France
Macedonia
Ethiopia
UK
United King.
GEO2
Montepl.
Skopje
14.41 E
03.53 E
42.50 N
52.15 N
41.52E
12.50 E
21.00 E
Reading
51.70 N
00.98 W
Georgia
Kasbegi
41.43 N
44.49 E
KAZ
Kasakstan
unknown t
48.00 N
68.00 E
RUS1
Russia
St. Petersburg
59.92 N
30.25 E
PI 319021
SPA
La Ercin
43.53 N
05.34 W
PI 319822
NOR1
09.40 E
NOR2
Oppland
Rosendal
61.10 N
PI 319823
Spain
Norway
Norway
59.59 N
06.01 E
PI 325369
PI 325379
RUS2
UKR
Russia
Ukraine
Stavrop
Yalta
45.02 N
44.30 N
PI 369278
RUS3
Russia
PI 464682
TUR
PI 384882t
PI 419228
PI 419233
PI 430546
1RA2
Turkey
Iran
GRE1
Greece
GRE2
Greece
RUS4
Russia
Novo-Siberski 55.02 N
Akdagma
36.60 N
Parvar
35.30 N
Nikitas
40.14 N
Kastaneai
41.38 N
Dedinovski
55.03 N
45.59 E
34.10 E
82.55 E
PI 93-21
GEO1
Georgia
Khulo
41.41 N
42.18 E
PI 485601
ITA2
Italy
45.23 N
11.41 E
PI 494653
ROM
Romania
Abbadia
Gheorghe
46.14 N
26.44 E
t Not original seed of the accession.
No collection site information available.
29.90 E
53.25 E
23.39 E
26.28 E
39.07 E
Table 3.2. Ecoclimatic range matrix description for the 28 accessions of birdsfoot trefoil.
Ecological descriptors
Ecoregiont
Identif.
Code
ETH
M262
L222
M314
L242
M243
L313
M262
M412
ITA1
L261
POL
L222
L243
M252
L343
L212
M243
MOR
CZE
IRA1
FRA1
SWI
FRA2
MAC
UK
GEO2
KAZ
RUS1
SPA
Domain
Humid temperate
Humid temperate
Dry
Humid temperate
Humid temperate
Dry
Humid temperate
Humid tropical
Humid temperate
Humid temperate
Humid temperate
Humid temperate
Dry
Humid temperate
Humid temperate
(cont'd on next page)
Elev.
Precip.
Division
Mediterranean
Hot continental
Tropical/Subtropical Steppe
Marine
Marine/Mountain
Tropical/subtropical/Steppe
Mediterranean/Mountain
Savanna/Mountain
Mediterranean
Hot continental
Marine
Mediterranean/Mountain
Temperate Desert
Warm/Continental
Marine/Mountain
Temperature
Snow Cloud.
Aver. Low High
m
mm
1680
460
1980
90
1580
30
770
11.4
3.6
22.0
581
6.9
-2.8
17.1
910
2130
300
90
90
800
370
100
700
298
696
1075
146
496
685
949
484
645
1695
245
466
909
cm
15.3
4.8
10.5
4.2
-1.8 -11.2
19.0 10.4
5.4
16.8
13.5
7.9
-5.6
12.9
4.1
-3.5
9.7
3.8
-0.1 -11.0
4.3 -14.0
3.6 -8.3
8.2
2.5
0.0
35.3
25.4
0.0
17.4
2.5
7.0 124.3
28.8
0.0
16.5 16.2
18.8
0.0
23.2 9.25
19.2 35.1
16.5
0.0
10.5 75.5
22.0 104.2
17.2 45.1
14.6
0.0
72.1
36.7
68.4
37.6
39.4
69.7
46.4
67.1
52.5
34.2
31.7
42.7
58.6
30.2
43.0
Table 3.2. (contd.)
Ecological descriptors
Ecoregiont
Identif.
NOR1
NOR2
RUS2
UKR
RUS3
TUR
IRA2
GRE1
GRE2
RUS4
GEO1
ITA2
ROM
Code
Domain
M242 Humid temperate
L244 Humid temperate
L343 Dry
M252 Humid temperate
L137 Polar
M262 Humid temperate
M312 Dry
Humid temperate
L261 Humid temperate
L212 Humid temperate
M252 Humid temperate
L243 Humid temperate
M252 Humid temperate
L261
Elev.
Precip.
Division
Marine
Marine
Temperate desert
Prairie
Subartic
Mediterranean/Mountains
Tropical/Subtropical/Steppe
Mediterranean
Mediterranean
Warm continental
Mediterranean/Mountains
Marine
Prairie
Temperature
Snow Cloud.
Aver. Low High
m
mm
910
760
30
60
120
1290
910
460
150
515
2532
267
452
388
333
842
513
580
533
637
150
900
60
910
751
525
°C
0.6
1.3
9.8
8.3
0.7
7.8
13.4
14.5
13.1
-9.5
-7.0
-6.2
-1.9
-18.0
-6.6
3.6
4.4
2.6
3.7 -13.0
2.8 -9.0
12.6
1.1
6.2 -5.2
cm
11.9 156.2
29.7
10.4
17.2
26.3
25.5
5.5
42.4
20.1
1.1
18.5 195.4
53.6
41.5
19.8
38.8
55.4
24.0
25.4
23.7
17.9
0
69.1
6.4
58.1
0.0
52.2
69.1
36.0
14.1 109.1
48.7
43.2
41.5
24.2
16.4
8.0
27.6
t From Bailey, 1989.
o.)
33
Original seeds of most accessions (see notation, Table 3.1) were
obtained from the USDA-ARS Plant Introduction Station at Pullman, WA. The
seeds were scarified using liquid nitrogen, germinated at 20° C in plastic boxes
on blue blotter paper, inoculated with an appropriate Rhizobium strain, and the
germinated seedlings transplanted to greenhouse flats. Fifteen plants of each
accession were grown in the greenhouse under 16 / 8 h (light / dark) conditions
at approximately 20° C. Plants were fertilized, watered, and treated for insect
and disease pests as needed. All of the genotypes, except PI 260268 from
Ethiopia which required vernalization at 2° C for four weeks, flowered at
ambient temperature and 16 / 8 h (light / dark) conditions in the greenhouse
over a period of three years.
Based on general morphological uniformity among all plants of each
accession, one genotype was chosen from each 15-plant population and
transplanted into a 2.8 I pots filled with commercial potting soil mix. All
morphological characterizations were based on this representative individual.
The remaining 14 genotypes of each accession were maintained in 10 cm
diameter pots. To assess intra-accession genetic variation and validate use of
the single genotype to represent the population of plants from each accession,
five to six plants were selected at random from each accession and analyzed
for RAPD variation (Appendix 1).
Genotype Descriptions
Morphological Characters
A list of 18 morphological characteristic comprised of 12 qualitative and
6 quantitative variables based on earlier descriptions by Chrtkova-Zertova
(1973) were used to assess the range of intraspecific morphological variation
among the genotypes (Table 3.3). Ten observations were measured per
morphological characteristic except for number of flowers per umbel which
used 20 observations per genotype.
Table 3.3. Plant morphological characters used for the birdsfoot trefoil accession descriptions.
Plant charactert
Method of measurement and units
Whole plant
Growth habit
Underground shoots
Colour of plants
(i) erect, (ii) ascending, (iii) decumbent
(i) present, (ii) no present
(i) glaucous, (ii) bright green, (iii) dark green, (iv) brownish green
Stems
Stem firmness
Length of peduncle
(i) solid, (ii) hollow (by transverse cuts)
mm; measurements on well developed secondary branches.
Leaves
Leaflets form
Thickness of leaflets
Leaflets indumentum
Central leaflet length
Central leaflet width
(i) narrow oblanceolate, (ii) oblanceolate, (iii) obovate, (iv) round,
and (v) obcordate.
(i) thin, (ii) slightly fleshy, and (iii) fleshy.
(i) glabrous, (ii) ciliate, (iii) hairy
mm; central leaf, well developed branches.
mm; central leaf, well developed branches.
Flower
Flowers per umbel
Inflorescence position
(cont'd on next page)
20 random undisturbed umbels.
axils of all leaves, axils of upper leaves
Table 3.3. (cont'd.)
Plant charactert
Flower size
Calyx size
Calyx teeth / calyx tube
Calyx teeth form
Calyx indumentum
Color of corolla
t After Chrtkova-Zertkova, 1973.
Method of measurement and units
mm; distance from calyx base to the corolla tip.
mm; random undisturbed umbels, pedicel included.
(i) shorter, (ii) of the same length, (iii) longer.
(i) awl shaped, (ii) lanceolate, (iii) narrow triangular,
(iv) widely triangular.
(i) glabrous, (ii) ciliate, (iii) hairy
(i) pale yellow, brigth yellow, dark yellow.
36
A binary data matrix was constructed to describe the qualitative and
quantitative morphological characteristics (a total of 71 character states) to
determine morphological distance among genotypes (Appendix 2). The
pairwise morphological distance (dmorph) among the 28 genotypes was
calculated using PAUP 3.1 software for the Macintosh (Swofford, 1993)
d morph =
1
a
b-1
[2]
where:
a is the number of shared characters (character state present or absent), and b
is the total number of character states scored.
Molecular Characters
DNA Extraction and Quantification
Plant DNA was isolated by the method described by Steiner et al.
(1995). Fresh leaves (approximately 3 cm2) were collected from each genotype
and put into 1.1 ml tubes, each containing five to six 3.0 mm diameter glass
beads, that were held in microtiter format racks, frozen in liquid nitrogen,
lyophilized, and ground into a fine powder at room temperature. Two replicates
of 30 to 40 mg ground material from each entry were incubated with occasional
gentle mixing in 1.0 ml 100 mM Tris-HCI pH 8.0, 1.4 M sodium chloride, 20 mM
EDTA (ethylenedinitrilo tetraacetic acid), 20 g L-1 CTAB
(hexadecyltrimethylammonium bromide), 10 g L 1 PVP-40, and 20 ml L-1 2­
mercaptoethanol at 60° C for 30 to 40 min. Chloroform (800 ml) was added to
each sample at equal volume, gently mixed, and centrifuged at 11,000 g for 2
min. In a new tube, the upper aqueous phase was added to 750 ml
isopropanol, gently mixed and incubated at -20° C for at least 15 min., and then
centrifuged at 11,000 g for 10 min. The supernatant was discarded and the
pellet washed in 1 ml 700 ml L-1 ethanol, air-dried, and resuspended in 125 ml
37
of 10 mM Tris-HCI pH 8.0 and 1 mM EDTA. The quantity, purity, and integrity of
the genomic DNA was determined by electrophoresis with known amounts of
lambda DNA in a 7 g L-1 Seakem agarose (FMC, Rockland, ME) gels in lx Tris­
acetate EDTA. The samples were diluted to approximately 1 ng m1-1 for use in
the RAPD reactions.
RAPD Analysis
Approximately 10 1A1 of the original extract was diluted 170-fold with 1690
I of water. Three microliters of diluted extract was used in a 15 µl reaction
mixture as template for the RAPD reactions containing 50 mM Tris-HCI pH 9, 45
mM ammonium sulfate, 1.5 mM magnesium chloride, 100 mM dNTP's, 0.2 mM
10-mer primer (Operon, Alameda, USA), 1U Tfl DNA polymerase (Epicentre
Technologies, Madison, USA), and overlaid with 50 RI mineral oil. The six
primers used in the experiment are known from prior experiments to be
polymorphic among birdsfoot trefoil genotypes. The primers OPA-8; OPA-10;
OPB-6,7, and 8; OPB-13; met the selection criteria and produced 31, 27, 21, 18,
15, and 19 polymorphisms, respectively. The RAPD products were selected if:
(i) an intense and unique band occurred in at least one accession, (ii) the band
did not occur in all accessions, and (iii) the band was repeatable in all
replications. The length of the RAPD product bands were determined by
comparison of their mobilities to those of known standards using third-order
polynomial regression equations. The oligonucleotide sequence of each primer
is given in Table 3.4.
The reactions were run in a MJ Research (Watertown, USA) 96-well
microtiter format thermocycler (PTC-100) using the temperature profile: 95°C for
2 min 30 sec, 46° C for 40 sec., 72° C for 1 min., 94° C for 40 sec, 46° C for 40
sec, 72° C for 1 min, 41 cycles of 40 sec each at 94, and a final 9 min at 72° C.
RAPD products were separated by electrophoresis using 11 RI of the reaction
mixture in a 17.5 g L-1 3:1 NuSieve agarose gel (FMC, Rockland, USA) in lx
Tris-borate EDTA The gels were run at a constant 90 mA, including
38
Table 3.4. List of primers used,oligonucleotide sequences, number of RAPD
product bands, and range of RAPD product size for 28 birdsfoot trefoil
genotypes.
Primert
Oligonucleotide sequence
Bands
Length
no.
by
293-1773
469-1720
499-1423
346-1710
431-1144
443-1880
OPA-8
5'-GTGACGTAGG-31
31
0 PA-10
5'-GTGATCGCAG-3'
27
OPB-6
5'-TGGTCTGCCC-3'
21
OPB-7
18
OPB-8
5'-GGTGACGCAG-3'
5'-GTCCACACGG-3'
OPB-13
5'-TTCCCCCGCT-3'
19
t Operon, Alameda, CA.
15
39
a 100 by DNA ladder standard (Gibco-BRL, Gaithersburg, USA) lane in each
gel. Following electrophoresis, the bands were visualized by ethidium bromide
staining and photographed under ultraviolet light with Polaroid 667 film.
Photographs of the gels with stained bands were scanned with a color
scanner (Epson ES-1200C), and the image analyzed with NIH Image (National
Institute of Health, Bethesda, MD) and Metaflo (The Valis Group, Richmond,
CA) software using a 7200 Power Macintosh computer (Apple Computer,
Cupertino, CA).
Genetic distance (dgenetic) was estimated using equation [2],
dgenetic = 1 - a ID
1
except where:
a is the number of shared RAPD product bands (bands present or absent), and
b is the total number of bands scored. Preliminary experiments showed that this
measure was more desirable compared to the Dice index for characterizing
intraspecific variation where the genetic lineages of accessions are known
(data not shown).
Statistical Analysis
Identification of Genetically Diverse Groups
The resulting pairwise morphological and genetic distance matrices
were analyzed by cluster analysis based on Euclidean distance and Ward's
(1964) clustering technique (Systat 5.2.1 for the Macintosh, Evanston, IL).
The congruence of the geographic, ecological, morphological, and
genetic classifications were determined by Mantel's test (Mantel, 1967) using
the MXCOMP command of NTSYS-pc program version 1.80 (Rohlf, 1993). In
performing this test, if both matrices being compared contain corresponding
distances estimates, then the value of the Ztest criterion is large compared to
chance expectation:
40
Z=
Exifyi;
where: X1j and Yij are two different measures relating to nth element of a
sample to the fth element in both matrices. This test required the calculation of
symmetric distance matrices for each combination of classification characters.
The relatedness among specific matrices was measured by the product
moment correlation value which is related to Z The estimated Z value is
compared with its permutational distribution obtained from 500 random
samples of all possible permutations of the matrices. The output of this test
provides an empirical probability of obtaining a random Z value in excess of the
estimated Z (Smouse et al., 1986).
The relatedness of the genetic, morphological, and geographic
distances among the genotypes with individual ecological variables was
measured using Mantel's Z test criterion. The relationships of individual
qualitative morphological traits with individual ecological variables were
determined using the character states for a qualitative trait as the independent
variable and the quantitative value of the ecological variable from each
genotype collection site as the dependent variable in a one-way analysis of
variance using the F-statistic criterion (Snedecor and Cochran, 1980) for
significance (Systat 5.2.1 for the Macintosh, Evanston, IL). The relationships of
the quantitative morphological traits with individual ecological variables was
tested using Pearson's correlation coefficient (Snedecor and Cochran, 1980).
The placement of genotypes into the four RAPD cluster groups were
validated using the RAPD cluster number as the classification factor in a
stepwise discriminant analysis (SPSS 6.1 for the Macintosh, Chicago, IL). The
test of significance of each RAPD product band was done using the univariate
F-ratio (Hair et al., 1997). Based on the resulting analysis, 19 significant RAPD
band products were identified (OPA-8, 293 bp, 1205 bp, 1263 bp; OPA-10, 971
bp, 1009 bp, 1110 bp; OPB-6, 726 bp, 764 bp, 834 bp, 938 bp, 1158 bp, 1423
bp; OPB-7, 783 bp, 836 bp; OPB-8, 595 bp, 627 bp, 830 bp, and OPB-13, 1125
bp, 1542 bp ) that correctly placed 100% of the genotypes.
41
Results and Discussion
Morphological Classification
The 28 genotypes displayed polymorphism for both qualitative (Tables
3.5) and quantitative (Table 3.6) morphological characteristics for the range of
traits reported by Chrtkova-Zertova (1973). The genotypes were classified into
five morphological cluster groups (Fig. 1). The general appearance of the
plants ranged from glabrous to pubescent leaves and erect to decumbent
growth habit. The genotypes mostly had solid stems, except for POL, RUS1,
SPA and RUS3, which had hollow stems. Leaf shape was variable, ranging
from narrow, to obovate, to round leaf forms. The genotypes were also variable
for leaf length and width with ETH, KAZ, RUS4, and GEO1 having the largest
leaves. The MOR and FRA1 genotypes were rhizomatous. Only MOR has been
previously described as having rhizomes (Li and Beuselinck, 1996). Rhizomes
in birdsfoot trefoil have been described for birdsfoot trefoil var. crassifolius, var.
norvegicus, and var. carnosus (Chrtkova-Zertova,1973).
Most of the inflorescence umbels were attached to the axills of the upper
leaves, except for the FRA2, ETH, ITA1, ITA2, GRE1, and GRE2 genotypes that
had the umbels located in the axills of all leaves. The genotypes were also
variable in calyx traits, including teeth form and indumentum. The corolla color
of most mature flowers was bright or dark yellow. Corolla color at the withering
stage of development was either orange yellow or orange pink. The number of
florets per umbel ranged from 1.6 (ETH) to 5.8 (RUS3). Length of the calyx
ranged from 5.6 to 9.8 mm for MOR and ETH, respectively. Total flower length
ranged from 10.0 to 15.5 mm for UKR and GEO1, respectively. The ETH
accession was autogamous (Chapter 4). All other accessions required hand
manipulations to produce self-pollinated pods, if any.
Morphological similarities among genotypes were related to the general
geographic proximities of their collection sites (Table 3.7). The closer the
genotype collection sites are to one another, the more similar the genotypes
are morphologically. Similarly, the more similar the ecological characteristics of
Table 3.5 Description of the 28 birdsfoot trefoil genotypes by 12 morphological qualitative traitst.
Whole plant
Identificat.
Growth Undergr
Stem
habit
shoots Color Firmn.
MOR
CZE
D
E
IRA1
FRA1
A
NP
NP
E
P
SWI
FRA2
MAC
ETH
A
ITA1
D
E
E
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
NP
POL
UK
GEO2
KAZ
RUS1
SPA
NOR1
NOR2
RUS2
UKR
RUS3
TUR
IRA2
A
D
A
A
E
E
D
D
D
A
E
E
D
A
(cont'd on next page)
P
G
BG
G
DG
DG
BG
BG
BG
BG
BG
DG
BG
BG
DG
DG
BG
QG
BG
BG
BG
BG
DG
Calyx
InfluorTube / teeth Teeth Indu- Corolla
Thickn. Indum. position ratio
form mentum color
Leaf
Form
S
S
S
OBC
OBL
NO
T
T
S
OBL
SF
S
S
S
S
S
R
R
T
NO
T
OBL
OBL
OBL
F
H
S
S
S
H
H
S
S
S
S
NO
R
OBL
NO
OBL
OBL
OBO
OBL
R
H
OBC
S
R
NO
S
SF
F
T
T
T
T
T
T
T
T
T
SF
T
T
T
T
C
G
C
C
G
C
C
G
C
G
G
H
C
G
H
C
G
C
C
C
C
C
AUL
AUL
AUL
AUL
AUL
AAL
AUL
AAL
AAL
AUL
AUL
AUL
AUL
AUL
AUL
AUL
AUL
AUL
AUL
AUL
AUL
AUL
SL
SL
SL
L
NT
AS
C
G
C
S
NT
H
SL
AS
AS
G
G
L
L
L
L
S
L
L
S
SL
L
SL
S
L
SL
SL
SL
S
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
WT
NT
NT
WT
H
C
C
G
H
H
C
C
H
C
C
H
H
C
C
C
PY
BY
PY
BY
DY
PY
BY
DY
BY
BY
PY
BY
BY
PY
DY
BY
BY
DY
BY
BY
BY
DY
Table 3.5. (contd.)
Whole plant
Identificat.
GRE1
GRE2
RUS4
GEO1
ITA2
ROM
Growth Undergr
Stem
habit
shoots Color Firmn.
A
A
E
E
A
A
NP
NP
NP
NP
NP
NP
DG
DG
BG
BG
DG
DG
S
S
H
S
S
S
Leaf
Form
OBL
OBL
R
R
OBL
OBL
Thickn. Indum.
T
T
T
T
T
T
C
C
C
C
C
C
Calyx
InfluorTube / teeth Teeth lndu- Corolla
position ratio
form mentum color
AAL
AAL
AUL
AUL
AAL
AUL
S
L
C
SL
L
NT
NT
H
H
SL
SL
L
SL
NT
NT
C
C
H
PY
BY
DY
BY
BY
BY
t Growth habit: E = Erect, A = ascendent, D = decumbent; Underground shoots: P = present, NP = not present; Color
of plant: G = glaucoish, BG = bright green, DG = dark green; Stem firmness: S = solid, H = hollow; Leaflets form:
NO = narrow oblanceolate, OBL = oblanceolate, OBO = obovate, R = round, OBC = obcordate; Leaf thickness: T =
thin, SF = slighly fleshy, F = fleshy; Leaf indumentum: G = glabrous, C = ciliate, H = hairy; Influorescence position:
AAL = axilla of all leaves, AUL = axilla of upper leaves; Calix tube/ calix teeth: S = shorter, SL = of the same length,
L = larger; Calix teeth form: AS = awl shaped, L = lanceolate, NT = narrow triangular, WT = widely triangular; Calix
indumentum: G = glabrous, C = ciliate, H = hairy; Colour of corolla: PY = pale yellow, BY = brigth yellow, DY = dark
yellow;
Table 3.6. Description of the 28 birdsfoot trefoil genotypes by six quantitative traits.
Quantitativ
Identificat.
MOR
Leaflet
width
NO R2
± 0.3
± 0.6
± 0.2
± 0.4
± 0.3
3.7 ± 0.4
3.4 ± 0.2
5.2 ± 0.3
2.8 ± 0.2
4.1 ± 0.6
4.0 ± 0.6
4.2 ± 0.5
7.1 ± 0.7
3.9 ± 0.4
2.4 ± 0.3
3.9 ± 0.4
3.6 ± 0.4
RUS2
4.1
UKR
4.2 ± 0.8
CZE
IRA1
FRA1
SWI
FRA2
MAC
ETH
ITA1
POL
UK
GEO2
KAZ
RUS1
SPA
NO R1
2.5
3.7
2.5
3.5
3.8
(cont'd on next page)
±0.6
Leaflet
length
6.4 -.1: 0.2
8.4 ± 0.4
7.6 ± 0.1
5.8 -.1- 0.1
7.2 ± 0.1
9.1 ± 0.3
7.3 ± 0.2
10.2 -±- 0.3
7.0 ± 0.2
8.7 ± 0.3
8.1 ± 0.6
7.8 ±- 0.2
12.1 ±- 0.3
9.0 ± 0.2
5.9 ± 0.1
8.8 -± 0.2
8.6 ± 0.2
9.0 ± 0.2
7.6 -± 0.2
e
Descr iptors
Floret
number
Peduncle
length
1.7 ± 0.1
30.3 ± 12.9
24.2 ± 11.5
48.5 ± 10.2
52.8 ± 11.2
33.8 ± 07.5
36.6 :t 05.2
48.2 ± 06.6
32.7 ± 07.5
77.8 ± 09.3
46.5 ± 13.0
49.3 ± 06.1
72.1 ± 07.4
81.8 ± 14.7
33.8 ± 06.7
40.6 ± 07.1
52.3 -± 06.7
63.6 ± 07.1
63.4 ± 05.8
91.1 ± 13.7
4.6 ± 0.3
1.8 -± 0.2
3.6 ± 0.2
2.4 ± 0.2
2.0 ± 0.2
3.9 ± 0.3
1.6 ± 0.1
4.1 ± 0.1
4.7 ± 0.2
3.4 ± 0.2
5.0 ± 0.2
4.3 ± 0.1
4.7 ± 0.4
3.2 ± 0.1
5.5 ± 0.2
4.8 ± 0.2
3.6 ± 0.3
2.8 ± 0.3
Flower
size
Calyx
size
11.5 ± 0.2
13.2 ± 0.2
10.6 ± 0.2
13.6 ± 0.2
13.8 ± 0.2
12.3 -±- 0.1
13.0 ± 0.2
11.4 -±- 0.2
14.8 ± 0.1
11.6 ± 0.1
12.8 ± 0.2
15.0 ± 0.1
14.0 ± 0.2
13.0 ± 0.2
12.2 ± 0.1
14.5 -±- 0.1
15.0 ± 0.1
12.6 ± 0.3
10.0 ± 1.5
5.6 ± 0.1
6.8 ± 0.1
5.7 ± 0.2
6.1
±0.2
6.4
8.4
9.2
9.8
7.8
6.5
7.6
8.8
7.9
8.5
7.8
7.9
7.6
7.3
6.7
± 0.1
± 0.3
± 0.1
± 0.1
± 0.1
± 0.1
± 0.1
± 0.2
± 0.2
± 0.1
± 0.2
± 0.2
± 0.2
± 0.1
± 0.2
Table 3.6. (cont'd.)
Quantitativ
Identificat.
RUS3
TUR
IRA2
GRE1
GRE2
RUS4
GEO1
ITA2
ROM
Leaflet
width
3.5 ± 0.6
4.6 ± 0.6
3.1 ± 0.6
3.1
±0.6
3.7 ± 0.4
4.5 ± 0.6
5.1 ± 0.8
3.4 ± 0.3
2.7 ± 0.3
e
Descr iptors
Leaflet
length
Floret
number
Peduncle
length
8.3 ± 0.4
5.8 ± 0.3
3.9 ± 0.2
51.5 ± 07.1
46.1 ± 08.4
42.2 ± 05.5
51.9 ± 04.8
46.6 -± 06.2
52.2 ± 04.2
63.7 ± 04.5
58.3 ± 03.3
53.9 ± 04.7
7.3 :4.-_ 0.3
7.9 ± 0.1
8.8 ± 0.4
8.8 -± 0.3
12.2 ± 0.3
9.6 ± 0.1
7.4 -± 0.2
7.0 ± 0.1
2.1 ± 0.1
3.1 ±0.2
3.3 ± 0.2
5.4 ± 0.2
4.1 ± 0.2
3.1 ± 0.2
4.3 ± 0.2
Flower
size
Calix
size
13.8± 0.2
7.1 ±0.2
6.1 ± 0.1
10.9 ± 0.2
12.9 ± 0.3
14.5 ± 0.3
14.8 ± 0.1
14.8 ± 0.1
15.5 ± 0.1
11.9 ± 1.6
14.2 ± 0.1
7.0
6.9
9.2
7.8
8.2
9.5
8.6
± 0.1
± 0.2
± 0.1
±- 0.1
± 0.2
± 0.1
± 0.1
46
Group 1
RUS4
GEO2
UKR
TUR
Group 2
POL
CZE
UK
SPA
FRA1
RUS2
GE01
MAC
ITA1
Group 3
KAZ
RUS3
NOR1
NOR2
.RUS1
ETH
Group 4
FRA2
MOR
IRA1
SWI
IRA2
GRE1
Group 5
GRE2
ITA2
ROM
I
0.0
I
0.17
I
0.35
I
0.52
I
0.70
Euclidian distance
Figure 3.1. Ward's cluster analysis classification of 28 exotic
old world birdsfoot trefoil genotypes based on 18
morphological traits described by 71 binary characters
states using Euclidian distance.
47
Table 3.7 Normalized Mantel statistic (2) from comparisons of different proximity
matrices using RAPD, combinations of morphological, ecological characters
and geographic, distances.
Descriptor
Genetict
Geographicalt
Ecological§
Z-value
Morphological°
Genetic
Geographic
1.6 *
2.4 ***
1.9 *
2.4 ***
1.5
4.2
t Matrix of genetic distances calculated in PAUP using 130 random amplified
polymorphic DNA bands.
Geographic distances.
§ Matrix of Euclidian distances(Swoffords's, 1993) constructed using six
ecological descriptors.
# Matrix calculated using 18 morphological traits (ChrtkovaZertova, 1973) in
71 binary characters states using Swofford's (1992) distance.
* ,** ,*** significant at P s 0.05, P s 0.01 and P s 0.001 level respectively.
11
48
the genotype collection site are, the more similar the genotype morphology.
The geographic and ecological classifications are highly similar (Z= 4.2; P s
0.001). The only ecological descriptors that are not related to geographic
distance are high temperature and precipitation (Table 3.8).
General morphological classification similarities were attributed to low
temperature, amount of sunshine, elevation, and latitude (Table 3.8). Among
the quantitative morphological traits, the expression of number of florets per
umbel was highly affected by environmental factors (Table 3.9). There is a great
deal of collinearity among many of the ecological characteristics (data not
shown). Using stepwise multiple correlation, the number of florets per umbel is
most significantly influenced by the amount of sunshine and low temperature of
the collection sites (R = 0.86; P s 0.0001). Sunshine and low temperature are
collinear (r = 0.53; P s 0.004). The only other quantitative morphological trait
associated with an ecological variable was leaf length with longitude. Leaf
length increased as the genotype collection site moved east (Table 3.9).
Genotypes collected at lower latitudes tended to have the umbels
located in the upper leaf axilla more than those collected at higher latitudes
which had the umbels distributed along the length of the stems (37° vs. 46° N,
respectively; Table 3.10). More upper leaf axilla-borne umbel genotypes are
found in warmer than cooler environments (14.9 vs. 6.2° C, respectively). This
may be an adaptive feature for cooler climates where the number of growing
degree days are limited but day length is long during the time of reproduction.
Such a mechanism could maximize the number of seeds that are produced by
producing inflorescences at all stem nodes, rather than only at the terminal end
of a stem.
Plants with decumbent growth form tend to be located at lower
elevations (233 m) than plants with ascending (832 m) and erect (926 m)
growth forms. Decumbent growth form is also found at higher latitudes (50.2° N)
than ascending plants (38.5° C). Erect plant growth form genotypes, with the
exception of NOR1 and NOR2, also tend to occur at lower latitudes. Glaucouscolored genotypes (MOR and IRA1) were only found at high altitudes (1830 m).
These findings are in general agreement with those of Chrtkova-Zertova
Table 3.8. Association among genetic, morphological, and geographic classifications with nine ecological descriptors
for 28 birdsfoot trefoil genotype collection sites.
Ecological descriptorst
Classification
Snow
Temperature
Average Low
High
Cloudiness
Precipitation
Elevation
Geography
Latitude
Longitude
Z-valuet
Genetics
Morphological
Geographic#
-1.4
1.2
1.8*
0.5
-0.1
4.4***
2.8**
4.1***
5.2***
1.8*
-0.5
2.6**
2.2 **
5.6***
3.9***
0.6
0.8
1.1
0.1
0.3
3.2***
3.0**
3.3***
4.6***
0.5
0.2
7.8***
t Matrices constructed from individual ecological characters.
t * ,** ,*** significant Mantel statistic at P s 0.05, P s 0.01 and P s 0.001, respectively.
§ Matrix of distances (Swofford's, 1992) constructed from 130 random amplified polymorphic DNA bands using PAUP
software.
Matrix calculated using 18 morphological traits (Chrtkova-Zertova, 1973) in 71 binary characters states using
Swofford's (1992) distance,
# Matrices constructed from geographic distances.
50
Table 3.9. Pearson's correlation coefficients (r) between four quantitative
morphological traits and nine ecological descriptors.
Morphological descriptors
Descriptor
Leaf
length
Leaf
width
Calyx
length
Floret
number
0.04
0.62***
-0.71***
-0.74***
-0.45**
-0.76***
0.14
-0.44**
0.75***
0.16
r
Snow
Aver.Temp.
Low temperature
High temperature
Cloudiness
Precipitation
Elevation
Latitude
Longitude
0.28
-0.11
-0.29
0.10
-0.05
-0.20
-0.16
-0.05
-0.16
-0.04
0.11
-0.01
0.42*
0.36
0.36
-0.26
-0.37
-0.11
0.01
0.01
-0.04
-0.06
0.09
-0.14
-0.10
0.07
* ,** and *** significant at P s 0.05, P s 0.01 and P s 0.001, respectively.
Table 3.10. Analysis of variance relationships for ten qualitative morphological traits with nine ecological descriptors.
Qualitative morphological descriptors
Descriptor
Snow
Temp. avr.
Low temp.
High temp.
Cloudiness
Precipitatation
Elevation
Latitude
Longitude
Umbel
position
Calyx
ratio
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
****
***
ns
ns
ns
ns
*
ns
Leaf
pubesc.
ns
ns
ns
**
ns
*
ns
ns
ns
Calyx
teeth
form
ns
ns
ns
ns
**
ns
ns
ns
ns
Corolla
color
Leaf
shape
Plant
color
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
Leaf
Calyx Growth
thickness pubesc. habit
ns
*
**
ns
****
*
*
ns
ns
ns
ns
*
ns
ns
*
ns
* ** ***, **** significant at P s 0.05, 0.01, 0.001, and 0.0001, respectively.
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
52
FRA2
MAC
Group 1
IRA1
ETH
MOR
CZE
'
KAZ
RUS3
UKR
NOR1
SWI --1
FRA1 --I
Group 2
SPA
GE01
RUS1
POL
UK
NOR2
ITA1
I
1
RUS2
GRE1
Group 3
GRE2
IRA2 1____
TUR
RUS4
Group 4 ITA2
I
ROM
1
I
I
I
I
I
0.0
0.15
0.30
0.45
0.60
Euclidian distance
Figure 3.2. Classification of 28 exotic old world birdsfoot
trefoil genotypes based on 130 RAPD product bands using
Euclidian distance and Ward's cluster analysis.
53
(1973). Light- and dark-green plants are found at low average altitudes with
the exceptions of SWI, ETH, and NOR2.
Leaves with ciliate indumentum are found in regions with lower
precipitation and higher average temperature (523 mm and 20.9° C,
respectively) than leaves with glabrous (924 mm and 15.2° C, respectively) and
hairy (1302 mm and 12.5° C, respectively) indumentum. There are no
difference between the ecological variables describing glabrous- and hairy-leaf
indumentum classes. This finding differs from the observation of Chrtkova-
Zertova (1973). More genotypes with thicker leaves are collected in warm and
sunny environments at low latitudes than at cool and cloudy envirnonments.
This finding also disagrees with the observations of Chrtkova-Zertova (1973).
The finding of associations between birdsfoot trefoil morphological traits
and ecological descriptions of genotype collection sites is supported by other
research. Associations between altitude and morphological traits (Small et al.,
1984), agronomic forage quality factors and geographic origins (McGraw et al,
1989), and seed proteins (Steiner and Poklemba, 1994) have been reported.
Re-analysis of the Chrtkova-Zertova (1973) data shows a trend of species
varietal development from warm, widely distributed genotypes to cooler, more
geographically restricted regions (Steiner, 1998). These findings disagree with
the conclusions of Grant and Small (1996) that geographic distance is not
associated with plant morphological, genetic, and chemical composition.
Genetic Classification
The genotype classification based on 130 polymorphic RAPD bands
produced four cluster analysis groups (Fig. 3.2). Several polymorphic bands
were observed for each primer, and most of them produced band sizes ranging
from 293 by to 1880 by (Table 3.4). The four RAPD groups were used as
classes in a stepwise multiple discriminant analysis to validate the cluster
analysis classification of the genotypes. Nineteen of the 130 bands were
identified as significant (P 0.05) descriptors that differentiated genotypes.
54
The four classification groups were described by three discriminant functions
using 19 RAPD band products as the primary distinguishing markers and
produced a canonical correlation value of 0.999. One-hundred percent of the
accessions were correctly placed by the stepwise discriminant analysis
function.
As with the relationship between morphological and geographic
similarities, the closer the geographic distance the genotypes are collected
from one another, the more similar their genetic distance (Table 3.7). There was
no general relationship between genetic and ecological distance. However,
RAPD Group-1 genotypes were all collected from the lowest average latitudes
(36.3° N). These sites also received the greatest amount of average annual
sunshine (60%) of the four RAPD classification groups and were mostly found
in countries near the Mediterranean Ocean and Asia Minor. In contrast, RAPD
Group-2 genotypes were collected at the greatest average latitude (50.1° N)
and had the least average sunshine (39%) of all RAPD classification groups.
These genotypes are primarily from northern and continental Europe.
Genotypes in RAPD Group-3 are primarily of southeastern European origin.
RAPD Group-4 genotypes are the most genetically diverse from the other
classification groups and are found in southern Europe. The greater the
collection site latitude, the lower the sunshine percentage (r = -0.785; P s
0.0001). Group-4 genotypes also were different from other RAPD classification
genotypes for cross-compatibility reproductive success (Chapter 4). This result
verifies the uniqueness of these genotypes from others based on RAPD
analysis. The lack of similarity between genetic and ecological classification
suggests that genotypes adapted to similar but geographically distant habitats
were derived from different progenitors but have acquired similar phenotypes.
Birdsfoot trefoil accessions from the NPGS collection were also shown to be
associated with geographic regions based on seed protein patterns (Steiner
and Poklemba, 1994).
Because RAPD descriptors are associated with geographic proximity of
genotypes collection sites but not with their ecological similarity, classifications
of germplasm should not solely rely on ecological descriptions. However,
55
because low temperature and cloudiness are associated with the genetic
classification of genotypes, these ecological descriptors may be important
criteria when selecting germplasm adapted to specific environments. Specific
traits may be associated with native habitat conditions and thus provide insights
into adaptive mechanisms that could be useful for crop improvement (Rick,
1973). Useful germplasm may be identified in regions that are environmentally
distinct from those where germplasm has previously been obtained (Steiner
and Poklemba, 1994; von Bothmer and Severg, 1995).
Classification Method Comparisons
The genetic and morphological classifications were associated with one
another (Table 3.7). The deviation in genetic and morphological classifications
may be due to conservation of morphological traits for adaptive reasons, in
spite of the occurrence of random mutations (Johns et al., 1997). It is believed
that populations exhibiting similar molecular composition but dissimilar
morphology have resulted from recent selection pressures that have resulted in
few gene changes (Crawford, 1990). Single genes can have a significant effect
on morphological trait expression (Beer et al,. 1993). However, the association
between morphological and genetic classifications of genotypes indicates that
phenotypic assessments can be an effective selection criteria for evaluating
birdsfoot trefoil germplasm.
Geographic distance among genotype collection sites is highly related to
ecological distance (Table 3.7). However, if geographic locations alone are
used to classify germplasm, then important adaptive features may be lost when
searching for useful genotypes. The genotypes NOR1 and NOR2 (which are
morphologically and genetically similar; Figs. 3.1 and 3.2) are from
geographically close proximity to one another, but are ecologically diverse due
to difference in collection site elevation, precipitation, and snowfall (Table 3.2).
Genotypes IRA1 and I RA2 are from sites that are of close geographic and
ecological proximity, are similar morphologically, but are genetically diverse.
56
Overall, using a combination of evaluation methods including RAPD,
morphological, and ecological descriptors reveals combinations of variation
among the birdsfoot trefoil genotypes that would not be apparent with any
single measurement. The utility of combining various kinds of descriptors
provides a more complete understanding about the diversity present in
birdsfoot trefoil germplasm collections and provides information that may
improve utilization of these resources.
Specific morphological traits were identified using specific combinations
of RAPD product bands as determined by stepwise discriminant analyses (data
not shown). However, further validation of these RAPD markers using individual
genotypes is needed to determine their usefulness as selection tools for
identifying potentially useful traits when screening birdsfoot trefoil germplasm.
Similarly, specific RAPD band products were identified that are associated with
ecoregion domains (data not shown). Associations between Trifolium
incarnatum L. influorescence color and specific RAPD product bands have also
been reported (Steiner et al., 1998). More detailed analysis may reveal other
associations between RAPD bands and birdsfoot trefoil traits of interest.
Conclusions
The 28 genotypes displayed polymorphism for both qualitative and
quantitative morphological characteristics and were classified into five
morphological cluster groups. Morphological similarities among genotypes
were related to the general geographic proximities of their collection sites.
Genotype morphology was also associated with the general ecology of the
collection site habitat and specific morphological traits were found related to
specific environmental characters. Floral morphological characteristics were
related to collection site latitude and average temperature. Plant growth form
was correlated with collection site elevation and latitude. There was no
relatioship between glabrous and hair leaf indumentum expression.
Geographic distance among genotype collection sites was associated
57
with genetic distance. The genotypes were grouped into four RAPD
classification groups that were related to their geographic regions of origin that
were Mediterranean basin/Asia Minor, northern and continental Europe,
southeast Europe, and southern Europe. However, ecological similarity of the
genotype collection sites is not related to their genetic similarity. The lack of
similarity between the genetic and ecological classifications suggests that
genotypes adapted to similar but geographically distant habitats were derived
from different progenitors but have acquired similar phenotypes. Because
RAPD descriptors are associated with the geographic proximity of genotypes
collection sites but not with their ecological similarity, classifications of
germplasm should not solely rely on ecological descriptions. The utility of
combining RAPD, morphological, and ecological descriptors reveals
combinations of variation among the birdsfoot trefoil genotypes that would not
be apparent with any single measurement and can provide a more complete
understanding about the diversity in birdsfoot trefoil germplasm collections.
58
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von Bothmer, R., and 0. Seberg. 1995. Strategies for the collecting of wild
species. p. 93-111. In L. Guarino et al. (ed.) Collecting plant diversity.
CAB Intern., Wallingford, UK.
Ward, J.H. 1964. Hierarchical grouping to optimize an objective function. J. Am.
Stat. Assoc. 58:236-244.
62
CHAPTER 4
ADAPTIVE ECOLOGY OF Lotus corniculatus L. GENOTYPES: II.
REPRODUCTIVE COMPATIBILITY AND INTROGRESSION ABILITY
Abstract
Birdsfoot trefoil (Lotus corniculatus L.) is a widely distributed polymorphic
perennial forage legume species found in wild and naturalized populations
throughout temperate regions of Europe, Asia Minor, North Africa, and North
and South America. Information about the reproductive compatibility among
genetically and ecologically diverse genotypes is scant. The objectives of this
research were to: (i) characterize the ease of introgression of 28 birdsfoot trefoil
genotypes into two genetically diverse hybridization testers, and (ii) determine if
the cross-compatibility among exotic genotypes is related to their genetic
background and ecological origins. The ease of introgressing 27 exotic
genotypes into other germplasms backgrounds was determined by using
bidirectional crosses with a domestic and an exotic genotype tester. A diallele
crossing matrix of eight genotypes was also used to determine crosscompatibility among exotic germplasm. Reproductive success was measured
as percentages of pods set and pollen viability. The self pod set and pollen
viability percentages of the genotypes were not correlated, but exhibited a
range of differences. Incompatibility among crosses was expressed as either an
inability to set pods or the production of progeny that had inviable pollen.
Intermediate crosses could be identified that could bridge any combination of
genotypes that were incompatible. Reproductive barriers to cross compatibility
in birdsfoot trefoil are environmentally neutral and distributed randomly
throughout the species. This differs from the findings that the distribution of
birdsfoot trefoil morphological characteristics are associated with the ecological
origins and genetic similarities of the genotypes. Even though birdsfoot trefoil is
63
origins and genetic similarities of the genotypes. Even though birdsfoot trefoil is
morphologically diverse, exotic germplasms can be utilized by conventional
breeding methods.
Introduction
Birdsfoot trefoil is a perennial, non-bloating, forage legume that has
valuable attributes making it a good alternative to other temperate region
legumes. It is better adapted to slightly acid, droughty, infertile, or wet soils that
do not support other forage legumes (Seaney and Henson, 1970; Marten et al.,
1987). Information about effective cross-compatibility of ecologically and
genetically diverse birdsfoot trefoil genotypes is needed to determine the
efficacy of introgressing traits from exotic germplasm sources. Incompatibility
barriers could limit the potential for utilizing traits from exotic germplasm
sources not presently used in commercial cultivars.
Birdsfoot trefoil generally exhibits poor self-compatibility (Miller, 1969),
but autogamous genotypes are available (Steiner, 1993; Steiner and
Poklemba, 1994). It was observed that some Lotus species do not produce
seed under greenhouse conditions without manipulation. Only about 3% of the
genotypes produce seeds when manipulations are made by hand (McDonald,
1946). However, successful tripping of flowers under greenhouse conditions
can be variable (McKee, 1949). The inheritance of male sterility in birdsfoot
trefoil may involve the cytoplasm and be controlled by several complimentary
genes (Negri and Rosellini, 1996). The percentage of seed pods set has been
used as a measure of cross compatibility success in birdsfoot trefoil (Miller,
1969).
Wild relatives of domesticated crops can be rich sources of valuable
traits. Even though plant introductions provide a diverse array of genes, this
diversity may not be easily incorporated into commercial-adapted materials
(Hallauer, 1978). One reason may be that mechanisms maintaining genetic
isolation exist between diverse germplasm pools. The potential value of wild
germplasm and the risk of extinction of uncollected types, creates an urgent
64
need for accelerating the collection and characterization of germplasm
(Paterson et al., 1991).
Extensive genetic variation has been reported within birdsfoot trefoil
(Chrtkova-Zertova, 1973). However, many cultivars of birdsfoot trefoil have
been developed from a narrow germplasm base (Steiner and Poklemba,
1994). Therefore, it would be useful to determine the degree of reproductive
compatibility among diverse genotypes and identify possible routes to
introgress traits from exotic germplasm not previously utilized in plant breeding
programs. Barrier to reproductive success may cause difficulties if exotic
genotypes are used for cultivar development (Schaaf and Hill, 1979).
Identifying possible crossing bridges to transfer characters also could allows us
to improve birdsfoot trefoil types where incompatibility among specific
genotypes precludes successful hybridization. Knowledge about the fertility
and crossing behavior of birdsfoot trefoil genotypes is scant, and little work has
been done to determine the genetic basis of taxonomic characters through
crossing studies (Negri et al., 1989).
In addition, scant information has been available describing the
association among different kinds of descriptors that could be used to
characterize birdsfoot trefoil germplasm collections. Many germplasm collection
classifications are based on a few traits with a limited understanding of their
geographic origins (Brown, 1989). In germplasm conservation, considering the
relationships among ecological and reproductive traits may be important.
Recently, it has been proposed that through understanding specific
associations among genetic traits and environmental descriptors it may be
feasible to describe and better utilize the intraspecific variation available within
germplasm collections (Beuselinck and Steiner, 1992). It would be valuable to
know whether relationships exist among reproductive traits affecting
hybridization and ecogeographic descriptors so that we can better characterize
and utilize birdsfoot trefoil germplasm. Differences in compatibility among
genotypes may suggest similar genetic origins. Differences in the degree of
genetic relatedness among genotypes and their congruence with reproductive
compatibility have not been reported. Studies like these are needed to provide
65
complementary information for a better characterization of germplasm
collections.
The objectives of this research were to: (i) characterize the ease of
introgression of 28 birdsfoot trefoil genotypes into two genetically diverse
hybridization testers, and (ii) determine if the cross-compatibility among exotic
genotypes is related to their genetic background and ecological origins.
Materials and Methods
Genetic Materials
Twenty-eight old-world exotic genotypes and one North American
germplasm of birdsfoot trefoil from the National Plant Germplasm collection
(Table 4.1) were chosen based on the ecological diversity of the collection site
origin (Chapter 3). Original seeds of most accessions were obtained from the
USDA-ARS Plant Introduction Station at Pullman, WA. The seeds were
scarified using liquid nitrogen, germinated at 20° C in plastic boxes on blue
blotter paper, and germinated seedlings transplanted to greenhouse flats.
Fifteen plants of each accession were selected at random, inoculated with an
appropriate Rhizobium strain and transplanted into flats in the greenhouse
under 16 / 8 h (light / dark) conditions at approximately 20° C. Based on plant
appearance among genotypes within an accession, a single genotype was
chosen from each accession and transplanted into 10 cm diameter pots.
Vegetative cuttings of all 28 genotypes were made to assure ample numbers of
plants to produce flowers for crossings. The plants were fertilized, watered, and
pests controlled as needed to maintain active growth.
All of the genotypes, except PI 260268 from Ethiopia (ETH) which
required vernalization at 2° C for four weeks, flowered at ambient temperature
and 16 / 8 h (light / dark) conditions in the greenhouse over a period of three
years.
66
Table 4.1. Pollen viability and self compatibility percentages of the 29 birdsfoot
trefoil genotypes.
Genotype
Accession
number
Pollen
Identification
Country
viability
Self
compatibility
-------%
PI 31276
PI 180171
PI 227512*
Fl 234670
P1234811
P1235525*
PI 251143*
PI 260268
Fl 260692*
PI 267060
PI 290717*
PI 93-94
P1315082*
Fl 315454
P1319021
PI 319822
P1319823
P1325369
P1325379
PI 369278
P1464682
PI 384882*
PI 419228
PI 419233
P1430546
PI 93-21
PI 485601
P1494653
NC-83
MORt
Morocco
Czech Republic
60.2
IRA1
Iran
FRA1
91.8
96.7
68.2
84.9
MAC
France
Switzerland
France
Macedonia
ETHt
Ethiopia
CZE
SWIt
FRA2t
ITA1
Italy
POL
UK
GEO2
KAZ
Poland
United Kingdom
Georgia
Kasakstan
RUS1
SPA
NOR1
NOR2t
RUS2t
95.1
97.1
97.1
94.6
98.6
96.5
99.7
13.1
97.1
19.3
Russia
Spain
Norway
94.0
78.7
Norway
0.0
0.0
0.0
10.7
3.6
0.0
0.0
8.0
0.0
8.0
3.7
12.0
0.0
8.0
10.0
4.9
85.1
UKR
RUS3
Russia
Ukraine
Russia
95.6
95.5
94.3
74.0
TUR.I.
Turkey
93.1
IRA2
GRE1
GRE2
RUS4
GEO1
ITA2
ROM
Iran
92.4
88.4
NC-83t
7.4
0.0
0.0
40.1
0.0
1.5
0.0
94.0
0.0
0.0
0.0
Greece
Greece
Russia
Georgia
Italy
Romania
USA
t Genotypes used for dialelle crossing analysis
t Original seed of the accession not available.
95.1
98.4
65.0
59.7
92.3
82.2
Measures of Reproductive Success
67
Reproductive success was determined as: (i) the percentage of pods that
set following crossing, (ii) the number of seeds produced per pod that
successfully set, (iii) pod length, and (iv) the pollen viability percentage of the
resulting F1 progeny from each cross.
Bee sticks (Williams, 1980) were used to collect pollen from
inflorescences and 70% ethanol used to kill pollen on the bee stick between
each cross. The selfing percentage of each genotype was determined using at
least 15 florets that were rolled between the fingers with slight pressure
(Seaney, 1962). At maturity, which occurred approximately 30 days after
pollination, the pods were harvested, the seeds threshed by hand, and the
number of fully-developed and shriveled seeds were counted.
Three randomly selected, well-developed florets collected during
anthesis from all parental genotypes and from florets of their F1 hybrid progeny
were squeezed to exude pollen onto a microscope slide. The pollen was
immediately stained with a mixture of 50% glycerol and 50% water containing
acetocarmin (1g/100 ml). The slide cover slips were sealed with clear nail
polish. At least five plants per cross were sampled and the average pollen
viability determined (Beuselinck et al., 1996). The reliability of this technique
was verified by comparison with an in vitro pollen germination method (Kariya,
1989) using a mixture of 20% sucrose, 1% agar, and 20 ppm boric acid. No
pollen germination occurred in the absence of boric acid. A correlation
coefficient of 0.66 (P s 0.001) was found between the two methods.
Ease of Introgression with USA and SWI Testers
The ease of introgressing exotic traits into other birdsfoot trefoil genetic
backgrounds was determined by using bidirectional crosses of USA and SWI
genotype testers with the remaining 27 exotic genotypes. The USA genotype
68
had an erect-growth habit and appearance of North American cultivars while
SWI has the morphological appearance of L. alpinus. A minimum of 15 flowers
were used for crossing in both directions using the USA and SWI testers. All
crosses, except for ETH as a female receptor, were done without emasculation
one to three days before pollen dehiscence. Flowers from the ETH genotype
used as female receptors were emasculated by removing the 10 stamens and
fused keel petals with forceps, and leaving intact the standard and two wing
petals (Seaney, 1962). Pollen was transferred after full expansion of the
emasculated flower (one day).
Cross-compatibility Among Exotic Genotypes
The USA, SWI, and six other of the 27 exotic genotypes (SWI, MOR,
FRA2, ETH, TUR, RUS2, and NOR2) selected for their genetic and ecological
diversity were included in a diallele crossing matrix to determine the crosscompatibility among exotic genotypes. The reciprocal crosses among all
genotypes were done using at least 80 flowers per cross in both directions. All
crosses were done as described above for determine ease of introgression.
Statistical Analysis
Analysis of variance was used to determine the effects of genotype,
crossing direction, and their interaction on reproductive success. The mean
comparisons were done using Fisher's protected LSD at P s 0.05 (Statview
512+, Brain power, Inc. Calabasas, CA.). Box plots were used to present
summaries of differences between USA and SWI parental testers and crossing
direction for percentage pod set, pod length, seeds per pod, and F1 pollen
viability. Box plots were also used to display the results from the diallele cross
among the eight genotypes.
69
Pearson's correlation (Snedecor and Cochran, 1980) was used to
determine associations between parental clone self-compatibility and pollen
viability percentages as well as the relationships among the measures of
reproductive success with ecological characteristics of the collection sites of the
genotypes. Only genotypes that set pods were used in the correlation analyses
for pod length, seeds per pod, and F1 pollen viability. The ETH genotype was
excluded from the correlation of percentages selfed pods set and pollen
viability because it is nearly autogamous.
The four genetic classification groups based on random amplified
polymorphic DNA (RAPD) from Chapter 3 were used as the independent
variable in an analysis of variance to determine the effect of genetic
classification on the percentage of pods set and pollen viability of the parental
clones. Mean separation was based on Fisher's protected LSD test (Systat
5.2.1 for the Macintosh, Evanston, IL).
Similarity matrices were constructed for the eight genotypes used in the
diallele crossing block for (i) reproductive distance, a Pearson correlation
matrix (Systat 5.2.1 for the Macintosh, Evanston, IL) of parental self
compatibility and pollen viability; and average percentage pod set, pod length,
seeds per pod, and F1 progeny pollen viability percentage of each genotype
used both as male and female parents crossed with the other seven genotypes;
(ii) genetic distance of the genotypes as determined in Chapter 3 using PAUP
3.1 (Swofford, 1993; and (iii) morphological distance as determined in Chapter
3. The congruence of the reproductive, morphological, and genetic
classifications were determined by Mantel's test (Mantel, 1967) using the
MXCOMP command of NTSYS-pc program version 1.8 (Rohlf, 1993) as done
in Chapter 3.
70
Results and Discussion
Characterization of Parental Genotypes
Characterization of the accessions for pollen viability and level of selfing
is shown in Table 4.1. In general, all genotypes had pollen viability
percentages greater than 70% except for MOR, SWI, GEO1, and ITA2. These
genotypes may be hybrids of birdsfoot trefoil that express reduced pollen
viability. The range of variation for selfing was from 94% for ETH to no selfing
for 14 of the 29 genotypes. The ETH genotype comes from a family of
accession that are the only know wild tetraploid genotypes of birdsfoot trefoil
(Steiner and Poklemba, 1994). The range of self compatibility of those
genotypes that were self compatible are greater than that described by
McDonald (1946). Chandler et al. (1986) have reported that wild Helianthus
genotypes with low pollen viability percentages sometimes indicate that the
individuals are actually natural hybrids. Because birdsfoot trefoil is comprised
of many highly variable morphological forms (Chrtkova-Zertova, 1973), it is
possible that some of the genotypes used are hybrids between birdsfoot trefoil
varieties. There was no relationship between percentages selfed pods set or
pollen viability (r = 0.20; P s 0.32; excluding ETH which is nearly autogamous).
The length of parental pods was positively correlated with the number of
seeds produced per pod, regardless of genotype or crossing direction (USA
female and male are 0.77 and 0.81, respectively, and SWI female and male are
0.82 and 0.81, respectively; P s 0.001). The correlations among all other
measures of reproductive success were not significant, regardless of genotype
or crossing direction (data not shown). There was no relationship between
parental pollen viability and selfed pod percentages with any of the collection
site ecological variables (data not shown).
71
Ease of Exotic Genotype Introgression with USA and SWI Testers
The measures of reproductive success for the USA and SWI testers with
the 27 exotic genotypes are given in Fig. 4.1. When introgressing exotic
germplasm, it is important to choose a partner that is reproductively crosscompatible. Attempts to transfer desirable genes from wild accessions into
cultivated genotypes can be accompanied by difficulties with crossincompatibility.
Differences in crossing direction exist, with USA serving equally well as
a female or male parent with the other 27 genotypes for percentage of pods set
(P s 0.10). The SWI tester performed better as a female parent than as a male
(P s 0.0001). However, the F1 progeny from the SWI female tester had lower
average pollen viability than the USA progeny (P s 0.05).
Two levels of incompatibility were observed. Some crosses among
genotypes were incompatible as a result of not setting any pods (Table 4.2).
Using USA as the female parent, only GEO1 pollen did not set pods. All
genotypes were compatible with SWI as the female parent. Ten of the
genotypes were not compatible with USA pollen (CZE, FRA2, GRE2, IRA2, KAZ,
POL, RUS1, RUS3, SPA, and UK) and six not compatible with SWI pollen
(CZE, KAZ, ROM, RUS4, SPA, and UK). Conversely, some crossing
combinations showed hyper-compatibility relative to the rest of the crossing
combinations (e.g., NOR and ITA2 female genotypes are very compatible with
USA pollen, Figure 4.1).
A second level of incompatibility resulted from crossing combinations
that successfully produced pods, but the resulting F1 progeny did not produce
viable pollen. These included the USA tester as the female with ETH and ROM
pollen, USA pollen with ETH as the female parent, SWI female with IRA1
pollen, and ETH and UKR females with SWI pollen (Figure 4.1). The fact that
the exotic genotypes did not show differences in crossing direction for most of
the reproductive characters when crossed with NC-83 may indicate that this
genotype could be used as a female or male parent. A high degree of
reproductive success can be obtained (percentage of pods set) using both
NC-83
100
ITA2
NOR2
A
e
0
80
-.5 60­
234811
NC-83
40
0
35.
234811
B
E 30
O
.c 25
tr) 20
ILD
15
'3 10
-0 40
20
a_
0
0
5=1;0 a
a
0
a
5.
0
M
F
M
F
M
F
M
F
M
100
--7; 90
80
,..?; 70
60
.5 50
40
a) 30
75
20
_ca=
a_
F
M
F
Crossing direction
M
10
0
Crossing direction
Figure 4.1. Female (F) and male (M) cross compatibility between NC-83 and PI 234811 with 27 exotic
genotypes measured as: A, percentage of pod set; B, pod length; C, seeds per pod; and D, Fi pollen
viability. Accession names at the top and bottom of A and D indicate extremes combination values for pod
set and pollen viability, respectively. Box plots labeled with different letters indicate mean comparison
between male and female parents are different at P s 0.05 according to LSD test.
73
USA and SWI as female parents (96 and 100%, respectively) (Table 4.2).
However, when using these two testers as pollen sources, fewer successful
crosses resulted (63 and 78% for USA and SWI, respectively). This indicates
that incompatibility barriers between the exotic genotypes and the two testers
can be overcome, but attention must be given to determine the optimal direction
to achieve successful hybrids. It can be assumed that similar responses would
be observed using different testers.
The characterization of reproductive compatibility and the identification
of complexes of compatible genotypes presents the possibility of using specific
accessions as bridges in breeding programs for incorporating of unique traits of
interest into birdsfoot trefoil (0' Donoughue and Grant, 1988). For example, if
traits in the ETH genotype were desired, USA would not be a satisfactory
crossing parent because the resulting progeny would be sterile (0% F1 pollen
viability by crosses in either direction) and the percentage of pods set would be
low (< 10%), regardless of direction of the cross. On the other hand, the SWI
genotype used as the female partner is very receptive to crossing with the ETH
genotype (75% pod set) and the resulting F1 progeny are fertile (54%). SWI is
compatible with USA (percentage pods set are 37 and 63% with USA as the
female and male parents, respectively, with F1 progeny having pollen viability
percentages in excess of 90%). Those crosses that demonstrate high levels of
compatibility may be due to high degrees of chromosome homology (Grant, et
al., 1962). The relationship between karyotype and cross compatibility among
these genotypes needs to be determined. Further studies are also needed to
determine the effect of F1 parentage on reproductive success in bridge crosses.
Identification of predictors of reproductive success would assist with
better utilization of exotic germplasm resources. Based on the genotype RAPD
classification (Chapter 3), genotypes from Group-4 (GEO2, ITA2, and ROM) had
the lowest percentage of pods set (5% compared to 33, 29, and 46% for groups
1, 2, and 3, respectively) when USA was the female parent (P s 0.03) and the
highest percentage of pods set (60% compared to 9, 18, and 11%, respectively)
when USA was the pollen source (P 5 0.01). RAPD Group-4 genotypes also
Table 4.2. Female (F) and male (M) cross-compatibility between NC-83 and SWI with the 27 exotic genotypes
measured as pod set and pollen viability percentages.
USA
Pod set
Accession
SW!
Pollen Viability
Pod set
Pollen Viability
number
Identif.
PI 31276
MO R
17.9
23.1
92.7
PI 180171
CZE
45.4
-t
92.9
PI 227512
IRA1
63.6
30.0
95.2
95.7
50.0
42.1
0.0
89.9
PI 234670
FRA1
10.0
20.0
95.4
83.5
40.0
50.0
89.2
93.0
PI 235525
FRA2
33.3
59.8
1.9
60.2
96.1
PI 251143
MAC
54.5
10.0
96.1
96.9
62.5
38.9
80.8
96.2
PI 260268
ETH
9.3
2.5
0.0
0.0
75.0
5.0
54.1
0.0
PI 260692
ITA1
63.6
8.3
95.7
97.8
73.3
11.1
81.4
93.0
PI 267060
PO L
20.0
95.9
73.3
20.0
86.3
96.3
PI 290717
UK
58.3
97.6
25.0
PI 93-94
GEO2
27.3
PI 315082
KAZ
11.0
94.3
73.3
PI 315454
RUS1
23.1
96.1
73.3
PI 319021
SPA
27.3
94.2
87.5
(cont'd on next page)
94.7
97.8
23.2
43.7
73.5
30.0
53.8
95.4
44.4
49.2
94.8
86.8
87.7
70.6
77.7
97.8
89.6
11.1
88.2
68.1
90.9
Table 4.2. (cont'd.)
USA
Pod set
Accession
SWI
Pollen Viability
Pod set
Pollen Viability
number
Identif.
PI 319822
NOR1
30.0
30.0
96.1
96.4
73.3
60.0
79.4
96.4
PI 319823
NOR2
6.2
83.7
98.0
98.1
51.9
69.5
88.4
96.9
PI 325369
RUS2
3.6
48.1
93.7
97.2
43.1
55.5
93.0
97.0
PI 325379
UKR
7.7
30.0
96.0
96.2
23.5
5.5
78.6
0.0
PI 369278
RUS3
25.0
75.0
50.0
82.2
96.2
PI 464682
TUR
43.6
60.0
27.7
76.8
79.2
PI 384882
IRA2
58.3
60.0
40.0
74.8
94.6
PI 419228
GRE1
50.0
60.0
11.8
74.9
91.0
PI 419233
GRE2
50.0
27.8
26.7
62.0
96.1
PI 430546
RUS4
27.3
P1 93-21
GEO1
94.7
7.0
94.5
91.3
92.3
30.0
95.7
97.5
97.3
20.0
97.5
70.0
96.3
38.9
93.4
25.0
52.9
81.1
72.4
73.3
83.3
75.4
PI 485601
ITA2
6.2
80.0
94.8
96.3
35.7
PI 494653
ROM
9.1
30.0
0.0
94.8
60.0
t Indicates no pod set therefore, no pollen was produced.
72.4
83.1
76
had the lowest F1 pollen viability (72% compared to approximately 90% for the
other three groups; P s 0.04). These results indicate that it may be possible to
use molecular markers to identify genotypes that will be cross-compatible with
specific germplasm sources.
Cross-compatibility Among Diverse Genotypes
A subset of eight ecologically diverse genotypes, including USA and
SWI, were used in a diallele crossing block. Means comparison for female
compatibility observed among the crosses are reported in Table 4.3. Male
means are not presented because there were no differences among
genotypes. There was no interaction effect between genotype and crossing
direction on F1 progeny pollen viablility (P s 0.91) (Table 4.3). However,
genotype and crossing direction interacted and affected the percentage of pods
set, pod length, and number of seeds per pod (P s 0.0001, 0.0001, and 0.05,
respectively; Figure 4.2).
The NOR and SWI genotypes were the most promiscuous and the FRA
genotype the least (Table 4.3). The NOR, SWI, and RUS genotypes were
generally superior as female parents for percentage of pods set (Fig. 4.2A). The
TUR and FRA genotypes performed better as male parents for pod set than as
female parents. Crossing direction had no effect on pod set of MOR, USA, ETH.
The ETH genotype had a lower F1 progeny pollen viablility than the rest of the
genotypes (Fig. 4.2D). Crossing combinations that resulted in non-viable pollen
include SWI as the male parent with ETH as female, ETH as male with USA as
female, RUS as female with FRA as male, and USA as both male and female
parent with ETH (Figure 4.2D).
No relationship was found among the eight genotypes for reproductive
cross compatibility with genotype genetic background (based on RAPDs) or
morphological similarities (Mantel t = 0.96; P s 0.17 and 0.06; P s 0.47,
respectively). This finding differs from that for birdsfoot trefoil morphological and
RAPD characteristics that were associated with the ecology of the genotype site
77
Table 4.3. Means of female parent diallele cross-compatibility averages among
eight birdsfoot trefoil genotypes.
Genotype
Pod
set
Pod
length
Seeds
per pod
Fl Pollen
viability
%
mm
no.
%
NOR2
68.7 a
26.3 a
12.6 ab
90.1
SWI
22.7 ab
22.9 ab
19.4 bc
20.3 bc
15.9 a
78.0
91.3
FRA2
58.1 ab
50.9 b
28.1 c
21.6 cd
15.7 cde
12.4 de
1.6 gc
LSD
13.2
RUS2
MOR
USA
TUR
ETH
15.0 bc
24.1 ab
9.3 bc
8.6 bcd
11.3 abc
7.6 cd
9.9 bc
5.9d
4.6d
5.8
4.6
85.5
78.0
84.1
81.0
64.3
NS
Means within columns followed by the same letter are not significant at the 0.05
level based on LSD test.
100
MOR SWI FRA2 ETH NOR2 RUS2 TUR USA
60
A
50
80­
0
.6 60.
o
a
40
ab
30
-8 40­
I
20
cc
lid
Le T
0
11 FM FM FM
30
-0 25
MOR SWI
M F M FMFM
C
a)
ocl" 15
V
1) 10
5 Is!
1
20
10
0
FRA2 ETH NOR2 RUS2 TUR USA
O
2" 20
0
MOR SWI FRA2 ETH NOR2 RUS2 TUR USA
3.011
FMFMFMFMFM
Crossing direction
100
90
80
70
60
50
40
30
20
10
0
FMFMFMFMFMFMFMFM
MOR SWI
aj
FRA2 ETH NOR2 RUS2 TUR USA
0
a
al
!I
0
ETH
USA
FRA
ETHETH
FMFMFMFMFMFMFMFM
Crossing direction
Figure 4.2. Female (F) and male (M) diallele cross compatibility among seven exotic and one North
american-adapted genotypes measured as: A, percentage of pod set; B, pod length; C, seeds per pod;
D, percentage of Fi pollen viability. Accessions used are: MOR, PI 31276; SWI, PI 234811; FRA2, PI
235525; ETH, PI 260268; NOR2, PI 319823; RUS2, PI 325369; TUR, PI 464682; and USA, 'NC-83'.
Accession names at the bottom of D indicate poor combination for pollen viability. Box plots labeled
with different letters have means significant different at P s 0.05 according to LSD test.
79
of origin (Chapter 3). It appears that reproductive barriers to cross compatibility
in birdsfoot trefoil are environmentally neutral and distributed randomly
throughout the species.
Conclusions
The 29 parental genotypes used in the experiment exhibited a range of
responses for self pod set and pollen viability percentages. There was no
correlation between these responses. The only measures of reproductive
success that were correlated were number of seeds per pod and pod length
Using the two hybrid testers (USA and SWI) and the diallele cross among the
eight genotypes, incompatibility among crosses was expressed either as a
failure to set pods or that produced progeny which did not produce viable
pollen. However, among the parental genotypes used, intermediate crosses
could be identified that could bridge any combination of parents that were
incompatible.
There was no relationship between the measures of reproductive
success with any of the ecological descriptions of the genotype collection site.
RAPD markers identified a group of genotypes with the lowest percentage of
pods set when USA was the female parent, the highest percentage of pods set
when USA was the pollen source, and the lowest F1 pollen viability of the four
classes. These results indicate that it may be possible to identify specific
markers to determine genotypes that will be cross-compatible with specific
germplasm sources. However, no relationship was found for the degree of
reproductive compatibility with genetic relatedness of the parental genotypes.
Similarly, there was no relationship between genotype reproductive
compatibility and morphological similarities. It appears that reproductive
barriers to cross compatibility in birdsfoot trefoil are environmentally neutral and
distributed randomly throughout the species. This differs from the findings that
the distribution of birdsfoot trefoil morphological characteristics are associated
with the ecological origins and genetic similarities of the genotypes.
80
References
Beuselinck, P.R., and J.J. Steiner. 1992. A proposed framework for identifying,
quantifying, and utilizing plant germplasm resources. Field Crops Res.
29:261-272.
Beuselinck, P. R., B. Li, and J. J. Steiner. 1996. Rhizomatous Lotus corniculatus
L. I. Taxonomic and cytological study. Crop Sci. 36:179-185.
:
Brown, A.H.D. 1989. Core collections: a practical approach to genetic
resources management. Genome 31:818-824.
Chandler, J. M., C. C. Jan, and H. Beard, 1986. Chromosomal differentiation
among the annual Helianthus spp. Systematic Botany 11:354-371.
Chrtkova-Zertova, A. 1973. A monographic study of Lotus corniculatus L. I
Central and Northern Europe. Rozpravy Ceskoslovenske Akademie Ved,
83, 1-94.
Grant, W. F., M. R. Bullen, and D. de Nettancourt,. 1962. The cytogeneties of
Lotus. I. Embryo-cultured interspecific diploid hybrids closely related to L.
corniculatus. Can. J. Genet. Cytol. 4:105-128.
Ha Hauer, A.R. 1978. Potential of exotic germplasm for maize improvement. p.
229-247./n D.B. Walden (ed.) Maize breeding and genetics. Jhon Wiley
& Sons, New York, N.Y.
Kariya, K. 1989. Sterility caused by cooling treatment at the flowering stage in
rice plants. Japanese J. of Crop Sci. 58:96-102.
Mantel, N. 1967. The detection of disease clustering and a generalized
regression approach. Cancer Res. 27:209-220.
Marten, G.C., F.R. Eh le, and E.A. Ristau.1987. Performance and
photosensitization of cattle related to forage quality of four legumes.
Crop Sci. 27:138-145.
MacDonald, A. 1946. Birdsfoot trefoil (Lotus corniculatus L.) Its
characteristics and potentialities as a forage legume. Cornell Agric Exp.
Stn. Memo 261. p.182.
.
McKee, R. 1949. Fertility relationships in the genus Lotus. Agron. J. 11:313-316.
Miller, J.D. 1969. Cross-compatibility of birdsfoot trefoil, Lotus corniculatus L.
Crop Sci. 9:552-555.
81
Negri, V., B. Romano, and F. Ferranti. 1989. Male sterility in birdsfoot trefoil
(Lotus corniculatus L. ). Sex Plant Reprod. 2:150-153.
O'Donoughue, L.S., and W.F. Grant. 1988. New sources of indehiscence for
birdsfoot trefoil, Lotus corniculatus (Fabaceae ) produced by interspecific
hybridization. Genome 30:459-468.
Paterson, A.H.,S.D. Tanksley, and M.S. Sorrells. 1991. DNA markers in plant
improvement. Advances in Agronomy. 46:39-90.
Rohlf, F.J. 1993. NTSYS-pc. Numerical taxonomy and multivariate analysis
system. Version 1.80 Applied Biostatistics Inc., N.Y.
Rumbaugh, M. D. 1991. Plant introductions: The foundation of North American
forage legume cultivar development. p. 103-114. In H. L. Shands and L.
E. Wiesner (ed.) Use of plant introductions in cultivar development.
CSSA Special publ. 17. ASA, CSSA, and SSSA, Madison, WI.
Seaney, R. R. 1962. Evaluation of methods for self- and cross-pollinating
birdsfoot trefoil, Lotus corniculatus L. Crop Sci. 2:81
Seaney, R.R., and P.R. Henson. 1970. Birdsfoot trefoil. Adv. Agron.
22:119-157.
Snedecor, G. W., and W. G. Cochran. 1980. Statistical methods Iowa State
University. Press, Ames, IA.
Steiner, J. J. 1993. Electrophoretic techniques for evaluating forage
germplasms. International Herbage Seed production Newsletter NO. 19.
Statview 512+, 1988. Statview 512+: The interactive statistics and graphics
package. Brain Power, Inc. Calabasas, CA.
Systat, 1992. Systat: Statistics, Version 5.2 Edition.Systat, Inc., Evanston, IL.
Williams, P.H. 1980. Bee-sticks, an aid in pollinating Cruciferae. Hart Sci.
15:802-803.
82
CHAPTER 5
ADAPTIVE ECOLOGY OF Lotus corniculatus L. GENOTYPES: III.
CHLOROPHYLL FLUORESCENCE AND ADAPTATION
Abstract
Birdsfoot trefoil (Lotus corniculatus L.) is a widely distributed species that
has mechanisms that allow it to be adapted to a wide range of habitats. Plant
optimal growth temperature characteristics based on the thermal response of
the photosystem II (PSII) variable fluorescence (Fv) reappearance following
illumination has been proposed as a means to differentiate germplasm and
identify genotypes adapted to specific environments. The objectives of this
research were to: (i) identify and describe the optimal temperature
characteristics of eight ecologically diverse birdsfoot trefoil genotypes using the
thermal response of PSII Fv, and (ii) determine if there are relationships
between the optimal temperature characteristics of the genotypes and
ecological characteristics of their collection sites. Chlorophyll fluorescence
transients ratios (FTR) were determined from eight exotic genotypes were
selected for their ecological diversity. Measurements were made for 10 to 40° C
in 5° C increments for 33 min. from the time of initial dark adaption in 3 min.
increments. The eight genotypes differed in their FTR responses to temperature
and were grouped into two classes. However, no associations were found
between similarities in genotype FTR and genetic or ecological similarities.
With the eight genotypes used in this experiment, chlorophyll variable
fluorescence does not appear to be suitable technique for identifying birdsfoot
trefoil genotypes adapted to specific environments.
83
Introduction
Birdsfoot trefoil (Lotus corniculatus L.) is a widely distributed species that
is adapted to the British Isles; throughout much of Europe; in several South
American countries including, Argentina, Brazil, Chile and Uruguay; in areas of
India, Australia, New Zealand (Beuselinck and Grant, 1995); and in North
America it has become an important forage species that grows from the Atlantic
Coast west to eastern Kansas, Nebraska, the Dakotas in the United States, and
the southern part of Ontario province in Canada (Kingsbury and Winch, 1967).
Birdsfoot trefoil ranges from sub-Arctic to sub-tropic regions (Widdup et al.,
1987).
The distribution of a species can be controlled by climate (Woodward,
1987). For widely distributed temperate-region species, climate is assumed to
be primarily responsible for the range of genotypic variation found between
species (Tinkle, 1972). Descriptions of relationships between birdsfoot trefoil
germplasm morphological and biochemical characteristics with ecological
descriptions of accession origins have been given elsewhere (ChrtkovaZertova, 1973; Small et al., 1984; McGraw et al., 1989; Steiner and Poklemba,
1994; Chapters 3 and 4). Studies describing whole-plant physiological
characteristics are very scarce (Gonzalez et al., 1995) and non-existent for
birdsfoot trefoil (Steiner, 1994).
Genetic variability studies among the different genetic pools of L.
corniculatus have been made, using a variety of plant descriptors, including,
qualitative and quantitative morphological traits (Crtkova-Zertova, 1973),
secondary plant product chemical composition (Harney and Grant, 1964a,
1964b), and seed proteins (Steiner and Poklemba, 1994).
The chlorophyll fluorescence technique has been proposed as a tool for
monitoring the extent of plant stress under various environmental conditions
such as chilling and drought stress (Larcher and Neuner, 1988; Ogren, 1990),
freezing stress (Oquist and Ogren, 1987; Somersalo and Krause, 1989), and
cold-climatization and extreme temperatures (Boese and Huner, 1990;
84
Somersalo and Krause, 1989). Chlorophyll fluorometry experiments
demonstrated differentiation of frost-sensitive Trifoliurn species and ecologically
diverse genotypes within T. alexandrinum and T. repens (Barnes and Wilson,
1984). It has been proposed that different thermally adapted species show
unique variable fluorescence recovery characteristics that reflect physiological
optimal temperatures. Photosystem II Fv can be easily monitored in leaves of
plants that have been dark-adapted (Moffat et al., 1990; Peeler and Naylor,
1988). Recently, methodology has been developed to identify optimal
temperature characteristics of species using the thermal response of the
photosystem II (PSII) variable fluorescence (Fv) reappearance following
illumination (Burke, 1990; Ferguson and Burke, 1991; Burke and Oliver, 1993;
Burke, 1995). It has been reported that the Fv dark recovery indicates that cool-
and warm-season species have unique temperature characteristics for the
recovery of Fv after illumination at 25° C (Burke, 1990).
As far as it is known, no published literature is available describing the
screening of germplasm using the chlorophyll fluorescence technique. Also,
apart from screening for frost- and freezing tolerance two genotypes of two
Trifolium species (Barnes and Wilson, 1984), no relationships have been
established for optimal variable flourescence temperature characteristics with
the ecological origins of diverse genotypes. Using variable fluorescence
recovery response measurements may provide mechanistic links to plant
growth within different environments and be able to predict the ecological
adaptation of germplasm. As with all artificial classifications, it is necessary to
establish functional relationships between physiological measures, plant
responses and other specific trait descriptor that can be associated with plant
ecological adaptation (Steiner, 1994).
The objectives of this research were to: (i) identify and describe the
optimal temperature characteristics of eight ecologically diverse birdsfoot trefoil
genotypes using the thermal response of PSI I Fv, and (ii) determine if there are
relationships between the optimal temperature characteristics of the genotypes
and ecological characteristics of their collection sites.
85
Materials and Methods
Selection of Genotypes
A preliminary screening, using random amplified polymorphic DNA
(RAPD) polymorphisms, of the 29 genotypes from the NPGS birdsfoot trefoil
collection was used to determine genetic and ecological diversity (Chapter 3).
A subset of eight diverse genotypes (USA, SWI, MOR, FRA2, ETH, TUR, RUS2,
and NOR2) were selected for further analysis (Chapter 4). A description of the
ecological characteristics of the genotype collection sites is given in Table 5.1.
A description of how the ecological characteristics were obtained is given in
Chapter 3. The ecological characteristics of USA were based on the site of
seed increase for this germplasm release (Miller et al., 1983).
Fluorescence Reappearance Measurement
The eight genotypes of similar age were grown in the greenhouse as
described in Chapter 3. Each genotype was maintained at 25°under 260
1,tmo les
m-2 s-1 constant light conditions for 14 h prior to analysis. The
characteristics of the chlorophyll fluorescence induction may be modified by
any factor involved directly or indirectly in the photosynthetic metabolism
(Walker, 1990). Therefore, to reduce the variability in the chlorophyll
fluorescence profile, attention must be given to physiological age and,
nutritional status and conditions of the experiment. Three to four excised leaves
from a genotype were placed on moistened 3MM chromatography paper in
contact with thermal controlled blocks of the thermal plate system (Burke and
Mahan, 1993). The leaves were then covered with CO2 permeable transparent
plastic film (Glad Cling wrap, First Brands Corporation, Danbury, CT) and
Table 5.1. Ecological descriptions of eight birdsfoot trefoil genotype collection sites.
Ecological descriptors
Entry
Identification
---no.---
PI 31276
PI 234811
PI 235525t
PI 260268
PI 319823
PI 325369
MOR
PI 464682
NC-83
TUR
SWI
FRA2
ETH
NOR2
RUS2
USA
Location
Temperature
Ecoregion
code
Elevation Precipit. Average Low
High
° lat.
° long.
33.63N
46.87N
43.36N
9.37N
59.59N
45.02N
36.60N
44.75N
4.87W
9.53E
3.53E
40.52E
6.01E
45.59E
M262
M243
29.90E
M261
1580
30
2130
760
30
1290
93.33W
M221
200
t No original seed of the accession available.
m
261
M412
244
332
1680
mm
770
1075
146
685
2532
267
333
53
°C
1.3
3.6
-11.2
10.4
12.9
-7.0
9.8
-6.2
7.8
-6.6
-11.2
11.4
-1.8
19.0
16.8
5.3
22.0
7.0
28.8
18.8
10.4
25.5
19.8
22.5
SnowCloudiness
cm
%
0.0
72.1
124.3
0.0
39.4
69.7
0.0
67.1
17.2
26.3
5.5
42.4
55.4
38.8
54.6
59.1
87
illuminated for at least ten min. at 25° C using a high-pressure sodium lamp with
a light intensity of 650 pmol rT1-2 S-1. After the illumination period, the thermal
cells were set to temperatures ranging from 10 to 40° C and the high-pressure
lamp turned off and chlorophyll fluorescence measurement immediately begun
using a Branker SF fluorometer (Richard Brancker Research, Ottawa, Canada)
and recorded at 3 min. intervals for 33 min. using a 10s excitation period with 5
W m-2 light (modified procedure from Burke, 1990). Each temperature condition
was replicated five times. Water vapor condensing on the fluorometer probe at
10 and 15° C was removed by wiping the probe with tissue paper and forcing
desiccated air across the probe surface and over the plastic wrap before each
fluorescence measurement was made. Similar problems were reported by
Huner et al., (1992), who recommended the application of a thin film of glycerol
on fluorometer probes to overcome the problem.
Data Analysis
The chlorophyll fluorescence transients ratios (FTR) were determined as:
FTR = Fv/ Fo
where Fo = initial fluorescence and Fv = Fmaximum
[1]
F0
.
The FTR measurements
were made from 10 to 40° C in 5° C increments (seven temperatures) and
plotted as functions of time from the initial Fo observation (12 measurements).
The 84 FTR obervations (seven temperatures and 12 time observations) were
used to calculate a Euclidean distance matrix. The resulting matrix was
analyzed by cluster analysis based on Euclidean distance and Ward's (1964)
clustering technique (Systat 5.2.1 for the Macintosh, Evanston, IL). The
congruence of the ecological, morphological, reproductive, and genetic
classifications (see Chapter 4) were determined by Mantel's test (Mantel, 1967)
using the MXCOMP command of NTSYS-pc program version 1.80 (Rohlf,
1993).
88
The optimal temperature for fluorescence transient ratios (FTRopt) was
defined as the maximal value among the summations of the 12 sequential time
FTR observations (FTRsum) for each of the seven temperatures:
33min
FTRsum =
E FTR
[2]
t =0
These values were used as quantitative representations of the FTR response
curves for each genotype.
Pearson's correlation analysis (Snedecor and Cochran, 1980) between
the seven FTR opt temperature response characteristics and individual
ecological descriptions (see Chapter 3) were also done to identify relationships
between whole-plant photosynthetic function collection site ecology of the
genotypes for significance (Systat 5.2.1 for the Macintosh, Evanston, IL). The
association of a Euclidian distance matrix based on FTRsum values with that of
the 84 FTR observation matrix was done with the NTSYS-pc program to
validate the use of FTRsum data as a quantitative representation of the
chlorophyll variable fluorescence response curves (Mantel t -= 3.9; P s 0.0001).
89
Results and Discussion
Fluorescence Reappearance Curves
The potential of the chlorophyll fluorescence technique in screening
germplasm accessions in exotic accessions of birdsfoot trefoil from different
geographical regions was assessed. The FTR response curves to temperature
for the eight genotypes are shown in Figs. 5.1
5.4. The FTR curves among
genotypes are different (P < 0.001). The FTRopt of each genotype is shown in
Fig. 5.5.
The FTRopt for MOR and FRA2 was 15° C. The optimum temperature for
ETH, NOR2, RUS2, TUR, and USA was 10° C. The SWI genotype reached an
optimal plateau between 10 and 30°. The wide temperature range for FTRopt is
due to an increase in the rate of FTR with a concomitant decrease in maximal
FTR at each temperature between 10 and 30° C (Fig. 5.1, SWI). A range of
FTRopt have been shown for Capsicum annuum L. and Lycopersicom
esculentum L. (30-35° C and 20-25° C, respectively), so this finding is not out of
the ordinary. The eight genotypes were placed into two cluster groups (Fig.
5.6).
Association of Chlorophyll Fluorescence Response with other
Classification Characteristics
Based on comparisons of matrices, there are no similarities between the
PSII Fv response with genetic, morphological, reproductive, and ecological
similarities of the eight genotypes (Table 5.2). Similarly, there were no
associations between the FTRopt with the individual ecological descriptions of
the collections sites or measures of reproductive success of each genotype
(data
MOR
1.0
15°
10°
20°
25°
30°
35°
40°
1.0
0.8
0.8
u° 0.6
0.6
.
0.4
0.4
rrei01.0.5r.,
0.2
0.0
0 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40
0.2
0.0
SWI
1.0
10°
15°
20°
25°
30°
0.8
LE
350
40°
1.0
0.8
.
0.6
0.6
.
LL 0.4
0.4
0.2
11.14A9111
00
0 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40
0.2
0.0
Time in darkness (min)
Figure 5.1. Fluorescence transient ratio (Fv/Fo) temperature response curves for
Morocco (MOR) and Switzerland (SWI) birdsfoot trefoil genotypes at seven
temperatures. Each point represents the mean of five replications ± standard error
of the mean.
FRA2
1.0
10°
15°
20°
0.8
LE 0.6
Le..
0.4
.
0.2
0.0
0
25°
r:
30°
35°
.
1.0
40°
0.8
0.6
0.4
_remp.
0.2
itessw*-
0.0
10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40
ETH
10°
15°
20°
25°
,
30°
35°
1.0
40°
0.8
.
7114.
_
0.4
:
0.2
lifigiV°. -14*/16411:
0.0
0.6
0.2
0.0
0 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40
Time in darkness (min)
Figure 5.2. Fluorescence transient ratio (Fv/Fo) temperature response curves for
France (FRA2) and Ethiopia (ETH) birdsfoot trefoil genotypes at seven
temperatures. Each point represents the mean of five replications ± standard error
of the mean.
NOR2
1.0
10°
20°
15°
.
25°
0.8
1.0
30°
35°
400
0.8
Le.
0.6
0.6
LL.
0.4
0.4
/00
0.2
.
0.0
0 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40
0.2
0.0
RUS2
1.0
10°
15°
.200
0.8
-
25°
30°
35°
40°
1.0
0.8
LL
0.6
0.6
LL
0.4
0.4
0.2
0.2
1.0
-.
0 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40
0.0
Time in darkness (min)
Figure 5.3. Fluorescence transient ratio (Fv/Fo) temperature response curves for
Norway (NOR2) and Russia (RUS2) birdsfoot trefoil genotypes at seven
temperatures. Each point represents the mean of five replications ± standard error
of the mean.
TUR
1.0
10°
.
15°
20°
25°
30°
35°
1.0
40°
0.8
0.8
IL) 0.6
0.6
LL 0.4
0.4
0.2
,,
4ros."d­
0.0
0 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40
0.2
V.
USA
1.0
10°
15°
20°
25°
30°
35°
40°
0.8
EL"'
0.6
1.0
0.8
_
_
0.6
LL 0.4
0.4
.
0.2
.
0.0
0 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40 10 20 30 40
0.2
0.0
Time in darkness (min)
Figure 5.4. Fluorescence transient ratio (Fv/Fo) temperature response curves for
Turkey (TUR) and USA birdsfoot trefoil genotypes at seven temperatures. Each
point represents the mean of five replications ± standard error of the mean.
0.7
0.7
PI 31276
0.6
MOR
0.5
PI 234811
- SWI
15°C
10 -30°C
PI 235525
P1260268
FRA2
ETH
15°C
10°C
0.5
0.4
0.4
-
0.3
Li
Li
0.6
0.2
-.
0.3
41fr\IIIND
0.2
.
0.7
0.7
Fri,c" 0.6
P1 319823
PI 325369
PI 464682
NC-83
NOR2
RUS2
TUR
USA
10°C
0.5
10°C
10°C
..
0.4
-
0.3
-
10°C
0.6
0.5
0.4
0.3
0.2
0.2
0.1
0.1
0
10 20 30 40 50 10 20 30 40 50 10 20 30 40 50 10 20 30 40 50
TEMPERATURE ( °C)
Figure 5.5. Optimum temperature for fluorescence transient ratios (FM optimum) for
eigth birdsfoot trefoil genotypes. Each point represents the mean of 12
observations per temperature taken every three minutes.
95
SW'
Group 1
FRA2
MOR
USA
ETH
Group 2
NOR2
TUR
RUS2
I
I
0.0
0.05
I
0.1
I
I
0.15
0.2
Euclidian distance
Figure 5.6. Classification of eight birdsfoot trefoil
genotypes based on all the chlorophyll fluorescence
transient, ratio measurements made from temperatures
ranging from 10 to 40° C using 12 times (0-33 min)
recorded at 3 min intervals with lOs exitation, using the
Euclidian distance and Ward's cluster analysis.
96
Table 5.2 Normalized Mantel statistic (Z) and probability values(P) from
comparisons of different Euclidian distance matrix for the eight birdsfoot
trefoil genotypes using chlorophyll fluorescence transient ratios.
Fluorometry Distancet
Distance
Genetict
-1.3
0.75
0.1
0.48
Reproductive
-1.6
0.81
Ecological#
-1.4
0.67
Morphological§
t Matrix calculated using the variable fluorescence ratios (Fv/Fo).
t Matrix of genetic distances from RAPD calculated in PAUP.
§ Matrix calculated using the 18 morphological traits (Chrtkova-Zertova, 1973).
Matrix constructed using reproductive traits (Chapter 4).
# Matrix of Euclidian distances constructed using seven ecological descriptors
11
97
not shown). These results differ from the findings of Barnes and Wilson (1984)
who found that two genotypes each of T. alexandrium and T. repens from warm
environments were more frost-sensitive than genotypes from cold
environments.
Conclusions
This study investigated the use of a chlorophyll variable fluorescence
measurement technique as a screening tool to differentiate ecologically diverse
genotypes of birdsfoot trefoil. The genotypes were grouped into two classes.
However, no associations were found between similarities in chlorophyll
variable fluorescence differential temperature response and genetic or
ecological similarities of the genotypes. With the eight genotypes used in this
experiment, chlorophyll variable fluorescence does not appear to be an
suitable technique for identifying birdsfoot trefoil genotypes adapted to specific
environments.
98
References
Bailey, R.G. 1989. Explanatory supplement to ecoregions map of the
continents. Environ. Conserv. 16:307-310
Barnes, J. D., and J. M. Wilson. 1984. Assessment of the frost sensitivity of
Trifolium species by chlorophyll fluorescence analysis. Ann. Appl. Biol.
105:107-116.
Beuselinck, P.R., and W.F. Grant. 1995. Birdsfoot trefoil. p. 237-248. In R.F.
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101
CHAPTER 6
COMMON CONCLUSION
The 28 genotypes displayed polymorphism for both qualitative and
quantitative morphological characteristics and were classified into five
morphological cluster groups. Morphological similarities among genotypes
were related to the general geographic proximities of their collection sites.
Genotype morphology was also associated with the general ecology of the
collection site habitat and specific morphological traits were found related to
specific environmental characters. Floral morphological characteristics were
related to collection site latitude and average temperature. Plant growth form
was correlated with collection site elevation and latitude. There was no
relationship between glabrous and hair leaf indumentum expression.
Geographic distance among genotype collection sites is associated with
genetic distance. The genotypes were grouped into four RAPD classification
groups that were related to their geographic regions of origin Mediterranean
basin/Asia Minor, northern and continental Europe, southeast Europe, and
southern Europe. However, ecological similarity of the genotype collection sites
is not related to their genetic similarity. The lack of similarity between the
genetic and ecological classifications suggests that genotypes adapted to
similar but geographically distant habitats were derived from different
progenitors but have acquired similar phenotypes. Because RAPD descriptors
are associated with the geographic proximity of genotypes collection sites but
not with their ecological similarity, classifications of germplasm should not
solely rely on ecological descriptions. The utility of combining RAPD,
morphological, and ecological descriptors reveals combinations of variation
among the birdsfoot trefoil genotypes that would not be apparent with any
102
single measurement and can provide a more complete understanding about
the diversity in birdsfoot trefoil germplasm collections.
The 29 parental genotypes used in the experiment exhibited a range of
responses for self pod set and pollen viability percentages. There was no
correlation between these responses. The only measures of reproductive
success that were correlated were number of seeds per pod and pod length.
Using the two hybrid testers (USA and SWI) and the diallele cross among the
eight genotypes, incompatibility among crosses was expressed either as a
failure to set pods or that produced progeny which did not produce viable
pollen. However, among the parental genotypes used, intermediate crosses
could be identified that could bridge any combination of parents that were
incompatible.
There was no relationship between the measures of reproductive
success with any of the ecological descriptions of the genotype collection site.
RAPD markers identified a group of genotypes with the lowest percentage of
pods set when USA was the female parent, the highest percentage of pods set
when USA was the pollen source, and the lowest F1 pollen viability of the four
classes. These results indicate that it may be possible to identify specific
markers to determine genotypes that will be cross-compatible with specific
germplasm sources. However, no relationship was found for the degree of
reproductive compatibility with genetic relatedness of the parental genotypes.
Similarly, there was no relationship between genotype reproductive
compatibility and morphological similarities. It appears that reproductive
barriers to cross compatibility in birdsfoot trefoil are environmentally neutral and
distributed randomly throughout the species. This differs from the findings that
the distribution of birdsfoot trefoil morphological characteristics are associated
with the ecological origins and genetic similarities of the genotypes.
This study investigated the use of a chlorophyll variable fluorescence
measurement technique as a screening tool to differentiate ecologicalally
diverse genotypes of birdsfoot trefoil. The genotypes were grouped into two
103
classes. However, no associations were found between similarities in
chlorophyll variable fluorescence differential temperature response and genetic
or ecological similarities of the genotypes. With the eight genotypes used in this
experiment, chlorophyll variable fluorescence does not appear to be an
suitable technique for identifying birdsfoot trefoil genotypes adapted to specific
environments.
104
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120
APPENDICES
121
TUR1
TUR2
TUR3
TUR4
TUR5
ETH1
ETH2
ETH3
ETH4
ETH5
NOR4
NOR3
NOR1
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SWI3
SWI2
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USA1
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I
0.01
0.3
0.7
1.1
1.4
Appendix 1 Intra-accession variation assessment of the eight
birdsfoot trefoil genotypes.
Appendix 2. Binary data matrix of the 18 morphological traits and the 71 characters states.
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