AN ABSTRACT OF THE THESIS OF
Sonali Dilip Gandhi for the degree of Doctor of Philosophy in Crop Science
presented on April 26, 2002. Title: Genetics of Low Erucic Acid and Cytologi
Analyses of Wide Hybrids in Meadowfoam
Abstract approved
Redacted for Privacy
Steven J. Knapp
Cultivated meadowfoam (Limnanthes a/ba Benth.) is an annual oil seed crop
native to southern Oregon. California and British Columbia. The genus
Limnanthes is composed of nine species and divided into two sections, Inflexae and
Reflexae. The seed oil of meadowfoam is a rich source of erucic acid and several
novel very long-chain fatty acids (VLCs). The former has been linked to increased
risk of heart disease. The safe limit of erucic acid for human consumption is upto
5% of total fatty acids. Because the erucic acid concentrations of wildtype lines
typically range from 9 to 23 % and low erucic acid variants have not been
discovered, chemical mutagenesis was used to develop a mutant line (LE76) with
greatly reduced erucic acid (3 %). The phenotypic distributions off2 progeny
from crosses between wildtype and mutant lines were continuous and differed
across genetic backgrounds. Quantitative trait loci (QTL) affecting erucic and
dienoic acid were mapped using
F23
progeny from a cross between LE76 and
Wheeler (a wildtype line) and a simple sequence repeat (SSR) map spanning the
meadowfoam genome. The domestication of meadowfoam was based on L. a/ha,
belonging to section Inflexac. The secondary and tertiary gene pools have not been
important to the domestication process and have not supplied diversity for
meadowfoam breeding. With the objectives of introgressing genes from wild
relatives and also producing cytoplasmic male sterile lines by inserting the nuclear
genome of L. a/ba into wild cytoplasm, inter-sectional crosses involving L. a/ba
and three subspecies of L. doug/ash and intra-sectional crosses involving L. a/ba
and two subspecies of L.floccosa were carried out. The isolation mechanisms
involved in keeping species apart from each other were found to be different within
and between sections. The study of partially fertile intra-sectional hybrids showed
that the reduced pollen viability (30-33%) was not due to structural differences
between the chromosomes of the two species, as normal meiotic behavior was
observed in PMCs. The inter-sectional crosses were found to be incompatible and
various abnormalities during pollen tube growth were observed.
Genetics of Low Erucic Acid and Cytological Analyses of Wide Hybrids in
Meadowfoam
by
Sonali Dilip Gandhi
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented April 26. 2002
Commencement June 2002
Doctor of Philosophy thesis of Sonali Dilip Gandhi presented on April 26, 2002
APPROVED:
Redacted for Privacy
Major Professor, representing Cr4p Science
Redacted for Privacy
Chair of the Department of Crop and Soil Science
Redacted for Privacy
Dean of Gaduate 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
Dilip Gandhi, Author
ACKNOWLEDGMENTS
I express my deep sense of gratitude and indebtedness to Dr. Steven J. Knapp,
my research guide and major professor, for his inspiring guidance, constant
encouragement and constructive criticism during the conduct of research work as well
as throughout the writing of this text. His keen and unfailing interest have made me
strive for perfection. I am also thankful to him for his trust in accepting me as a part of
his highly esteemed team and providing me with the financial support throughout my
doctoral program. I am grateful to members of my graduate committee, Dr. Oscar
Riera-Lizarazu, Dr. Tony Chen, Dr. Shawn Mehlenbacher and Dr. Timothy
Schowalter for their time and willingness to help.
I would like to extend special thanks to Jimmie Crane whose greatness lies in
not only having an enormous amount of knowledge about meadowfoam but also
sharing it with whomever approaches him. He shared all the information right from
cultivating meadowfoam to doing fatty acid extraction in meadowfoam. Special thanks
are also extended to Dr. Mary Slabaugh, who taught and helped me extensively in all
aspects of molecular techniques. Her concern, encouragement and gentleness will be
appreciated forever. Thanks are due to my colleagues in lab Kishore. Pablo, Ju-Kyung,
Shunxue, Felix. Judy. Cate. Carrie and Adam whose friendship and warmth has made
every day memorable. I am also grateful to Dr. Pablo Velasco-Pazos for being
generous with his time in helping me especially during the time of manuscript
preparation. I wish to express my gratitude towards Dr. Isabel Vales for her
unreserved help in making cytological preparations. The faculty and staff of the
Department of Crop Science who directly or indirectly contributed to the successful
completion of this work are also gratefully acknowledged. I would also like to take
this opportunity to thank Caprice Rosato who assisted in fragment analysis of
SSRmarkers.
The funding and support from USDA grant #
99-34407-7509
to Dr. S. J.
Knapp is also acknowledged.
Though miles away from here, the love and concern of my parents and family
members have always been the sources of inspiration. Last but not least,
encouragement, support and love given by my husband 'Krishnakishore" are beyond
appreciation. His love has given me the strength and courage needed for successful
completion ofthis journey. Nothing was really possible without his help and
understanding.
CONTRIBUTION OF AUTHORS
Dr. Steven J. Knapp initiated, advised and supervised all aspects of the
projects. He substantially assisted in formulating the hypotheses and revising the
manuscripts. Jimmie Crane in association with Dr. Steven J. Knapp developed the low
erucic mutant line (LE76), Wheeler, 0MF64. OMF4O-1 1, OMFIO9-3, and OMF156.
He also helped in fatty acid profiling. Dr. Venkatakrishnakishore developed the SSR
markers used in this study for construction of the genetic map. He also helped in
collection of flowers and further processing for pollen tube observation. Dr. Oscar
Riera-Lizarazu provided the facilities for microscopy and also guided in making
cytological preparations for pollen tube observation as well as meiotic analysis of the
pollen mother cells.
TABLE OF CONTENTS
Page
CHAPTER 1
INTRODUCTION........................................................................................................
1
CHAPTER 2
MAPPING GENETIC FACTORS UNDERLYING LOW ERUCIC ACID
CONCENTRATION IN MEADOWFOAM ............................................................... 6
ABSTRACT ............................................................................................. 7
INTRODUCTION ................................................................................... 9
MATERIALS AND METHODS ........................................................... 14
RESULTS.............................................................................................. 20
DISCUSSION........................................................................................ 37
REFERENCES ....................................................................................... 45
CHAPTER 3
DIFFERENTIAL REPRODUCTIVE ISOLATION MECHANISMS WITHIN AND
BETWEEN SECTIONS OF GENUS LIMNANTHES .............................................. 53
ABSTRACT ........................................................................................... 54
INTRODUCTION ................................................................................. 55
MATERIALS AND METHODS ........................................................... 58
RESULTS.............................................................................................. 62
DISCUSSION ........................................................................................ 77
REFERENCES ....................................................................................... 87
TABLE OF CONTENTS (CONTINUED)
CHAPTER 4
CONCLUSIONS ......................................................................................................... 93
BIBLIOGRAPHY ....................................................................................................... 98
LIST OF FIGURES
Figure
Page
2.1. Erucic and dienoic acid concentrations ............................................................ 21
2.2. Erucic and dienoic acid concentration .............................................................. 24
2.3. Frequency distribution of 20:1A5 concentration................................................ 27
2.4. Linkage map of meadowfoam based on F2 progeny of Wheeler x
LE76.................................................................................................................. 31
2.5. Frequency distribution of erucic and dienoic acid concentration for
94 F23 families of the cross Wheeler x LE76 ................................................... 33
2.6. LOD plots of the QTL identified for erucic and dienoic acid
concentration ..................................................................................................... 34
3.1. Pollen grains of OMF1O9-3 and LFP x OMF1O9-3 ........................................... 64
3.2. Diakinesis, Metaphase I and Anaphase I observed in the PMCs of
OMF1O9-3 and LFP x OMF1O9-3 .................................................................... 64
3.3. Style morphology for 0MF156 (L. alba), OMF1O9-3 (L. alba), L.
douglasii ssp. nivea, L. douglasii ssp. rosea, L. douglasii ssp.
douglasii ............................................................................................................ 66
3.4. Pollen germination, penetration, and tube growth in compatible and
incompatible crosses ......................................................................................... 68
3.5. Heavy callose deposition on the stigmatic surface in LDD x
0MF156 (A), pollen tube turning back in LDD x 0MF156 (B),
swollen tip pollen tube in LDR x 0MF156 (C), burst pollen tube in
LDD x 0MF156, forked pollen tube in OMF1O9-3 x LDD (E),
coiling pollen tube in 0MF156 x LDN (F) ...................................................... 72
3.6. Schematic representation of checkpoints to prohibit the growth of
pollen tube in incompatible crosses ................................................................... 82
LIST OF TABLES
Table
Page
2.1. Fatty acid concentrations for wildtype (WT) and low erucic acid
(LE76 M7, M. M4. and M) meadowfoam lines ............................................... 20
2.2. Mean fatty acid concentrations for Wheeler, OMF40-1 1, 0MF64 and minimum,
maximum, and mean fatty acid concentrations for the F2 progeny of the three
crosses ...................................................................................... 23
2.3. Phenotypic correlations between major fatty acids of meadowfoam ................. 28
2.4. Summary of simple sequence repeat map .......................................................... 30
2.5. Mean fatty acid concentration for Wheeler and LE76 and minimum,
maximum fatty acid concentrations for 94 F23 derived families ..................... 32
2.6 Summary of the QTL detected for erucic and dienoic acid
concentrations .................................................................................................... 35
3.1. Summary of crosses attempted ........................................................................... 62
3.2. Number of ring and rod bivalents and chiasma frequency observed
for n cells at diakinesis of meiosis I ............................................................... 65
3.3. Pollen germination, penetration and pollen tubes in compatible and
incompatible crosses ......................................................................................... 69
3.4. Inhibition sites for incompatible crosses ........................................................... 73
3.5. Abnormalities observed in pollen tube growth of in-compatible
crosses............................................................................................................... 76
Genetics of Low Erucic Acid and Cytological Analyses of Wide Hybrids in
Meadowfoam
CHAPTER 1
INTRODUCTION
Cultivated meadowfoam (Limnanthes alba Benth.) is an annual oil seed crop
native to southern Oregon, California and British Columbia (Mason, 1952; Kahn,
1971; Jam, 1986). The genus Limnanthes comprises of nine species. The genus has
been divided into two sections, Jnflexae and Reflexae based on the morphological
characters of petals folding inward or outward during seed maturation (Mason,
1952). The entire genus Limnanthes is diploid (2n=1O), hermaphroditic and selfcompatible (Mason, 1952).
The extent of genetic variation available in a crop is a prerequisite for its
improvement due to the fact that efficiency of selection mainly depends upon it.
The two major approaches employed by plant breeders in order to widen the
genetic base of a crop are i) accessing the genetic diversity present in wild relatives
of crop plants by distant hybridization and, ii) inducing mutations using chemical
or physical agents. We used the approach of induced chemical mutagenesis for
development of low erucic meadowfoam due to the lack of natural variation for
erucic acid in meadowfoam. We also tried to make inter and intra-sectional hybrids
2
to expand the gene pooi by introducing alien genes carried by wild species in
meadowfoam.
Meadowfoam seed oil is very unique in many aspects. It contains substantial
amounts of unsaturated very long chain (C20 and
C22)
fatty acids VLCFs (Earl et
al., 1959; Bagby et al., 1961; Smith et al., 1960; Miller et al., 1964; Higgins et al.,
1971). The concentration of VLCFs in meadowfoam seed oil ranges from 94 to 96
% compared to 45 to 56 % in rapeseed. Meadowfoam seed oil has less than 2 %
saturated fatty acids (Knapp and Crane, 1995), which is significantly less than
rapeseed (6 to 8 %) and other widely consumed seed oils (Weiss, 1983). The
principal fatty acids found in meadowfoam seed oil are erucic acid (22:1A13), cis-5eicosenoic acid (20:1A5), cis-5-docosenoic acid (22:1A5) and cis-5, cis-13eicosenoic acid (22:2 A5A13) or dienoic acid (Earl et aL, 1959; Smith et al., 1960;
Bagby et al., 1961). The seed oil of meadowfoam also has very high oxidative
stability (Muuse et al., 1992; Isbell, 1997; Mund and Isbell, 1999). This, together
coupled with a low general toxicity has made meadowfoam seed oil important for a
number of non-food applications (Isbell, 1997, 1999), in particular the cosmetic
industry (Dworak, 1994).
One of the major factors that may prevent the entry of meadowfoam oil into
food, medical, nutritional, and pharmaceutical markets is the presence of high levels
(8 to 23.2 %) of erucic acid in generic (wild type) meadowfoam. High levels of
erucic acid in the diet have been associated with health problems (Borg, 1975;
McCutcheon et al., 1975). The United States Food and Drug Administration
3
currently permits dietary oils containing no more than 2% of erucic acid for human
consumption (Federal register,
1985).
Whereas, European union food commission
allows 5% erucic acid of total fatty acids in rapeseed oil meant for human
consumption (Official Journal of the European Community, 2001). The
development of low erucic acid meadowfoam cultivars may open the doors of the
edible, medicinal and pharmaceutical markets for meadowfoam seed oil.
Meadowfoam is a recently domesticated oilseed crop. Limnanthes a/ba was
identified by Gentry and Miller (1965) as the most promising species for
domestication. The domestication process, which began more than thirty years ago
(Crane and Calhoun,
1974)
has been entirely based on L. alba. The secondary and
tertiary gene pools have not been important to the domestication process and have
not supplied diversity for meadowfoam breeding.
Limnanthes alba belongs to section Inflexae. The section Inflexae is comprised
of four species L. a/ba, L. floccosa, L. gracilis and L. montana. The two
subspecies of L. alba ssp. a/ba and L. a/ba ssp versico/or form the primary gene
pool of Limnanthes whereas the secondary gene pool is composed of L. floccosa, L.
montana, and L. gracilis. All the species belonging to section Reflexae (L. bakery,
L. douglasii, L. macounii, L. striata, and L. macu/ans), are classified as the tertiary
gene pool of Limnanthes based on the fertility of inter sub-specific and interspecific
hybridization with L. alba (reviewed by Knapp and Crane,
1999).
The wild relatives of crop plants have been important sources of genetic
variability for economically important traits such as disease/insect resistance and
4
male sterility. Similarly wild species of meadowfoam possess genes with potential
utility in breeding.
Besides combining useful genes from different species, interspecific
hybridization is an important tool for developing cytoplasmic male sterility (CMS)
by inserting the nuclear genome of a cultivated species into wild cytoplasm. This
approach has resulted in the identification of CMS-inducing cytoplasms in a
number of genera (Schable and Wise, 1998). However very few attempts have
been made previously to make distant hybrids in meadowfoam. Mason (1952)
made limited attempts to make inter-sectional crosses. He crossed L. alba ssp.
versicolor with L. douglasii ssp. douglasii in both directions and L. alba ssp. alba
with L. douglasii ssp. rosea in one direction. Crosses with L. douglasii ssp. nivea
were never attempted. All crosses in his study failed to set seed. Also, intrasectional, interspecific hybridization involving L. alba and two sub-species of L.
floccosa, pum i/a and bellingeriana, has never been reported.
The two long range goals of this research are to i) open the doors to food,
pharmaceutical, and nutritional markets for meadowfoam seed oil and, ii) access
the genetic diversity in wild species belonging to section Inflexae and Reflexae and
develop cytoplasmic male-sterile lines by substituting the nuclear genome of L.
alba into alien cytoplasm. In order to build the basic framework to achieve the
above long range goals the present study with the following specific objectives was
undertaken in meadowfoam.
i)
To develop low erucic acid meadowfoam lines using mutagenesis.
ii)
Study the performance of the low erucic mutant in different genetic
backgrounds.
iii)
Map the genetic factors underlying fatty acid composition differences in
mutant and wild type meadowfoam.
iv)
Assess the feasibility of producing hybrids between cultivated
meadowfoam (L. alba) and L. floccosa ssp. pumila, L. floccosa ssp.
bellingeriana, L. douglasii ssp. douglasii, L. douglasii ssp. nivea, and L.
douglasii ssp. rosea.
v)
Understand the nature of barriers involved in making the above
mentioned inter- and intra-sectional hybrids.
CHAPTER 2
MAPPING GENETIC FACTORS UNDERLYING LOW ERUCIC
ACID CONCENTRATION IN MEADOWFOAM
S.D. Gandhi1, V.K. Kishore1, J.M. Crane', and S.J. Knapp'
'Department of Crop and Soil Science, Oregon State University, Corvallis, OR
97331, USA
7
Abstract
The seed oil of meadowfoam (Limnanthes
alba
Benth.) is very unique as it
contains substantial amounts of unsaturated very long chain (C20 and C22) fatty
acids VLCFs. Meadowfoam seed oil has less than 2 % saturated fatty acids. The
principal fatty acids of meadowfoam seed oil are erucic acid (22:1A13), cis-5eicosenoic acid (20:1A5), cis-5-docosenoic acid (22:1A5) and cis-5, cis-13eicosenoic acid (22:2 A5A13) or dienoic acid. This fatty acid composition provides
the oil with unusual properties such as high oxidative stability, which has made
meadowfoam seed oil valuable for a number of non-food applications. The
development of low erucic cultivars is essential for penetrating pharmaceutical and
edible oil markets. High levels of erucic acid in the diet have been associated with
health problems. The safe limit of erucic acid for human consumption is upto 5% of
total fatty acids. Because the erucic acid concentrations of wildtype lines typically
range from 9 to 23 % and low erucic acid variants have not been discovered,
chemical mutagenesis was used to develop a mutant line (LE76) with greatly
reduced erucic acid. LE76 produced three-fold less erucic acid than the wildtype
(3 as opposed to -9 %). We postulated that the erucic acid phenotype of LE76
was caused by a single mutation; however, the phenotypic distributions ofF2
progeny from crosses between wildtype and mutant lines were continuous and
differed across genetic backgrounds. Quantitative trait loci (QTL) affecting erucic
acid and dienoic acid were mapped using F2:3 progeny from a cross between LE76
and Wheeler (a wildtype line) and a simple sequence repeat (SSR) map spanning
the meadowfoam genome.
Keywords: QTL, erucic acid, dienoic acid, chemical mutagenesis
Introduction
The seeds of meadowfoam (Limnanthes alba Benth.) are rich source of oil and
novel fatty acids (Earl et al., 1959; Bagby et al., 1961; Smith et al., 1960; Miller et
al., 1964). Meadowfoam is a distant relative of Arabidopsis thaliana (Wheeler et
al., 2000). The seed oils of taxa from both families (Limnanthaceae and
Brassicaceae) are rich sources of erucic (22:1A13) and other very long chain fatty
acids (VLCFs), primarily C20 and C22 compounds. Although VLCFs are produced
by rapeseed (Brassica napus L.), crambe (Cram be anthelminica L.) (Downey and
Craig, 1964; Harvey and Downey, 1964) and several other crucifers, the VLCFs of
the Limnanthaceae and Brassicaceae differ in a few important ways. First,
meadowfoam is a richer source of VLCFs (Earl et al., 1959; Bagby et al., 1961;
Smith et al., 1960; Miller et al., 1964; Higgins et al., 1971) than the Brassicaceae.
VLCF content in meadowfoam oil ranges from 94 to 96 % compared to 45 to 56 %
in rapeseed.
Second, meadowfoam is a source of "A5 unsaturated" fatty acids not found in
the Brassicaceae. The primary A5 fatty acids produced by meadowfoam are cis-5eicosenoic acid (20:1A5), cis-5-dococsenoic acid (22:1A5), and cis-5, cis-13-
eicosenoic acid (22:2 A5i13) or dienoic acid (Earl et al., 1959; Smith et al., 1960;
Bagby et al., 1961). The unique physical and chemical properties of meadowfoam
oil can primarily be traced to the unique fatty acid composition (Isbe!!, 1997,
1999). One of the unique properties of meadowfoam oil is extraordinary oxidative
10
stability (Muuse et al., 1992; Isbell, 1997, 1999; Mund and Isbell, 1999). The
uniqueness of the oil when coupled with low general toxicity, has created various
non-food market niches for meadowfoam oil (Burg and Kleiman, 1991; Isbell,
1997, 1999), especially for cosmetics (Dworak, 1994).
Third, less than 2 % of the fatty acids in meadowfoam oil are saturated (Knapp
and Crane, 1995), which is significantly less than rapeseed (6 to 8 %). The
saturated fat content of meadowfoam oil is the lowest reported thus far among
locally and globally important vegetable oils (Weiss, 1983). Because saturated fatty
acids comprise less than 2 % of the total fatty acids, meadowfoam oil seems to be
an excellent candidate for nutritional and medical applications and markets.
Virtually no research has been done on the specific nutritional and medical effects
and properties of meadowfoam oil.
One of the major factors that is preventing the entry of meadowfoam oil into
food, medical, nutritional, and pharmaceutical markets is the presence of high levels
(8 to 23.2 %) of erucic acid in generic (wild type) meadowfoam. High levels of
erucic acid in the diet have been associated with health problems (Borg, 1975;
McCutcheon et al., 1975). Diets containing rapeseed oils with greater than 10%
erucic acid resulted in accumulation of erucic acid in tissue lipids, reduced growth,
myocardial lipidosis and necrosis in rats. The maximal safe intake of erucic acid that
does not lead to lipidosis in rat heart has been estimated to be 2 to 10% dietary fatty
acids (Beare et al., 1972; Abdellatif and Vles, 1973; Kramer, 1973; Christophersen
et al., 1982). The United States Food and Drug Administration currently permits
11
dietary oils containing no more than 2% of erucic acid for human consumption
(Federal register, 1985). Whereas, European Union Food Commission allows 5%
erucic acid of total fatty acids in rapeseed oil meant for human consumption
(Official Journal of the European Community, 2001). The development of low
erucic acid cultivars may open the doors of the edible, medicinal and pharmaceutical
markets for meadowfoam seed oil. An understanding of the biosynthesis and
genetics of erucic acid in meadowfoam would provide a major boost to the efficient
development of low erucic acid meadowfoam cultivars.
Inmost seed oils the end product of de novo fatty acid synthesis are 16- and 18carbon fatty acids. The synthesis of fatty acids is carried out in the chioroplast and
other plastids. Palmitic (16:0), stearic (18:0), and oleic (18:1A5) acids are produced
through sequential condensation of two carbon units into C16 and C18 fatty acyl
chains. These fatty acids are released from acyl carrier protein through the activities
of thioesterases. In some higher plants such as Arabidopsis thaliana L., rapeseed,
and many other species, longer chains are produced through the activities of
elongases (Cassagne et al., 1994; Bao et al., 1998). In these species, the acyl
residues freed from the carrier protein are carried to the cytoplasm and converted to
the acyl-CoA esters by acyl-CoA synthetases, which serve as substrates for further
elongation into very long chain fatty acids (VLCFs) and desaturation in species
producing polyunsaturated fatty acids. These processes and assembly of
triacylglycerols are carried out in the endoplasmic reticulum (Bao et al., 1998).
12
InArabidopsis thaliana L. and rapeseed 18:1 substrate is elongated to 20:1A11
and 20:1A11 to erucic acid through the activity of elongases (Bao etal., 1998;
Harwood, 1996; Kunst et al., 1992; Cassagne et al., 1994). However Pollard and
Stumpf (1980) proposed a variant of this pathway for VLCF synthesis in
meadowfoam. They suggested the two branches in pathway where one branch
produces 20:0 and 22:0 by elongating 16:0. The elongated fatty acids are further
desaturated by A5 desaturase, thereby yielding 20:1A5 and 22:1A5. The other
branch produces 20:1A1 1, erucic acid (20:1A13) and dienoic acid (20:2A5A13).
Sandager and Styme (2000) suggested the involvement of two different thioesterases
FatA and FatB in VLCF synthesis of developing seeds of L. douglasii. FatA is
primarily active on 18:0 and 18:1A9 and FatB is primarily active on 14:0 and 16:0.
Knapp and Crane (1998) have showed that erucic acid concentration is
controlled by a dominant gene (E-locus), which decreases erucic acid content and
increases dienoic acid content in meadowfoam. The E-locus has been recently
mapped using a population derived from a cross between L. alba ssp. versicolor
and L. alba ssp. alba (Katengam et al. 2001). L. alba ssp. versicolor typically
produces more erucic and less dienoic acid than L. alba ssp. alba.
The lowest erucic acid concentration in meadowfoam reported from nature is
6.5% by Knapp and Crane (1995). The understanding of the basic genetics and
biosynthesis of erucic acid in meadowfoam coupled with the lack of natural
variation for low erucic acid encouraged us to develop low erucic acid lines in
13
meadowfoam using mutagenesis. The major objective of this study was to develop
a low erucic meadowfoam using chemical mutagenesis and to map the genetic
factors underlying the differences in the erucic acid concentrations in wild type and
mutant meadowfoam lines.
14
Material and Methods
Fatty acid profiling
The chemical analysis of seeds using destructive or non-destructive (half- seed)
method was carried out as described by Knapp and Crane (1998). The method
described by Knapp and Crane (1995) was used for extraction of fatty acid methyl
esters from seed samples and measurement of fatty acid concentrations using gas
chromatography. Half seed (non-destructive method) to whole seeds (destructive
method) were placed in a test tube having 0.5 ml hexane and crushed with Teflon
rod. The sample was incubated for 15 mm and evaporated to dryness with a stream
of N2 at 500 C. 0.1 ml of ethyl ether and 0.1M KOH were added to the sample.
After incubating the sample for 5 mm at 50° C, 0.1 ml of 0.15 M HCL and 0.5 ml
of hexane were added and mixed. The top layer was used to measure the fatty acid
concentrations using a Hewlett-Packard 6890 gas chromatograph (Hewlett-Packard
Inc., Avondale, CA, USA). Standard samples with known 18:1A5, 18:1A9, 18:3
(linolenic acid), 20:0, 20:1A1 1, 20:1A5, 22:1A5, erucic and dienoic acid
concentrations were used to identify peaks and check measurements (the standards
were obtained from Terry Isbell, USDA-ARS, NCAUR, Peoria, IL).
15
Low erucic acid mutant development
A low erucic acid mutant (LE76) of meadowfoam was developed by exposing
seeds of the open pollinated cultivar Mermaid to methanesulfonic acid ethyl ester
(EMS). Two hundred seeds (Mo) of Mermaid were soaked in distilled water (300 g
seeds! liter of water) for 18 hrs at 4°C. The imbibed seeds were then soaked in 0.04
M EMS (300 g seeds! liter of solution, pH adjusted to 7.0 using 0.1 M phosphate
buffer) for 8 h at 4° C. The chemically treated seeds were rinsed four times with
distilled water and soaked for 18 h in distilled water at 4°C. Finally the seeds were
germinated at 4° C on a moist blotter paper in covered 11 x 11 x 3 cm plastic boxes.
Seedlings were transplanted to potting soil (pumice: peat moss: sandy loam) in 7.5
x 7.5 cm2 plastic pots and grown in a growth chamber (Model CEL 37-14, Sherar-
Gillette Co., Marshall, MI) for 25 to 28 days at 15° C with 18 h of fluorescent light.
These plants were subsequently transplanted in bigger pots and transferred in a
greenhouse at 22° C with 16 h of fluorescent light. M1 plants were grown in
greenhouse and manually self-pollinated. M2 seeds were collected and fatty acid
content was measured non-destructively (as mentioned above). Only M2seeds
selected for low erucic acid were germinated and M2 plants were grown in the
greenhouse. Selfing and selection were repeated through the M5 generation.
16
Crosses of the mutant with wild type lines
The low erucic acid mutant of meadowfoam (LE76) was crossed with three
different meadowfoam lines having wild type erucic acid concentrations (Wheeler,
OMF4O-1 1, and 0MF64). OMF4O-1 1 is an S5 inbred developed from Mermaid and
Wheeler is an open pollinated cultivar (Knapp and Crane, 1999). They both belong
to L.
alba ssp. alba.
0MF64 is a self-pollinated S5 inbred belonging to L.
versicolor (Crane and Knapp, 2000).
F1
alba ssp.
plants were grown in greenhouse and
selfed to produce F2 seeds. F2 seeds from the crosses were non-destructively
analyzed for fatty acid content. In case of Wheeler x LE76, all F2 plants were selfed
and F2: 3 seeds were collected. Bulk samples of twenty seeds (F2:
3)
from each F2
plant of the cross Wheeler x LE76, LE76, Wheeler, OMF4O-1 1 and 0MF64 were
analyzed destructively for their fatty acid content.
Genetic mapping
The genetic map was constructed using 94 (Wheeler x LE76) F2 progeny
derived from a single
F1
seed. The young leaves from LE76, Wheeler and 94 F2
plants were harvested and frozen at 80°C prior to DNA extraction. Genomic DNA
from the frozen leaf tissue was extracted according to Lodhi et al. (1994) with some
modifications. One to two grams of frozen tissue was ground in the presence of
liquid nitrogen and incubated with 2% CTAB (cetyltrimetylammonium bromide)
17
extraction buffer for 1 h at 65°C. Chloroform extraction was carried out and the
aqueous phase was transferred and mixed with 0.5 volume of 5M NaC1. The DNA
was precipitated with 2 volumes of cold 95% ethanol and refrigerated at 4°C
overnight. The DNA pellets were dissolved in TE (10 mM Tris-HCL and 0.1 mM
EDTA, pH 8.0) buffer. Finally the dissolved DNA samples were treated with
RNase (10
jig/.tl)
for 1 h at 37°C.
Total of 369 simple sequence repeat (SSR) markers (Kishore, 2002) were
screened using the genomic DNA of LE76 and Wheeler to identify polymorphic
SSRs. Only the polymorphic SSRs were assayed using the genomic DNA of 94 F2
progeny as described by Kishore (2002), using polyacrylamide gels and
fluorescently labeled amplicons on an ABI Prism 377 DNA sequencer (Applied
Biosystems, Perkin-Elmer, Foster City, CA, USA). Filter set C and the genescan
500 TAMRA internal standard were used for assays performed with 6FAM, TET,
and HEX labeled amplicons. The amplicons for each SSR marker were separately
produced. They were pooled post-PCR, and loaded into each lane: each amplicon
in each pool lane was labeled with a different fluorophore (e.g. 6FAM, HEX and
TET). Three-color multiplexes were used for unambiguous identification of the
DNA fragments produced by each SSR primer pair. GeneScan ver. 2.1 and
Genotyper ver.2.0 (Applied Biosystem, Perkin-Elmer, Foster City, CA, USA)
software were used for recording allele lengths, which were also checked manually.
The individual loci amplified by multilocus SSR markers were labeled with
consecutive letters such as A, B, C etc.
Genetic maps were constructed using G-Mendel (Holloway and Knapp, 1993).
Tests for segregation distortion were performed for each locus using log-likelihood
ratio (G) tests. The observed ratio was significantly different from the expected
ratios of 1:2:1 for co-dominant and 3:1 for dominant markers when G> x2
(1,0.01),
where G is a log likelihood test statistic and x2 is a random variable from the x2
distribution with one degree of freedom. The markers were assigned to linkage
groups with the criteria of likelihood odds (LOD) threshold of 5.0 and a
recombination frequency threshold of 0.25. In case ofF2 population, it is often
difficult to order dominant marker loci linked in repulsion phase (Knapp et al.,
1995). Hence only dominant markers linked in coupling phase along with co-
dominant markers were used for construction of maps. Loci were ordered using
ORDER function of G-MENDEL. Map distances (cM) were calculated using
Kosambi (1944) mapping function.
Mapping of QTLs
Trait means and correlations were calculated using SAS (SAS institute, Cary,
NC). The broad sense heritability of the traits was calculated on the basis of parentoffspring correlation (Frey and Homer, 1957). The erucic and dienoic acid
concentrations measured from the bulk samples of 20 F2 seeds of the 94 F2 plants
along with the linkage map were used to perform composite interval mapping. This
19
was done using the ZmapQTL mapping method in QTLCartographer version 1.3
(Wang et al., 2002). The null hypothesis of no QTL was tested for positions
throughout the genome by comparing LOD scores to an empirical genome-wide
significance threshold calculated from 1000 permutations for P=0.05 (Doerge and
Churchill, 1996). Interactions between significant QTL for each trait were tested, as
the interactions between the closest loci for two different QTL. This was performed
using PROC ANOVA in SAS.
20
Results
The low erucic acid mutant
Three M2 lines with altered fatty acid profile were
identified amon
200 M2
lines produced by EMS mutagenesis. The M2 line LE76 produced less erucic acid
than the wild type (WT) (Table
Table
2.1).
The fatty acid composition of LE76 M2
Fatty acid concentrations for wildtype (WT) and low erucic acid (LE76
M4, and M5) meadowfoam lines.
2.1.
M2, M3,
Fatty acid
± SE*
(%)
Line
20:1A5
22:1A5
22:1A13
22:2A5A13
WT
65.5±0.46
3.4±0.16
9.4±0.38
17.3±0.62
LE76-1M2
62.2
2.5
2.7
28.2
LE76-1M3
55.8±0.51
2.9±0.09
4.9±0.21
30.6±0.45
LE76-1-11M3
43.3
3.0
1.3
30.6
LE76-1-1 1M4
54.6±0.42
3.0±0.09
3.3±0.16
34.8±0.49
LE76-1-11-1M4
51.24
2.34
2.65
39.88
LE76-1-1 1-1M5
56.8±0.52
2.8±0.1
2.9±0.19
32.8±0.52
SE* standard errors are not given for phenotypes of selected single individuals.
21
individuals were screened using half seed samples. One M2 individual (LE76-1)
from the low end of the erucic acid distribution was selected, germinated, and
selfed to produce an M3 line. The erucic acid concentration of LE76-1M2 was 2.7
%. One hundred and five LE76 M3 individuals were non-destructively assayed
using half seed samples (Fig. 2.1). The erucic acid concentration of the M3 progeny
45
NI3
1
40
0
0
S
00
0
00
0
.
0
30
00
0c0,0
25
20 I0
0 0
5
10
15
0
5
10
15
0
5
10
15
Erucic acid (%)
Figure 2.1 Erucic and dienoic acid concentration for 105 LE76-1 M3 progeny, 28
LE76-1-1 1 M4 progeny and 40 LE76-1-1 1M5 progeny (selected individuals
are represented by solid circles).
ranged from 0 to 10.8 %. One M3 individual (LE76-1-11) from the low end of the
erucic acid concentration was selected and selfed to produce an M4 line. The erucic
acid concentration of the selected M3 individual was 1.3 % The erucic range among
22
twenty eight M4 individuals was much narrower than among M3 individuals. One
M4 individual was selected (LE76-.1-1 1-1) and selfed to produce the M5 line. The
erucic acid concentration of selected individual (LE76-1-1 1-1) was 2.65 %. The
erucic acid concentrations of forty M5 individuals fell in the same range (0 to 5.5
%) as M4 individuals. The erucic phenotypes of the selected M4 and M5 lines were
not significantly different and the erucic acid phenotypes of M5 individuals fell in a
narrow range (0 to 5 %) (Fig. 2.1); thus, M5 progeny and beyond were bulked. The
M5 line produced '-3 % erucic acid, three-fold less than the wildtype, and -3O %
dienoic acid, two-fold more than the wildtype (Table 2.1 and Fig. 2.1). Because of
the lack of obvious segregation among M5 individuals, we concluded that the M5
line was homozygous for induced mutations affecting fatty acid composition.
Segregation of the Low Erucic Phenotype in Three Genetic Backgrounds
The genetic basis of the low erucic acid phenotype of LE76-1-1 1-1 M5
(hereafter LE76) was assessed by screening F2 intercross progeny from crosses
between LE76 and three genetically diverse wildtypes (Wheeler, 0MF40- 11, and
0MF64). The phenotypes of the parent lines are shown in Table 2. Wheeler is an
open-pollinated L. alba ssp. alba cultivar, OMF4O-1 1 is an inbred line isolated
from the L. alba ssp. alba cultivar Mermaid, and 0MF64 is an inbred line isolated
from a wild L. albassp. versicolor population. The 20:1A5, erucic, and dienoic acid
Table 2.2. Mean fatty acid concentrations for Wheeler, OMF4O-11, 0MF64 and minimum, maximum and mean fatty acid
concentrations for F2 progeny of the three crosses.
Mean fatty acid ±SD (%)
20:1A5
22:1A5
22:1A13
22:2A5A13
LE76
53.74±3.6
3.49±0.6
3.28±1.8
33.60±3.4
Wheeler
61.34±4.6
4.62±1.0
13.28±3.7
14.70±5.5
OMF4O-1 1
63.34± 1.45
3.52±0.51
8.73±1.2
22.88±1.9
0MF64
57.97±2.4
3.14±1.8
21.04±1.7
9.39±1.6
WheelerxLE7ó
64.67±2.0
4.60±0.6
8.02±1.8
20.29±2.8
OMF4O-1 1 x LE76
66.34±1.96
3.35±0.64
6.02±1.3
22.31±2.0
OMF64xLE76
64.41±2.3
5.10±0.9
13.61±4.5
15.20±3.8
24
concentrations of LE76 and the wildtype parents were significantly different (p <
0.0001). 22:1 A5 concentration of LE76 was only significantly different (p <
0.000 1) from Wheeler (Table 2.2). Fatty acid profiles of 135, 127 and 201 F2
individuals derived from the crosses Wheeler x LE76, OMF4O- 11 x LE76 and
0MF64 x LE76 respectively were analyzed to study the performance of the mutant
in different genetic backgrounds. The erucic acid distributions for the three crosses
were continuous, normal, and spanned different phenotypic ranges (Fig. 2.2).
Crosses to
Wheeler x LE76
35
OMF4O-11 x LE76
0MF64 x LE76
0'
';' 30
0
0c0
Y 25
0 00
c
0
o1c8
I
0
0
4
8 12 16
0
4
8 1216
0
4
8
0
0
12 16 2024 28
Erucic acid (%)
Figure 2.2. Erucic and dienoic acid concentrations for F2 progeny of three crosses.
25
L. alba ssp. alba wildtypes (Wheeler and OMF4O-1 1) produced F2 progeny from
the lower end of the distribution with erucic acid phenotypes (3.6 and 2.9 %) close
to the low erucic acid parent (3 %), whereas the cross to L. alba ssp. versicolor
(0MF64) produced F2 progeny from the lower end of the distribution with a
minimum of 5.8 % erucic acid; thus, the 0MF64 x LE76 cross did not produce any
erucic acid segregates in the low erucic acid range (3 % or less). The upper ends of
the erucic acid distributions for the OMF4O- 1 x LE76 and 0MF64 x LE76 crosses
surpassed the erucic acid concentrations of the respective wildtype parents. The
OMF4O-11 x LE76 F2 cross produced progeny with a maximum of 9.6 % erucic
acid, whereas the 0MF64 x LE76 cross produced F2 progeny with a maximum of
24.8 % erucic acid. The upper end of the erucic acid distribution for the Wheeler x
LE76 cross (13.4 %) did not surpass the wildtype parent (13.2 %). Thus, the lower
and upper ends of the 0MF64 x LE76 erucic acid distribution were -3 % greater
than the phenotypes of the parents.
Similarly, the dienoic acid distributions for the three crosses were continuous,
normal, and spanned different phenotypic ranges (Fig. 2.2). The maximum dienoic
acid concentrations ofF2 progeny from each cross were less than LE76. The
dienoic acid minimums and maximums were lowest for F2 progeny from the L.
alba ssp. versicolor cross. Several 0MF64 x LE76 F2 progeny from the upper end
of the erucic acid distribution had dienoic acid concentrations in the 6.3 to 8.0 %
range, significantly less than wildtype germplasm from both subspecies (Knapp and
Crane, 1995).
While no low erucic acid progeny were produced by the 0MF64 x LE76 cross,
20 'high erucic acid' progeny were produced. Three out of 135 Wheeler x LE76
F2 progeny and four out of 127 OMF4O-1 1 x LE76 F2 progeny had low erucic acid
('-1 % or less). None of the crosses produced transgressive segregates for low
erucic acid. Conversely, two crosses (OMF4O- 11 x LE76 and 0MF64 x LE76)
produced transgressive segregates for high erucic acid. Finally, each cross
produced transgressive segregates with dienoic acid concentrations less than the
wildtype parents (Fig. 2.2).
F2 progeny from the three crosses had 20:1 A5 concentrations greater than or
equal to the upper (wildtype) parent, e.g., 0MF64 produced 58.0 % of 20:1AS,
whereas F2 progeny produced 57.1 to 70.1 % 20:IAS (Table 2.2 and Fig. 2.3).
Erucic acid concentrations were significantly negatively correlated with
dienoic acid concentrations in all the three crosses, but most strongly in the cross to
L. alba ssp. versicolor
(Table 2.3). Erucic acid also showed significant negative
association with 20: lA5 concentration in the progeny of OMF4O-1 1 x LE76 and
0MF64 x LE76. However, erucic acid was positively associated with 22:1A5
concentration in the progeny of OMF4O-11 x LE76 and 0MF64 x LE76. Dienoic
acid showed a significantly negative association with 20:1A5 among the progeny of
Wheeler x LE76 and OMF4O-1 1 x LE76. However, dienoic acid showed a
marginal positive correlation with 20:1 AS concentration in the progeny of 0MF64
x LE76. Dienoic acid was also significantly negatively associated with 22:1A5
concentration in all three crosses.
27
Wheeler xLE76
80
60
C
40
Ii
20
0
OMF4O-ll xLE76
80
60
Ci
R
r.
40
C--
0
II
II
a)
1
0MF64 X LE76
80
60
40
20
0
I
III
C-- C-I
"000
I
I
C
I
I
I
00
2O:15 (¼)
Figure 2.3. Frequency distribution of 20:1 A5 concentration for
crosses.
F2
progeny of three
28
Table 2.3. Phenotypic correlations between major fatty acids of meadowfoam in
progeny of crosses Wheeler x LE76, OMF4O-1 1 x LE76 and 0MF64 x
LE76
Cross
Trait
20:IAS
22:IAS
22:1A13
WheelerxLE76
20:1A5
22:1A5
22:1A13
22:2A5A13
1.00
0.35**
1.00
-0.05
0.14
1.00
OMF4O-11 xLE76
20:1A5
22:1A5
22:1A13
1.00
0.12
1.00
22:2A5M3
OMF64xLE76
20:1A5
22:1A5
22:1A13
22:2A5M3
1.00
-0.13
1.00
F2
22:2A5A13
0.63**
Ø59**
0.65**
1.00
0.36**
0.21*
1.00
0.69**
0.45**
Ø33**
0.63**
0.31**
1.00
0.19*
0.51**
0.85**
1.00
1.00
** significant at P<0.0001
* significant at P=0.05
Linkage map
Since a continuous phenotypic distribution for erucic and dienoic acid
concentrations was observed in all three crosses, we decided to map the genetic
factors underlying the erucic and dienoic acid concentration differences between
the mutant and wild type meadowfoam by using a quantitative trait loci (QTL)
mapping approach. The F2 progeny derived from the cross Wheeler x LE76 was
29
used as the mapping population to make a linkage map and the fatty acid profiles of
F2:3 seeds were used for the QTL analysis. Out of 369 SSR markers screened on
genomic DNA samples of the mapping parents (Wheeler and LE76), 74 loci were
polymorphic. Of the polymorphic SSR loci, 48 were co-dominant and 26 loci were
dominant. The codominant SSR markers (48) and dominant SSR markers lacking
null alleles in Wheeler (11) were used to construct the genetic linkage map. Of the
59 segregating SSR marker loci, 54 coalesced into five linkage groups. The map
was 370.9 cM long and had a mean density of 7.1 cM per SSR marker locus (Table
2.4). Five (ORM 343, 0RM544, 0RM585, 0RM604, ORM613) SSR loci were
unlinked. Presence of unlinked loci can be explained theoretically as either due to
Type II errors or are located 27.5 cM distal to terminal SSR marker loci on the
genetic linkage map. As all the unlinked markers were singletons, we predicted that
at least 137.5 cM of the genetic linkage map is missing. The actual genetic linkage
map of meadowfoam based on this mapping population should be 508.4 cM long or
longer. Hence, we estimated that the genetic linkage map in present study spans
72.9 % of the meadowfoam genome.
Table 2.4. Summary of the meadowfoam simple sequence repeat map.
Linkage Number
group
of
markers
Number
of loci
Distance between loci
Mm.
Mean Max.
(cM)
(cM)
(cM)
group
Length of
Linkage
1
12
12
1.6
5.1
13.8
60.6
2
12
12
1.6
6.9
18.4
83.5
3
09
10
1.1
8.8
22.4
87.5
4
13
13
1.7
6.3
20.2
75.8
5
07
07
0.5
9.1
21.1
63.5
Total
54
54
1.1
7.1
22.4
370.9
The SSR markers were randomly distributed across the genome (Fig. 2.4). The
widest gap was 22.4 cM on linkage group 3. Of the 54 mapped SSR marker loci,
only four showed significant segregation distortion. 0RM371 and 0RM281 had an
excess of LE76 alleles, whereas 0RM466 and ORM6 14 had an excess of Wheeler
alleles.
We mapped 34 new SSR loci as 20 out of 54 mapped SSR loci have also been
previously mapped by Kishore (2002) in inter-subspecific mapping population.
Based on the common markers we could confirm the grouping and ordering of loci
between the two maps.
31
2
I
0.0 -f--ORM147
33 .-cM39
3
0.0
0RM424
12.1
0RM575
23.7
ORM196
0.0
4
ORMIO8
8.4 _f-f_URMS71
14.8
ORu83
20.2
ORMIU7
34.0
36.6
39.0
41.8
16.4
URMIOS
42.1
0RM307
66.1
56.7
0RM189
0RM427
48.2
52.7
60.6
07,5
0RM464
72.0
75.7
0Rv90
0RM265
79.5
ORPl5
83.5
0RM322
22.4
0Ru91A
5
0.0
ORMII0
6.7
9.8
0Rtu43
17.7
0RM477
26.3
28.5
30.2
34.3
0RM381
0.0
0RM482
19.3
URIu14
0RM166 ORMIO8
0RM164
ORM17I
0RM268
42.6
0RIu20
41.7
0RM371
40.4
0RM406
50.8
53.6
54.6
0RM136
0RM333
0RM173
40.7
0RM281
508
0RM122
66.6
ORfl82
57.0
57 5
ORM131
63.9
0RvV48
83.6
0RM324
74.2
0Ru21
87.5
ORM291B
77.4
0RM187
76.8
0RM563
ORMIO9
Figure 2.4. Linkage map of meadowfoam based on F2 progeny of Wheeler X
LE76.
Mapping QTL Underlying Fatty Acid Composition Differences
Broad-sense heritabilities were 0.43 for erucic and 0.27 for dienoic acid
concentration among F3 families. Erucic and dienoic acid were significantly
negatively correlated (p = 0.00 1) (r = 0.63) in F23 families. The erucic acid
32
concentrations for F2:3 families ranged from
2.9
to
12.6
% with a mean of 6.6
%
(Table 2.5). The distribution of the F2:3 progeny for erucic acid concentration was
Table
Mean fatty acid concentration for Wheeler and LE76 and minimum,
maximum fatty acid concentrations for 94 F2:3 derived families.
2.5.
Trait
Wheeler
Mean±SD (%)
LE76
F2:3 families
F2:3 families
Minimum Maximum
20:1A5
61.35±4.6
53.75±3.6
61.50±3.0
53.20
68.70
22:1A5
4.62±1.0
3.49±0.6
5.32±0.8
4.07
8.72
22:1A13
13.28±3.7
3.28±1.8
6.59±2.0
2.85
12.58
22:2A5A13
14.70±5.5
33.60±3.4
25.14±3.8
16.01
37.90
normal (Fig. 2.5A).
A
single transgressive family was observed for low erucic acid
phenotype. The distribution of the F2:3 families for dienoic acid concentration was
also normal (Fig.
2.5B).
with mean of 25.1
%
The dienoic acid concentration ranged from
(Table 2.5).
16.0
to 37.9 %
33
25
2U
15
0
5F
22:1M3 (%)
22:2E5M3 (%)
Figure 2.5. Frequency distribution of erucic and dienoic acid concentration for 94
F23 families of the cross Wheeler X LE76.
Composite interval mapping identified a total of four QTL (Fig. 2.6) underlying
the differences in the concentrations of the erucic and dienoic acid in the seed oil of
the mutant and wild type meadowfoam.
0
0
"-4
0
20 40 60 0
20 40 60
80 0
20 40 60 80
0
20 40 60
Map position (cM)
80 0
20 40 60 80
Erucic acid
Dienoic acid
Figure 2.6. LOD plots of the QTL identified for erucic and dienoic acid concentrations.
Table 2.6. Summary of the QTL detected for erucic and dienoic acid concentrations.
Trait
Erucic Acid
(er)
Dienoic Acid
(dien)
QTL
Linkage
group
LOD
0RM427
PVE
(%)
a
d
dla
ORM51S
0RM333
16.9
8.3
3
5.22
3.04
7.45
24.0
1.46
-0.83
1.50
0.54
0.92
-0.48
0.36
-1.11
-0.32
3
0RM187
7.78
29.6
-2.95
-1.89
0.63
cr2.2
2
2
cr3.1
dien3.1
er2.1
NML*
*NML= nearest marker locus to the QTL
%PVE= percentage of phenotypic variation explained
a= additive gene action
d= dominant gene action
Erucic acid (er): A total of three QTL were detected underlying the erucic acid
concentration differences between LE76 and Wheeler (Fig. 2.6). Two of these QTL
(er2.1 and er2.2) were located on linkage group 2 whereas one QTL (er 3.1) was
located on linkage group 3. A total of 49 % of the phenotypic variance was
explained (PVE) by the three QTL detected. The maximum amount of phenotypic
variance explained by a single QTL (er 3.1) was 24 % of phenotypic variance
(Table 2.6). As expected the alleles from the low erucic parent (LE76) were found
to be associated with two QTL (er 2.1 and er 3.1) having larger PVE, for decrease
in erucic acid content. Additive effects were observed for alleles at these QTL for
the differences in the erucic acid concentration. The allele from the high erucic
parent (Wheeler) was responsible for decreasing the erucic acid concentration at
QTL er 2.2.
Dienoic acid (diene): One QTL (dien 3.1) was identified on linkage group 3
(Fig. 2.6) to account for the differences in the concentration of dienoic acid in the
seed oil of Wheeler and LE76. The amount of phenotypic variance explained by
dien 3.lwas 29.6 % (Table 2.6). The allele from the low erucic parent (LE76)
contributed towards an increase in the dienoic acid at this QTL with additive gene
action. None of the interactions between the QTL for either single trait or two traits
was significant.
37
Discussion
The genetic variation available in a crop is a prerequisite for its improvement
due to the fact that efficiency of selection mainly depends upon it. Lack of natural
variation in a crop system leads the plant breeders to induce mutations in the crop
of interest and exploit useful mutants in the plant breeding programs. Plant breeders
for various agronomic as well as qualitative traits have exploited induced
mutagenesis using chemical or physical agents for crop improvement. We discuss
here the development of low erucic acid meadowfoam line through mutation
breeding, its performance in different genetic backgrounds and mapping the genetic
factors underlying the differences in the fatty acid concentrations from wild type
meadowfoam.
Induced mutagenesis to manipulate the fatty acid profiles of seed oil have been
used in Brassica species (Rakow, 1973; Robbelen and Nitsch, 1975; Auld et al.,
1992; Kott et al., 1996; Velasco et al., 1995, 1998), soybean (Erickson et al., 1988;
Bubeck et al., 1989; Wilcox and Cavins, 1990; Burton et al., 1994), Arabidopsis
(Browse et al., 1986) and many other oilseed crops. A chemical mutagen EMS is
usually a preferred agent in these studies, because of the chromosomal damage
caused by radiations in the target organism (Medrano et al., 1986). Most of the fatty
acid mutations developed by chemical mutagenesis were recessive and obtained
after screening several thousands of M2 and M3 progeny in Brassica species.
Hence, very few mutants in fatty acid biosynthesis of Brassica species have been
38
identified (Auld et al., 1992). However, the frequency of mutation seemed higher in
meadowfoam than that reported for other plants. We could identify three
individuals out of 200 M2 with different fatty acid profile in this study. In another
study in our lab we identified two mutants showing decreased erucic acid content
out of 205 M2 plants (data not shown). Thus confirming the higher frequency of
fatty acid mutations in meadowfoam compared to Brassica species.
The development of seed oils having a low or high percentage of erucic acid for
various edible and industrial applications is currently a topic of research (Barro et
al., 2001). The low erucic acid mutant identified in our study showed a three-fold
decrease in erucic acid content when compared with the wild type meadowfoam.
Similar results were shown in Brassica carinata where they recorded two-fold
decrease in erucic acid content (Velasco et al., 1995; Barro et al., 2001) in mutants
identified through chemical mutagenesis. In Brassica species, cultivars virtually
free of erucic acid have also been developed as a result of coordinated research
using chemical mutagenesis, recurrent selection and hybridization (FernandezEscobar et al., 1988; Velasco et al., 1995; Getinet et al., 1997). The near or total
absence of erucic acid in rapeseed is caused due to a mutation in the fatty acid
elongation 1 gene (Roscoe et al., 2001). This mutation is characterized by a great
decrease in quantities of VLCFs. A large decrease in the quantity of 20:IAII and
concomitant increase in
C18 lipid
species along with absence of erucic acid was
observed for erucic acid free cultivars in rapeseed. This mutation also resembles the
seed fatty acid elongation (FAEJ) mutants of A. thaliana (James and Dooner, 1990;
Lemieux et al., 1990 Kunst et al., 1992). However, in this study as the total
concentration of VLCFs was not reduced we assume that the low erucic acid
mutant was not caused by elongase lesions as that of rapeseed and Arabidopsis.
The erucic and dienoic acid are sequentially produced in one branch of the
pathway from elongated 18:1 substrate from oleate (18:1 A9) as per the pathway
proposed for very long chain fatty acid synthesis in meadowfoam (Pollard and
Stumpf, 1980). Erucic acid is further desaturated by A5 desaturase to produce
dienoic acid. While 20:1 A5 and 22:1 A5 are sequentially produced in another
branch of the pathway from elongated 20:0 substrate from palmitate (16:0).
Sandager and Stymne (2000) suggested the involvement of two different
thioesterases FatA and FatB type in long chain fatty acid synthesis in developing
seeds of meadowfoam. Where FatA type enzyme has a principle activity on 18:0
and 18:1A9 and FatB type enzyme primarily acts on 14:0 and 16:0. The low erucic
mutant in our study showed significantly increased dienoic acid with reduction in
20:1A5 and 22:1A5 than wild type meadowfoam. Hence we speculate that the
mutation might be affecting the activity of enzyme FatB that is responsible for
hydrolyzing the acyl chains of palmitate (16:0) in the process of producing 20:1A5
and 22:1A5. Consequently, increased activity of elongase and especially A5
desaturase due to lack of proportionate substrates leads to increased dienoic acid
with reduction in erucic acid. However this speculation is based on the assumption
that the same A5-desaturase enzyme is involved in both branches of pathway. The
discovery and cloning of a A5 desaturase from L. douglasii (Cahoon et al., 2000)
opens the way to study this possibility.
As the mutant identified in this study was developed by chemical mutagenesis
using EMS, it was expected that the mutant phenotype would be caused due to a
single gene mutation and we could map this mutation as a Mendelian factor.
However, a continuous phenotypic distribution for erucic acid was observed in the
F2
progeny of all the three crosses studied. The cross between LE76 and 0MF64,
an L. alba ssp. versicolor line, failed to produce low erucic acid progeny.
Conversely, crosses between LE76 and wild type L. alba ssp. alba lines (e.g.,
Wheeler and OMF4O-l1) produced progeny with low erucic acid (3.5%). Hence
we speculate that 0MF64 and other L. alba ssp. versicolor lines carry a linked
mutation (E locus
Knapp and Crane, 1998) that interacts with the low erucic acid
mutation(s) in LE76. The strong selection against mutant locus! loci in this genetic
background is also possible. However the occurrence of low erucic individuals in
segregating population of other two crosses where LE76 was crossed with
individuals belonging to L. alba ssp. alba substantiates the former possibility.
Extreme transgressive segregants were observed for higher concentration of
20:1 A5 in all three crosses studied. Presence of such outliers can be explained on
the basis of epistasis or overdominanace. Also when the parental lines fixed for set
of alleles having opposite effects are crossed, extreme phenotypes appear (Lynch
and Walsh, 1997). Phenomenon of overdominanace seems logical for 20: 1A5 as
extreme phenotypes only towards higher concentration were observed.
41
The initial strategy was to use F2 progeny of the cross between 0MF64 and
LE76 for mapping the genetic factors underlying the differences in the erucic acid
concentrations of the mutant and wild type meadowfoam, as they had the highest
difference for erucic acid concentration. However, the failure to recover low erucic
parental type in the segregating progeny of this cross led us not to use the
F2
progeny from 0MF64 x LE76 as the mapping population. The erucic acid
concentration difference between OMF4O-1 1 and LE76 was lower compared to the
difference between Wheeler and LE76. The polymorphism at DNA level was also
lower between OMF4O-11 and LE76 as both of them were derived from cultivar
Mermaid by selection and mutagenesis respectively (data not shown). Hence the
F2
progeny of Wheeler x LE76 was used for mapping the genetic factors underlying
the erucic acid concentration differences between the mutant and wild type
meadowfoam.
SSR markers have become the marker system of choice in many plant species
because of the high level of polymorphism (Wang et al., 1994). They are PCR
(polymerase chain reaction) based and suitable for high throughput genotyping.
Genetic maps based on SSRs have been developed for wheat (Roder et al., 1998),
rice (Tenmkykh et al., 2000), barley (Ramsay et al., 2000), sunflower (Tang et al.,
2002) and meadowfoam (Kishore, 2002). We used the SSR markers developed by
Kishore (2002) to construct the linkage map of meadowfoam. A stringent LOD
score (5.0) was used to construct the linkage map so as to increase the efficiency of
detecting true linkage and minimizing false positives in assigning markers to
42
linkage groups (Type I errors). All the 54 SSR loci that coalesced into five linkage
groups were randomly distributed across the genome of meadowfoam, which is in
agreement with the results of Kishore (2002). There was no clustering of the SSR
markers in our linkage map unlike the SSR maps in several other crops such as
barley and wheat (Ramsay et al., 2000; Roder et al., 1998) that showed
considerable clustering of SSR markers especially near centromeric regions. With
an average inter locus spacing of 7.1 cM the linkage map developed in this study is
an excellent framework map for mapping genetic factors underlying the differences
in the fatty acid concentrations of the mutant and wild type meadowfoam. The
genetic linkage map constructed in this study was second genetic linkage map for
meadowfoam comprised of SSRs. Hence, it gave us a powerful tool for comparing
and confirming the grouping and ordering of loci based on common markers
between this and previous map.
A total of three QTL were identified for the difference in the erucic acid
concentration, whereas one QTL was identified for the difference in dienoic acid
concentration between the mutant (LE76) and wild type (Wheeler) meadowfoam.
Alleles from LE76 contributed for decreasing the erucic acid concentration at two
(er
2.1 and er 3.1) out of three QTL. Whereas, an allele from Wheeler was
responsible for decreasing the erucic acid concentration at er 2.2. Hence we
speculate that the QTL detected in our study for erucic acid concentration
differences in the mutant and wild type meadowfoam include the mutated locus by
EMS along with some other modifier loci, which may or may not be targeted by
43
EMS. The presence of modifier alleles for erucic and dienoic acid was also
suggested by Knapp and Crane (1998) while studying the genetic basis of fatty acid
profile differences between the two subspecies of meadowfoam. They observed the
continuous variation for these two traits among E and ee progeny in segregating
populations. Further Katengam et al., (1999) empirically tested for the presence of
genetic effects other than the E locus by selecting and crossing E_ individuals from
the tails of dienoic acid distribution from a backcross population. They found a
continuous distribution for both erucic and dienoic acid in the 100
F2 progeny
derived from the cross between two E_ individuals suggesting the presence of
modifier alleles. A total of 49 % of the phenotypic variance was explained by the
QTL identified in this study for erucic acid concentration differences. The
heritability of erucic acid calculated by the parent-offspring (F2
F2
3)
correlation
method was 0.43. Heritability of dienoic acid was low (0.27) which is also
reflected in the low total phenotypic variance (29 %) explained by the QTL
(dien
3.1) identified in this study.
The two fatty acids erucic and dienoic were moderately but significantly and
negatively correlated (r =-0.63) in this study. The presence of two QTL er 3.1 and
dien
3.1 for erucic and dienoic acid respectively in close proximity of each other
suggesting the relatedness of these traits. Occurrence of QTL for related traits in
close proximity is a common phenomenon. Diers and Shoemaker (1992) also
mapped quantitative trait loci conditioning five major fatty acids mainly on two
linkage groups in soybean.
44
Despite providing excellent information regarding the genetic factors
underlying the fatty acid concentration differences between mutant and wild type
meadowfoam there are some limitations to this study. The modest size of the F2
mapping population (n = 94) may have limited our ability to identify QTL and to
estimate correctly the magnitude of their effects (reviewed in Lynch and Walsh,
1998). When interactions between significant QTL were tested it was found that
none of the interactions were significant. Thus suggesting that epistasis was not
involved in the QTL identified in this study. However we need to recognize that
interactions may be present between the QTL and the loci without significant
main effects (Knapp, personal communication), which was not tested.
Despite the limitations mentioned above, the development of near-isogenic
lines (NILs) and low erucic acid cultivars should proceed more rapidly and
efficiently by using flanking DNA markers to introgress the underlying mutation(s)
once the QTL are validated. DNA fingerprinting of advanced backcrossed
generations of lines selected for low erucic acid would provide a reliable and rapid
tool for validation of QTL identified in this study. In conclusion, this study has
elucidated the development of the first low erucic acid meadowfoam along with the
genetic factors underlying the differences in the fatty acid concentration of the
mutant and wild type meadowfoam.
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53
CHAPTER 3
DIFFERENTIAL REPRODUCTIVE ISOLATION MECHANISMS WITHIN
AND BETWEEN SECTIONS OF GENUS LIMNANTHES
S.D. Gandhi', J.M. Crane', 0. Riera-Lizarazu', V.K. Kishore' and S.J. Knapp'
'Department of Crop and Soil Science, Oregon State University, Corvallis, Or
97331 USA
54
Abstract
Cultivated meadowfoam (Limnanthes alba Benth.) is an annual oil seed crop
native to southern Oregon, California and British Columbia. The genus Limnanthes
is composed of nine species and divided into two sections, Inflexae and Reflexae.
Meadowfoam has been domesticated since 1973. The domestication of
meadowfoam was based on L. alba, belonging to section Inflexae. With the
objectives of introgressing genes from wild relatives and also producing
cytoplasmic male sterile lines by inserting the nuclear genome of L. alba into a
wild cytoplasm, inter-sectional crosses involving L. alba and three subspecies of L.
douglasii and intra-sectional crosses involving L. alba and two subspecies of L.
floccosa were carried out. The isolation mechanisms involved in keeping species
apart from each other were found to be different within and between sections. The
intra-sectional, inter-specific hybrids could be made by making the involved
species coincide in their flowering time. The study of partially fertile intra-sectional
hybrids showed that the reduced pollen viability (3 0-33%) was not due to structural
differences between the chromosomes of the two species, as normal meiotic
behavior was observed in PMCs. The inter-sectional crosses were found to be noncompatible and various abnormalities during pollen tube growth were observed.
Keywords: Inter-specific hybrids, meiosis, Brassicales
55
Introduction
Cultivated meadowfoam (Limnanthes alba Benth.) is an annual oil seed crop
native to southern Oregon, California and British Columbia (Mason, 1952; Kahn,
1971; Jam, 1986). The seed oil of meadowfoam contains unique unsaturated very
long chain fatty acids
(C20
and C22) with outstanding oxidative stability (Smith et
al., 1960; Bagby et al., 1961; Miller et al., 1964; Knapp and Crane, 1995, 1998;
Isbell, 1997). Limnanthes (Order Brassicales) is a distant relative of Arabidopsis
(Wheeler et al., 2000). The genus Limnanthes comprises of nine species. The genus
has been divided into two sections, Inflexae and Reflexae based on the
morphological characters of petals folding inward or outward during seed
maturation (Mason, 1952). The entire genus Limnanthes is diploid (2n=10),
hermaphroditic and self-compatible (Mason, 1952).
Limnanthes alba was identified by Gentry and Miller (1965) as the most
promising species for domestication. The domestication process, which began more
than thirty years ago (Crane and Calhoun, 1974) has been entirely based on L. alba.
The secondary and tertiary gene pools have not been important to the domestication
process and have not supplied diversity for meadowfoam breeding.
Limnanthes alba belongs to section Inflexae. This section is comprised of four
species viz., L. alba, L. floccosa, L. gracilis and L. montana. The two subspecies of
L. alba ie. alba and versicolor form the primary gene pooi of Limnanthes whereas
the secondary gene pool is composed of L. floccosa, L. montana, and L. gracilis.
The species belonging to section Reflexae include L. bakery; L. douglasii; L.
macounii; L. striata and L. maculans, and are classified as the tertiary gene pool of
Limnanthes based on the fertility of intersubspecific and inter specific hybridization
with L. alba (reviewed by Knapp and Crane, 1999).
The major reason for using interspecific hybridization in plant breeding is to
expand the gene pooi by introducing alien genes carried by wild species. The wild
relatives of crop plants have been important sources of genetic variability for
economically important traits eg., disease/insect resistance and male sterility.
Similarly wild species of meadowfoam possess genes with potential utility in
breeding.
Besides combining useful genes from different species, interspecific
hybridization is an important tool for developing cytoplasmic male sterility (CMS)
by inserting the nuclear genome of a cultivated species into wild cytoplasm. This
approach has resulted in the identification of CMS-inducing cytoplasms in number
of genera. The best characterized CMS-inducing cytoplasm of sunflower, PET1
and ogu cytoplasm of radish, which confers male sterility upon B. napus were
derived from distant hybridization (Reviewed by Schnable and Wise, 1998).
Meadowfoam is naturally allogamous heterotic species. The heterotic potential
of this crop can be best exploited by making single cross hybrids. The most
efficient way of doing this will be having CMS system developed in this crop. As
this is a new crop no hybrid seed production system has been developed yet for
meadowfoam.
57
Very few attempts have been made previously to make distant hybrids in
meadowfoam. Mason (1952) made limited attempts to make inter-sectional crosses.
He crossed L. alba ssp. versicolor with L. douglasii ssp. douglasii in both
directions and L. alba ssp. alba with L. douglasii ssp. rosea in one direction.
Crosses with L. douglasii ssp. nivea were never attempted. All crosses in his study
failed to set seed. Also, intra-sectional, interspecific hybridization involving L. alba
and two sub-species of L. floccosa viz., pumila and bellingeriana has never been
reported. The two long-range goals of this research are to access the genetic
diversity in the secondary and tertiary gene pooi of Limnanthes and to develop a
cytoplasmic male sterility system for efficient hybrid seed production in
meadowfoam. I order to tap the possibility of making hybrid seed and to
understand the nature of barriers involved, the present study of interspecific
hybridization was undertaken in meadowfoam.
Materials and Methods
Plant Material
This study was performed using two lines of L. alba viz., OMFIO9-3 and
OMF1 56 to cross with randomly chosen plants of L. floccosa ssp. pumila (LFP,
P128372 1) and L. floccosa ssp. bellingeriana (LFB, P1283 720), to produce intra-
sectional hybrids, and with L. douglasii ssp. douglasii (LDD, P1278170), L.
douglasii ssp. nivea (LDN, P1283713), L. douglasii ssp. rosea (LDR, P1283715) to
produce inter-sectional hybrids. The seeds of LFP, LFB, LDD, LDN and LDR are
held by the United State Department of Agriculture (USDA) National Plant
Germplasm System (NPGS) and the Oregon State university (OSU) Centre for
Oilseed Research (CORE) (Knapp and Crane, 1999). OMF1O9-3 is highly self-
pollinating line, developed by hybridizing an L. alba ssp. alba individual (derived
from a cross between a high oil line and open pollinated cultivar Mermaid) with
0MF64 (a self pollinating L. alba ssp. versicolor inbred line, Knapp and Crane,
2000) and selecting for high autonomous seed set. 0MF156 is an outcrossing line
which was developed by inter-pollinating an open pollinated population based on a
collection of L. alba ssp. alba and L. alba ssp. versicolor. Both sub-species of L.
floccosa are highly self-pollinated due to partial cleistogamy, whereas, all three
sub-species of L. douglasii are cross-pollinated due to protandry and heterostyly.
59
Pollination and crosses
Meadowfoam seeds were germinated at 4°C on moist blotter paper in covered
11- by 11 by 3 cm plastic boxes. Seedlings were transplanted to potting soil
(pumice: peat moss: sandy loam) in 7.5 cm2 plastic pots and grown in a growth
chamber (Model CEL 37-14, Sherar-Gillette Co., Marshall, MI) for 25 to 28 days at
15°C with 18 h of fluorescent light. These plants were subsequently transplanted in
bigger pots and grown in a greenhouse at 22°C with 16 h of fluorescent light.
The flowering times of three species were synchronized by transferring late
flowering lines to warm temperatures first to induce flowering. Early flowering
lines were transferred 10 to 15 days later.
L.
a/ba plants were transferred from low
to high temperature 10 to 15 days earlier than L. floccosa plants. Similarly L.
douglasii plants were transferred from low to high temperature 10 to 15 days earlier
than L. a/ba.
Flowers were emasculated one to two days pre-anthesis and manually
pollinated one to three days post-anthesis. Stigmas become receptive 12-36 h postanthesis in L. floccosa and L. alba and 48-72 h post-anthesis in L. douglasii. Pollen
from freshly dehisced anthers was used.
L.
floccosa x L. a/ba crosses were only
produced using L. floccosa as the female. We crossed L. a/ba and L. doug/asii
reciprocally.
Meiotic analysis
Unopened flower buds of OMF 109-3, LFP and LFP x OMF 109-3 were
collected and fixed in three parts absolute ethanol: one part 45% acetic acid
fixative for analyses of pollen mother cells (PMCs). Anthers were isolated and
squashed in 2% acetocarmine. Crystals of FeSO4 were added to squashes to
enhance staining. PMCs were observed at diakinesis, metaphase I, and anaphase I
of meiosis to observe chromosome pairing and separation under light microscope
(Zeiss Axioscope 2).
Bulk pollen samples were collected from parents and hybrids at anthesis,
stained with 121(1 (Jensen, 1989) and scored for stainability under a light micrscope.
The percentage of stainable pollen was estimated from 1000 pollen grains per
entry.
Pollen tube growth evaluation
Pollen tube growth was evaluated as described by Martin (1959) in all of the
inter-sectional crosses and five controls OMF1O9-3 x OMF1O9-3; LDD x LDD;
LDN x LDN; OMF1 56 x OMF1O9-3; LDD x LDN. Flowers were manually
pollinated at 24 to 36 h post-anthesis in L.
douglasii.
alba
and 48 to 72 h post-anthesis in L.
Flowers were harvested at 6, 12, 18 and 24 hrs post pollination Twenty
61
flowers were emasculated and pollinated per cross combination per time point.
Flowers were fixed in FAA (1 Formalin: 8 absolute ethanol: 1 glacial acetic acid)
for 24 hrs. Styles were isolated, rinsed in tap water, cleared and softened in 8N
NaOH for 14 h, and stained for callose with 0.1% solution of aniline blue dye in
0.1N K3PO4 for 4 h. Treated styles were mounted in a drop of Vectashield, an
antifade (purchased from Vector Laboratories Inc. Burlingame, CA-94010) and
covered with a cover slip with gentle squashing. The slides were observed using
epifluorescence under Zeiss Axioscope 2. The styles of L. douglasii lines were
divided into five sections, whereas the styles of OMF1O9-3 and 0MF156 were
divided into three and four sections respectively for the quantification of pollen
tube presence. A total of 960 styles for 12 non-compatible and 400 styles for five
compatible crosses were observed at 6, 12, 18 and 24 hrs after pollination for
pollen germination, penetration, and pollen tube elongation.
62
Results
The inter and intra-sectional crosses carried out are listed in Table 3.1.
Table 3.1. Summary of crosses attempted.
Cross
OMF1O9-3
LDD
LDN
LDR
0MF156
x
x
x
x
OMF1O9-3
x
x
x
x
x
x
-
x
-
OMF 156
LDD
x
x
LDN
x
x
LDR
x
x
LFP
x
x
LFB
x
x
-
LFP
LFB
-
-
-
-
-
-
-
-
-
-
-
Section Inflexae hybrids
The use of L. floccosa plants as the male parent was impeded by difficulty in
isolating minute pollen grains. The crosses carried out within section Inflexae
63
involving LFP, LFB and 0MF156, OMF1O9-3 produced seed. From nine LFP
flowers pollinated with OMF1O9-3 pollen five seeds were obtained. Eleven LFP
flowers, pollinated with 0MF156 produced 4 seeds. Eight and nine LFB flowers
were pollinated with OMF1O9-3 and 0MF156 respectively. Cross between LFB
and OMF1O9-3 produced 3 seeds. LFB x OMF 156 produced two seeds. Seeds from
only LFP x OMF1O9-3 germinated.
The hybrid plants resembled to their pollen parent OMF1O9-3 in branching
pattern and other characteristics such as hairy leaves and hairy sepals. The flower
size of hybrids was intermediate to both the parents. The hybrid progeny was male
and female fertile. This was tested by selfing the hybrid and backcrossing it to
OMF1O9-3. Approximately 30-40 seif-pollinations and backcrosses were sufficient
to produce 70 and 50 seeds respectively. However the hybrid showed very low
pollen viability (33%) (Fig. 3.1) as compared to both parents (97.4, 90.8 % in
OMF1O9-3 and LFP, respectively).
Meiotic behavior of F1
No meiotic abnormalities were observed in the parents or hybrids. Every cell
observed at metaphase I and diakinesis of first meiotic division had five bivalents
65
than the parents (Table 3.2). Similarly, the chiasma frequency of the hybrid (6.86)
was significantly (p<O.001) less than the parents. A total of 100 PMCs in anaphase
I were observed for LFP, OMF1O9-3, and the hybrids. It was found that the
Table 3.2. Number of ring and rod bivalents and chiasma frequency observed for
'n' cells at diakinesis of meiosis I.
Population
Number of
cells
LFP
100
OMF1O9-3
LFPxOMF1O9-3
Rings
(Mean)
351
Rods
(Mean)
Chiasma
frequency
149
8.51
(3.51)
(1.49)
100
396
(3.96)
(1.04)
104
8.96
110
205
(1.87)
345
(3.13)
6.87
chromosomes disjoined normally in parents as well as hybrids. No irregularities
were observed in the distribution of the chromosomes (Fig.3.2).
Inter-sectional crosses
Total of-250 pollinations were done for 12 inter-sectional crosses. None of the
inter-sectional crosses were successful. To dissect the reasons behind the failure, a
thorough examination of these crosses was done. The variation in length of the
styles between different species is known to play a role in successful pollen tube
elongation and fertilization of the ovule. Species belonging to section Inflexae
differ in their style length from species belonging to section Reflexae. The average
style length for OMF1O9-3 was 4.7 mm and for 0MF156 was 5.8 mm. The styles
of LDD, LDN and LDR are 7.9, 6.4 and 7.0 mm long, respectively (Fig. 3.3).
TWfl
LDD
Figure 3.3. Style morphology for 0MF156 (L. alba), OMF1O9-3 (L. alba), L.
douglasii ssp. nivea, L douglasii ssp. rosea, L. douglasii ssp. douglasii
Because of style length differences and the tendency of long styled species to fail as
females, one would expect that the pollen from Reflexae individuals should easily
reach the base of the short styles of Inflexae individuals. However, it was observed
67
that none of the pollen tubes reached the base of the style, irrespective of the
direction of the crosses, suggesting involvement of factors other than the length of
the style.
Pollen germination and penetration
Pollen germination for compatible crosses (Fig. 3.4) (the intra-specific crosses
analyzed as controls, which produced seed) ranged from 77 to 92%. However,
pollen germination in incompatible crosses (all the inter-sectional crosses which
failed to produce seed) ranged from only 32 to 69%. The mean pollen germination
for compatible crosses was 84%, which was significantly different (p<O.0001) from
the mean pollen germination of 48% in incompatible crosses.
The pollen penetration for incompatible crosses varied from 7 to 23%, whereas
for the compatible crosses it varied from 18 to 48% (Table 3.3). The mean pollen
69
Table 3.3. Pollen germination, penetration and pollen tubes in compatible and
non-compatible crosses
Cross
Compatible crosses
OMF1O9-3xOMF1O9-3
Number Number Germinated Penetrated Number
of
of
pollen
pollen
of
styles
pollen grains
grains
pollen
grains
(%)iiji
tubes
80
1575
91.93
39.49
453
LDDxLDD
80
2207
78.61
47.76
460
LDNxLDN
80
2171
87.93
32.01
512
OMF156xOMF1O9-3
80
2502
85.57
38.88
595
LDDxLDN
80
4168
77.25
17.97
407
LDDxOMF156
80
1124
43.5
8.27
22
LDDxOMF1O9-3
80
1018
32.02
15.60
23
LDNxOMF156
80
1111
38.70
13.23
56
LDNxOMF1O9-3
80
734
61.58
21.25
51
LDRxOMF156
80
946
33.80
11.80
23
LDRxOMF1O9-3
80
1049
45.09
12.20
16
OMF1O9-3xLDD
80
1553
68.83
12.56
61
OMF1O9-3xLDN
80
1056
56.62
7.38
7
OMF1O9-3xLDR
80
1247
46.83
8.18
8
OMF156xLDD
80
1679
56.99
22.57
76
OMF156xLDN
80
545
54.31
17.60
28
OMF156xLDR
80
889
42.96
15.07
50
In-compatible crosses
=Percentages of total number of pollen grains
=Percentages of total number of pollen grains
70
penetration of non-compatible crosses was almost three times significantly
(p<O.000l) lower than the mean pollen penetration of compatible crosses.
Pollen tube elongation
In in-compatible crosses a total of 45 pollen tubes were found in 240 styles,
observed at 6 hrs after pollination. When the same number of styles were observed
at 12, 18 and 24 hrs after pollination, they had 100, 127 and 150 pollen tubes,
respectively. There was no significant difference (p = 0.31) between the number of
pollen tubes observed at 18 hrs and 24 hrs after pollination. From the total 421
pollen tubes observed in 960 styles, not one pollen tube reached the base of the
style. For compatible crosses, 476 pollen tubes were found in 100 styles observed
at 6 hrs after pollination. The number of pollen tubes increased to 618 at 24 hrs
after pollination. There was no significant difference (p = 0.69) between the
number of pollen tubes observed at 18 and 24 hrs after pollination for compatible
crosses. In compatible crosses, all the pollen tubes observed at 6 hrs after
pollination had already reached the base of the style (Fig. 3.4). Thus suggesting that
in regular cases it would not take longer than 6 hrs for the germinated pollen tube
to reach the base of the style. Although not a single pollen tube was found at the
base of the styles for all the in-compatible crosses, the combinations 0MF156 x
LDD and OMF1 09-3 x LDD were found to be most promising based on the total
71
number of pollen tubes (76 for 0MF156 x LDD and 61 for OMFIO9-3 x LDD)
observed in 80 styles.
Sites of inhibition
The mechanism of inhibition for pollen development in inter-sectional crosses
was found to operate at two different sites, the stigma and the style. At the
stigmatic level, pollen development was arrested either by preventing germination
or preventing penetration of germinated pollen due to heavy callose deposition
either on the stigmatic surface (Fig. 3.5A) or in growing pollen tubes of germinated
pollen (Fig. 3.4). Out of the 12 incompatible crosses, six crosses viz., LDD x
0MF156, LDD x OMF1O9-3, LDR x OMF1O9-3, OMF1O9-3 x LDN, OMF1O9-3 x
LDR, 0MF156 x LDN showed callose deposition on the stigmatic surface (Table
4). In case of the pollen, which germinated and penetrated the stigmatic tissue,
most of the pollen tubes traveled successfully through the stylar neck. However
they were arrested in the lower portion of the styles (Fig. 3.4). In L.
douglasii
individuals, the pollen tubes were arrested in the lower two fifth portion of the
style, whereas for L. alba individuals the pollen tubes were arrested in the lower
one third or one fourth region of the styles (Table 3.4). The only cross showing
arrested pollen tubes in the first one fifth part of the style was LDN x 0MF156. In
the cross LDD x 0MF156, 100% of the pollen tubes were arrested in the lower
Table 3.4. Inhibition sites for incompatible crosses.
Cross
Number
of styles
Number
of
pollen
Callose
deposition
on stigma
styles)
tubes(%
% Pollen tube stopped*
lstsection
2'section 3rdsection 4thsection 5thsection
LDD x 0MF156
80
22
6.25
0.0
0.0
0.0
0.0
100.0
LDD x OMF1O9-3
80
23
16.25
0.0
4.54
4.54
77.27
13.6
LDNxOMF156
80
56
0.0
1.85
1.85
0.0
11.11
85.19
LDNx OMF1O9-3
80
51
0.0
0.0
0.0
0.0
2.5
97.5
LDRx 0MF156
80
23
0.0
0.0
20.68
13.79
31.03
34.48
LDR x OMF1O9-3
80
16
18.75
0.0
5.26
5.26
10.52
78.95
OMF1O9-3 x LDD
80
61
0.0
0.0
15.0
85.0
NA
NA
OMF1O9-3 x LDN
80
7
15.0
0.0
37.5
62.5
NA
NA
OMF1O9-3 x LDR
80
8
12.5
0.0
40.0
60.0
NA
NA
0MF156 x LDD
80
76
0.0
0.0
6.3
19.05
74.65
NA
OMF156xLDN
80
28
11.25
0.0
2.78
16.67
80.55
NA
OMF 156 x LDR
80
50
0.0
0.0
4.0
24.0
72.0
NA
* The styles of LDD, LDN, LDR were divided in five sections and the styles of OMF 109-3 and OMF 156 were divided in
three and four sections respectively.
74
one-fifth (near base) portion of the style. LDN x OMF1O9-3 and LDN x 0MF156
were other two crosses showing the maximum proportion of total pollen tubes (97.5
and 85.2% respectively) arrested in the lower one fifth region of the style. In case
of crosses where L. alba individuals were used as females, OMF1O9-3 x LDD and
0MF156 x LDN showed the highest percentages (85.0 and 80.6) of pollen tubes
arrested near the base.
Pollen tube growth abnormalities
The nomenclature for abnormalities in pollen tube growth observed in intersectional, and interspecific crosses in this study is based on the categories defined
by Willliams et al. (1982) and Rego et al. (2000) with some modifications. The
following five categories of abnormalities were observed:
i)
Disoriented or loss of direction pollen tube grows either backward or
horizontally instead of growing towards the base of the style (Fig.5B)
ii)
Swollen tip pollen tube the tip of pollen tube is swollen and ceased to
grow (Fig.3.5C)
iii)
Burst pollen tube - the tip of the pollen tube burst after swelling
(Fig.3.5D)
iv)
Forked pollen tube - the tip of the pollen tube is bifurcated (Fig.3.5E)
v)
Coiling pollen tube the pollen tube coils around itself (Fig.3.5F)
75
Among the five abnormalities listed above, swollen tip pollen tubes (ii) and loss
of directional growth (i) were the most prevalent classes observed in all the incompatible crosses. OMF1O9-3 x LDD and 0M156 x LDD showed the highest
percentages (88.52 and 78.95, respectively) of pollen tubes with swollen tip
(Table 3.5). Coiling pollen tubes were observed in only three crosses viz., LDD
x OMF1O9-3, LDN x 0MF156 and LDN x OMF1O9-3. Forked pollen tube
(OMF1O9-3 x LDD) and burst pollen tubes (OMF 156 x LDD) were observed in
only one cross each.
Table 3.5. Abnormalities observed in pollen tube growth of non-compatible crosses.
Cross
% Pollen tubes
Number of
pollen tubes
Burst
Swollen tip
Disoriented
Forked
Coiling
LDD x 0MF156
22
0.0
54.54
45.45
0.0
0.0
LDD x OMF1O9-3
23
0.0
43.48
39.13
0.0
17.39
LDNxOMF156
56
0.0
27.78
46.29
0.0
5.56
LDN x OMF1O9-3
51
0.0
54.90
37.25
0.0
7.84
LDR x 0MF156
23
0.0
69.56
30.43
0.0
0.0
LDR x OMF1O9-3
16
0.0
62.50
37.50
0.0
0.0
OMF1O9-3 x LDD
61
0.0
88.52
8.19
3.28
0.0
OMF1O9-3 x LDN
7
0.0
71.42
28.57
0.0
0.0
OMF1O9-3 x LDR
8
0.0
62.50
37.50
0.0
0.0
0MF156 x LDD
76
1.32
78.95
19.73
0.0
0.0
0MF156 x LDN
28
0.0
42.86
57.14
0.0
0.0
0MF156 x LDR
50
0.0
70.00
30.00
0.0
0.0
77
Discussion
Gene transfer from one species to another is prevented by many isolation
mechanisms in nature these include spatial isolation, non-overlapping flowering
time, and cleistogamy, which act as barriers only in nature but not in breeding
programs (Hermsen, 1992). On the other hand various pre-fertilization and postfertilization barriers due to genetic differences hinder the process of bringing
different species together even in breeding programs. It was found that successful
crosses could be made between L. floccosa and L. alba individuals that belong to
same section Inflexae. The reproductive isolation mechanisms evolved to keep
these two species apart in nature were non-overlapping flowering time and partial
cleistogamy in L. floccosa. However, both these barriers in nature could be
overcome in the breeding program. In crosses between the species L. douglasii and
L.
alba that belong to different sections, non-overlapping flowering time was the
only obvious barrier in nature. However, detailed studies have shown that nature
has ensured the separation of these two species by evolving pre-fertilization
barriers such as lack of pollen-pistil recognition and defective pollen tube growth.
Hence it is equally difficult even in breeding programs to bring these two species
together.
Intra-sectional crosses
Successful hybrid production between LFP and OMF1O9-3 has given us the
hope of utilizing species from the secondary gene pool of Limnanthes for
improvement of cultivated meadowfoam. However, failed germination of hybrid
seeds from other intra-sectional crosses listed in Table 1 demonstrates that the seed
set in inter-specific and inter-generic crosses is a product of interaction between
both, species as well as the individual genotypes involved (Bozorgipour and Snape,
1990). Similar observations were reported by Choudhari and Joshi (2001) in
Brassica where they found a marked difference in crossability of two genotypes
(RBT63 and RBT58) of B. tournefortii with one B. rapa genotype (BRBS). RBT63
produced 3.7% hybrids when crossed with BRBS whereas only 0.9% hybrids were
produced when RBT58 was crossed with BRBS. Rescuing embryos through
embryo culture in intra-sectional crosses where seeds failed to germinate was
beyond the scope of this study. However, the technique of embryo rescue for
immature embryos is well documented in meadowfoam (Southworth and
Kwiatkowski, 1995) and should be employed in order to recover these intra-
sectional hybrids.
The presence of characteristics from both parental species in the hybrid
indicates that the F1 plants had genes of both parents combined. The hybrids in the
present study showed considerably reduced pollen viability (30-33%) as compared
to both parents. Many of the previous investigations involving interspecific or
79
distant hybrids attributed the phenomenon of reduced pollen viability or male
sterility to various meiotic irregularities such as prevalence of univalents
accompanied by erratic segregation in Cuphea (Ali and Knapp, 1995) and Brassica
(Choudhari and Joshi, 2001); chromosomal bridges, fragments and sticky
chromosomes in Pisum (Nirmala and Kaul, 1994); absent or defective cytokinesis
in Glycine (Skorupska and Palmer, 1990), while cell fusion was considered to be
one of the causes of male sterility in Pennisetum (Rao et al., 1989). Such meiotic
abnormalities in inter-specific hybrids arise due to structural differences between
the chromosomes of parental species leading to abnormal microspore development.
Perfect chromosome pairing with five bivalents in each cell observed for the hybrid
in this study suggests that the genome of L. floccosa and L. alba are homologous.
Hence the low fertility of hybrids is not due to structural differences in the
chromosomes of parental species as shown by Adayapalam et al. (1999) in
Lathyrus. However, reduced homology between the chromosomes of the two
different species was evident in present study by the reduced number of ring
bivalents in hybrids as compared to both parents, leading to lower chiasma
frequency.
There are a number of causes for male sterility in higher plants. The male
sterility genes exhibit a wide array of action ranging from complete absence of the
androecium to the production of inviable pollen grains due to abnormal
microsporogenesis. They act with marked precision on various stages of
microsporogenesis (pre-meiosis, meiosis, post-meiosis) and prevent the normal
development of sporogenic tissue, tapetum cells, pollen mother cells and
microspores (Singh, 1993). As no meiotic irregularities were observed in this study,
the mechanism leading to male sterility or pollen inviability must be operating in
post-meiotic stages of microspore development. Post-meiotic degeneration of
microspores was also observed by Chhabra et al. (1997) in Pennisetum while
studying the effects of different cytoplasms on micro sporo genesis and anther
development using iso-nuclear A-lines. Degenerated pollen grains in a
morphologically normal anther is considered as gametophytic male sterility (Kaul,
As both of the parental species involved in hybridization showed full male
fertility and the hybrid showed 3:1 ratio of dead to viable pollen, we speculate that
pollen death may be occurring due to the interaction between nuclear genes and the
cytoplasm during pollen development. The nucleo-cytoplasmic interaction was also
very well documented in genus Limnanthes by Kesseli and Jam (1984) to account
for gynodioecy observed in natural populations of L. douglasii. The most plausible
explanation for the 3:1 ratio for dead to viable for the hybrid (LFP x OMF1O9-3) in
this study is the possible involvement of two unlinked loci (from L. floccosa),
which are required for compatible (viable) development of a microgametophyte
(pollen grain) in the cytoplasm of L.floccosa as in the case of maize T-cytoplasm,
sunflower PET-cytoplasm and onion T-cytoplasm (Schnable and Wise, 1998).
However, further backcrosses are necessary to substitute the nuclear genome of L.
floccosa with L. alba into L. floccosa cytoplasm to understand the exact genetic
nature of the phenomenon observed in this study.
Inter-sectional crosses
The results of the present study suggest that the barriers to inter-sectional
hybridization are bi-lateral where inhibition mechanism operates at four stages
indicated in Fig. 3.6 with some degree of independent control. The stigmatic and
stylar inhibition to normal pollen development reported in this study is in
agreement with previous studies carried out to analyze the pre-fertilization barriers
in non-compatible crosses such as Williams et al. (1982) in the Ericaceae family,
Kandasamy et al. (1994) in interspecific crosses between Brassica and Arabidopsis,
Rego et al. (2000) in yellow passion fruit, Tamari et a! (2001) in Tumeracea family.
The presence of either germinated or un-germinated pollen on the stigmatic
surface (Fig. 3.4, 3.5A) in all the non-compatible crosses in this study suggest that
the wet stigma of Limnanthes (classified according to Heslop-Harrison and
Shivanna (1977), does support pollen adhesion to some extent due to nutrient rich
exudates that promotes pollen hydration in a somewhat indiscriminate manner
(Wilhemi and Preuss, 1997).
The mechanism for inhibiting the penetration of the stigmatic surface by the
pollen tube was more specific. The rejection response was manifested either by
Pollen deposition on stigma
Pollen germination
$t
Pollen penetration
into papillar tissue
5
Pollen tube elongation
into st/le
5
Pollen tube reaches the
base and enters the
ovule
'5
Pollen Tails to
germinate
Pollen tails to
penetrate
Pollen tube Tails to
elongate
Pollen tube doesnt
reach the base
Fertilization takes place
Figure 3.6. Schematic representation of checkpoints to prohibit the growth of
pollen tube in incompatible crosses.
callose deposition on the stigmatic surface (Fig. 3.5A) adjacent to incompatible
pollen or within the germinating pollen tubes, especially at the tip of these rejected
pollen tubes (Fig. 3.4). This cell-to-cell reaction between incompatible pollen and
papillar cells of the stigma was also observed in previous studies carried out in
yellow passion fruit by Rego et al. (2000). They attributed this reaction to a protein
produced by the S-locus and categorized the inhibition as homomorphic
sporophytic incompatibility. The observation of arrested pollen tube growth with
abnormalities mostly in the lower part of the style in our study is also in agreement
with previous study by Gaget et al. (1984). Gaget et al. (1984) reported abnormal
pollen tube growth arrested in the lower part of styles while studying inter-sectional
crosses in Populus. In the case of yellow passion fruit, stylar inhibition arrested
pollen tubes in the upper one third of the style (Rego et al., 2000) for selfincompatible crosses. Gaget et al. (1984) and Rego et al. (2000) both speculated on
the presence of an interaction between pollen tube and stylar components as in the
classical gametophytic self-incompatibility system. All of the species in the present
study are self-compatible and no evidence for presence of an S-gene controlled
self-incompatibility system of either the sporophytic or gametophytic type has been
presented yet for genus Limnanthes. However, inhibitory responses in the form of
either callose deposition or arrested pollen tube growth suggest the presence of an
S-allele variant that has lost the function of self-recognition. Considering the
complex structure of the S-locus, Trognitz and Schmiediche (1993) suggested a
model for a common basis of intra-specific self-incompatibility and inter-specific
incompatibility. They suggested that the S-locus has two major components-style
and pollen specific. Stylar protein inhibits the pollen tube growth, and vice versa.
The process of inhibition is modified based on the incompatibility/compatibility
system. In the case of self-compatible species, recognition of S-allele does not
occur due to missing or non-expression of S in either style or pollen or both. Hence,
pollen tube suppresses the stylar products and fertilize the ovule. However, for
incompatible, inter-specific crosses where S alleles are different, the process of
recognition is meaningless. The interaction between stylar and pollen products is
not affected by either matching or differing factors. In this case varying degrees of
inhibition at various sites in the style is observed. The reaction depends upon the
ability of the two components to inhibit each other. Although this model is based
on the assumption of interaction between style and pollen genotypes, it seems to fit
well for the phenomenon observed in our study. Speculation of presence of
modified S alleles seems also justifiable because of the close relationship of genus
Limnanthes with crucifers (Rodman et al., 1998). The crucifer family includes selfincompatible genera, such as Brassica, and self-fertile genera such as Arabidopsis.
The self-incompatibility system is controlled genetically by the multifunctional
gene complex of the S locus in Brassica is very well studied (Boyes et al., 1997).
Conner et al. (1998) identified an Arabidopsis homeolog of the Brassica S locus
region. However, no sequence similar to the Brassica S locus gene those are
required for the self-incompatibility responses were detected in the Arabidopsis
genome. Thus absence of self-recognition genes at the S locus in Arabidopsis is a
very well documented phenomenon, which might also be true in the genus
Limnanthes.
The species belonging to section Reflexae have considerably larger styles than
those of species belonging to section Inflexae as mentioned earlier in this study.
However, crosses in both directions i.e. involving short styled female parent
(Inflexae) with long styled pollen parent (Reflexae) and vice versa failed. Also, in
all the non-compatible crosses studied, the major proportion of pollen tube arrested
was in the lower part or near the base of the style. These two observations rule out
the possibility of either lack of intrinsic ability of pollen tubes to grow beyond the
length of their own pistils as suggested by Shivanna (1996) or pollen from long
styled individuals can easily reach and fertilize the ovules of short styled
individuals as shown in Lycopersicon (Liedl et al., 1996); Leucaena (Sorensson and
Brewbaker, 1994) and Bromeliads (Vervaeke et al. 2001).
The various abnormalities such as lack of direction, arrested pollen tube growth
or even burst pollen tubes observed in this study basically suggest the lack of
signals or guidance cues necessary to direct the pollen tube into the transmitting
tissue of the style and finally towards the ovule. Although the exact nature of these
guidance cues is yet to be known, glycoproteins secreted by styles (Cheung et al.,
1995); carbohydrate-rich molecules secreted by ovules (Herrero, 2000), and a high
calcium ion concentration in synergid cells (Cheung and Wu, 2001) have all been
suggested as possible guidance cues for pollen tubes as they approach the ovules.
However, specificity must exist among guidance cues. As pollen tubes from one
species do not usually target ovules of other or related species (Shimuzu and
Okada, 2000). A single genetic mechanism cannot be used to explain the
phenomenon observed in this study. It is equally possible that the pollen-pistil
recognition in two groups i.e. long-styled individuals of section Reflexae and shortstyled individuals of section Inflexae may have evolved separately in each floral
morph. Thus both the long style and the short styled species may not share a
common molecular basis involving the recognition of the pollen by the style (Lloyd
and Webb, 1992).
Pre-fertilization barriers can be overcome using methods such as bud or delayed
pollination combined with embryo rescue techniques if necessary. There are equal
numbers of successful and unsuccessful examples for overcoming the prefertilization barriers by bud pollination (Lewin, 1993; Westwood et al., 1997;
Kandasamy et al., 1994). The success and failure largely varies from species to
species. Hence, further attempts to access the diversity present in section Reflexae
can be made using bud pollination for the promising crosses mentioned earlier in
this study.
In conclusion, the mechanisms evolved for isolation of species in the two
sections of genus Limnanthes are different. Hence the diversity present within the
section Inflexae seems to be more accessible for enhancing meadowfoam
improvment further by not only combining the best genes from different species
but also recovering male sterile cytoplasm in future backcross generations.
However, further attempts in terms of overcoming pre-fertilization barriers are
necessary to access the diversity present in section Reflexae.
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with Hordeum bulbosum and their potential for haploid production. Cereal
Res. Commun., 18: 203-208.
Cheung AY, Wang H and Wu HM (1995) A floral transmitting tissue-specific
glycoprotein attracts pollen tubes and stimulates their growth. Cell, 82:
383-393.
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93
CHAPTER 4
CONCLUSIONS
Meadowfoam (Limnanthes alba Benth.) is an annual oil seed crop native to
southern Oregon, California and British Columbia (Mason, 1952; Kahn, 1971; Jam,
1986). Meadowfoam was originally developed as a source of chemical feedstocks
for industrial markets (Isbell, 1997). The major fatty acids found in meadowfoam
seed oil are cis-5-eicosenoic (20:1 A 5), erucic (22:1 A 13), cis-5, cis-13-eicosenoic
(22:2 A 5 A 13), and cis-5-docosenoic (22:1 A 5) acid (Earle et al., 1959; Bagby et
al., 1961)
The consumption of erucic acid (22:1 A 13), a fatty acid found in the seed oils
of meadowfoam, rapeseed (Brassica napus L.), and several Brassicaceae, has been
linked to increased risk of heart disease (Borg, 1975; McCutcheon et al., 1975).
The United States Food and Drug Administration currently permits dietary oils
containing no more than 2% of erucic acid for human consumption (Federal
Register, 1985). Whereas, European Union Food Commission allows 5% erucic
acid of total fatty acids in rapeseed oil meant for human consumption (Official
Journal of the European Community, 2001).
The development of low erucic acid meadowfoam cultivars may open the doors
of the edible, medicinal and pharmaceutical markets for meadowfoam seed oil. We
used the approach of induced chemical mutagenesis using EMS for the
development of low erucic meadowfoam due to the lack of natural variation (the
lowest erucic acid concentration in meadowfoam reported from nature is 6.5% by
Knapp and Crane, 1995) for erucic acid in meadowfoam.
The low erucic acid mutant identified in our study showed a three-fold decrease
in erucic acid content (-P3
%
vs --9 %) when compared with the wild type
meadowfoam. The mutant phenotype does not seem to be caused by elongase
lesions as that of Brassica and Arabidopsis (Roscoe et al., 2001; James and Dooner,
1990; Lemieux et al., 1990 Kunst Ct al., 1992) as the total concentration of VLCFs
was not reduced. The performance of the mutant differed in different genetic
backgrounds.
F2
progeny of Wheeler x LE76 was used for mapping the genetic
factors underlying the erucic acid concentration differences between the mutant and
wild type meadowfoam. The linkage map comprised of 54 SSR loci was
constructed. A total of three QTL were identified for the difference in the erucic
acid concentration, whereas five QTL were identified for the difference in dienoic
acid concentration between the mutant (LE76) and wild type (Wheeler)
meadowfoam. The development of near-isogenic lines (NILs) and low erucic acid
cultivars should proceed more rapidly and efficiently using flanking DNA markers
to introgress the underlying mutation(s) once the QTL are validated.
Cultivated meadowfoam (Limnanthes alba Benth.) is one of eight species of
Limnanthes split between two sections: [nflexae (L. alba, L. gracilis, L. floccosa,
and L. montana) and Reflexae (L. douglasii, L. bakeri, L. striata, and L. macounii).
95
Various subspecies of L. alba, L. gracilis, L. floccosa, and L. douglasii have been
described (Mason, 1952). The domestication of meadowfoam began around thirty
years ago (Crane and Calhoun, 1974). The domestication process has been entirely
based on L. alba. The secondary and tertiary gene pools have not been important to
the domestication process and have not supplied diversity for meadowfoam
breeding. Very few attempts have been made previously to make distant hybrids in
meadowfoam. (Mason, 1952). No successful inter-sectional hybrid has been
reported till date in meadowfoam. Also, intra-sectional, inter-specific hybridization
involving L. alba and two sub-species of L. floccosa viz., pumila and bellingeriana
has never been reported. The specific aim of this study was to assess the feasibility
of producing hybrids between cultivated meadowfoam (L. alba) and L. floccosa
ssp. pumila, L. floccosa ssp. bellingeriana, L. douglasii ssp. douglasii, L. douglasii
ssp. nivea, and L. douglasii ssp. rosea for the purpose of accessing the genetic
diversity in these species and developing cytoplasmic male-sterile lines by
substituting the nuclear genome of L. alba into alien cytoplasms.
We found that the differential isolation mechanisms are involved within and
between sections of Limnanthes. Where the barriers of partial cleistogamy and nonoverlapping flowering could be overcome and hybrid seed was produced in intrasectional crosses. Whereas non-overlapping time was found not to be the only
barrier involved in making inter-sectional hybrids. Out of four intra-sectional
crosses, seeds of only LFP x OMF1O9-3 geminated. The hybrid showed reduced
pollen viability (3O-33 %). However the low fertility of the hybrid was not due to
structural differences in the chromosomes of parental species as the meiotic
analysis of PMCs showed normal chromosome pairing and disjunction in
metaphase I and anaphase I, respectively. As no meiotic irregularities were
observed in this study, the mechanism leading to male sterility or pollen inviability
must be operating in post-meiotic stages of microspore development. On the other
hand, all the inter-sectional crosses studied showed abnormal pollen tube growth.
The mechanism of inhibition of pollen development in inter-sectional crosses was
found to operate at two different sites, the stigma and the style. At the stigmatic
level, pollen development was arrested either by preventing germination or
preventing penetration of germinated pollen due to heavy callose deposition on the
stigmatic surface. Pollen tube growth was arrested mainly in lower part of the style
due to various abnormalities. Abnormalities observed in this study were
disoriented or loss of direction, swollen tip, burst, forked, coiling pollen tubes.
Swollen tip pollen tube and loss of directional growth were the most prevalent
classes observed in all the incompatible crosses.
Hence it seems that the diversity present within the section Inflexae is more
accessible for enhancing meadowfoam breeding further by not only combining the
best genes from different species but also recovering male sterile cytoplasm in
future backcross generations for efficient hybrid seed production. However, further
attempts in terms of overcoming pre-fertilization barriers are necessary to access
the diversity present in section Reflexae.
97
We reported here
i)
The development of the first low erucic acid meadowfoam.
ii)
The genetics underlying the differences in the erucic and dienoic acid
differences in low erucic mutant and wild type in meadowfoam.
iii)
Fertility of intra-specific hybrids and pre-fertilization barriers involved
in making the intra-sectional hybrids in meadowfoam.
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