Genetic studies of cold tolerance in barley
by Somvong Tragoonrung
A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in
Crop and Soil Science
Montana State University
© Copyright by Somvong Tragoonrung (1992)
Abstract:
The poor efficiency of selection for improved cold tolerance in winter barley has been attributed to a
combination of complex inheritance and difficulties in trait assessment. Several physiological
parameters have been used as selection criteria for cold tolerance. However, definitive analysis of the
Mendelian factors controlling these parameters has not yet been performed. The objectives of this
research were to genetically investigate the relationships among characters associated with winter
survival and to identify the chromosomal locations of genes controlling those traits.
One hundred doubled haploid lines derived from F1 plants from a cross between Dicktoo (winter) x
Morex (spring) were used to study the genetics of field survival, LT50, fructan content, flowering date
and prostrate growth habit. A medium density linkage map was constructed from these doubled haploid
lines using DNA, protein isoenzyme, and morphological markers. This map was then utilized to
identify the location of quantitative trait loci (QTL) associated with these traits. Sequence-tagged-sites
(STS) were developed for polymerase chain reaction as DNA (PCR) markers.
Significant correlations between crown fructan content and winter hardiness parameters were detected.
These results suggested that fructan content may be one factor contributing to cold tolerance. However,
selection based on high fructan content did not positively improve other winterhardiness associated
traits.
The STS-PCR system facilitated map construction in this cross. A set of 77 DNA markers were
resolved into eight linkage groups and distributed to seven chromosomes. Large QTL effects for
fructan content and all winter hardiness parameters except prostrate growth habit were detected in a
region of approximately 20 centiMorgans on the long arm of chromosome 7. Careful analysis of lines
carrying recombination breakpoint within this 20cM interval suggested that at least 2 genes affecting
measured parameters were located within this interval. This interval analysis suggested that the
association among at least some of these traits is due to linkage rather than pleiotropy. Smaller QTL
effects modifying characters measured in this study were detected on chromosome 2 and chromosome
5. GENETIC STUDIES OF COLD TOLERANCE IN BARLEY
by
Somvong Tragoonrung
A th e s is su b m itte d in p a r t ia l f u l f i l l m e n t
o f th e re q u ire m e n ts f o r th e degree
of
D o cto r o f P h ilo so p h y
in
Crop and S o il Science
MONTANA STATE UNIVERSITY
. Bozeman, Montana
J u ly , 1992
ii
APPROVAL
o f a th e s is subm itted by
Somvong Tragoonrung
T h is t h e s is has been read by each member o f th e t h e s i s committee
and has been found to be s a t i s f a c t o r y re g a rd in g c o n te n t, E nglis h usage,
fo r m a t, c i t a t i o n s , b i b l i o g r a p h i c s t y l e , and c o n s is te n c y , and is ready
f o r submission to th e C o lle g e o f Graduate S tu d ie s .
/a te
)
C h a irp ers o n , Graduate Committee
Approved f o r i h e M ajor Department
Date
Head, M ajor D
Approved f o r th e C o lle g e o f Graduate S tu d ie s
— Da
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In p re s e n tin g t h i s th e s is in p a r t ia l f u l f i l l m e n t o f th e
re q u ire m e n ts f o r a D o c to ra l degree a t Montana S ta te U n iv e r s it y ,
t h a t th e L ib r a r y s h a ll make i t
I agree
a v a ila b le t o b o rrow e rs under r u le s o f th e
L ib r a r y . B r ie f q u o ta tio n s from t h i s th e s is are a llo w a b le w ith o u t s p e c ia l
p e rm is s io n , p ro v id e d t h a t a c c u ra te acknowledgment o f source is made.
P e rm issio n f o r e x te n s iv e q u o ta tio n from o r re p ro d u c tio n o f t h is
t h e s is may be g ra n te d by my m ajor p r o fe s s o r , o r in h is absence, by th e
Dean o f L ib r a r ie s when, in th e o p in io n o f e it h e r , th e proposed use o f
th e m a te ria l is f o r s c h o la r ly purposes. Any co p yin g o r use o f th e
m a te ria l in t h i s th e s is f o r f in a n c ia l g a in s h a ll n o t be a llo w e d w ith o u t
my w r it t e n p e rm is s io n .
S ig n a tu re
________
Date
-
" 7 - / 7
7 2-
iv
ACKNOWLEDGMENTS
I
w ish to express my deep g r a t it u d e to D r. Tom B lake f o r h is
guidance and encouragement th ro u g h o u t th e course o f my g ra d u a te s tu d y .
S p e c ia l th a n ks and acknowledgment t o D r. P.M. Hayes, D r. J.
M a rtin , D r. T. McCoy, and D r. C. Bond f o r s e rv in g as my g ra d u a te
com m ittee and f o r t h e i r a d v ic e .
I
am a ls o th a n k fu l t o my c o -w o rke rs and a l l th e peo ple t h a t work
in Leon Johnson H a ll f o r t h e i r f r i e n d l y r e la t io n s h ip s .
V
TABLE OF CONTENTS
Page
APPROVAL............................................................................
STATEMENT OF PERMISSION TO USE...............................................
ii
iii
ACKNOWLEDGEMENTS...................................................................................
iv
TABLE OF CONTENTS.............................: ..................................................
v
LIST OF TABLES.......................................................................................
v ii
LIST OF FIGURES...................
ABSTRACT.....................................................................................................
v iii
ix
CHAPTER
1 INTRODUCTION..........................................................................
I
2 GENETICS OF FRUCTAN CONTENT AND WINTERHARDINESS
IN A SPRING x WINTER BARLEY CROSS............................
I n t r o d u c t io n ..................................................................
M a te r ia ls and M ethods..............................................
P la n t M a t e r ia l.........................................................
T r a i t Assessm ent..................................................
RFLP and C o r r e la tio n A n a ly s is ........................
R e s u lts and d is c u s s io n ............................................
3
3
4
4
5
6
7
3 STS FACILITATED PCR FOR BARLEY GENOME MAPPING..
I n t r o d u c t io n ........... ............................................
M a te r ia ls and M ethods................................
G e n e tic S to c k s ...................... ..................................
DNA I s o la t io n ......................................................
DNA S equencing.........................................................
P rim er S y n th e s is .................. ..................................
PCR A m p lif ic a t io n ..................................................
D e te c tio n o f Polym orphism s..............................
R e s u lts .............................................................................
STS P ro d u c tio n ...................... ..................................
Chromosomal L o c a tio n ............................................
E f f ic ie n c y o f PCR A n a ly s is ..............................
D is c u s s io n ......................................................................
16
16
17
17
18
18
19
19
19
20
20
21
22
30
vi
4 MAPPING OF TRAITS ASSOCIATED WITH WINTERHARDINESS
IN A WINTER x SPRING BARLEY CROSS.....................
I n t r o d u c t io n ....................................................
M a te r ia ls and M ethods..............................................
Germplasm....................................... ; ..........................
T r a it A sse ssm e n t....................................................
Map C o n s tru c tio n and QTL A n a ly s is ................
R e s u lts and D is c u s s io n ............................................
Map C o n s tr u c tio n ..................................................
Cold T o le ra n ce and QTL A n a ly s is ..................
Heading D a te .............................................................
F ru cta n C ontent o f Crown T is s u e ....................
P r o s tra te Growth H a b it........................................
36
36
38
39
39
42
52
53
54
5 SUMMARY.....................
60
REFERENCES CITED
33
33
35
63
vii
LIST OF TABLES
T able
1
Page
Mean, minimum, and maximum va lu e s o f p h y s io lo g ic a l
t r a i t s f o r w in te rh a r d in e s s ....................................................
8
2
C o r r e la tio n c o e f f ic ie n t s between fr u c ta n c o n te n t
in, 1991 and 1992 and p h y s io lo g ic a l p a ra m e te rs ........... 10
3
Percentage o f lin e s in common among p h y s io lo g ic a l
t r a i t s a t 15 and 25% s e le c tio n i n t e n s i t i e s ..................11
4
C o r r e la tio n c o e f f ic ie n t s o f m arkers t h a t showed
s ig n if ic a n t in t e r a c t io n s w ith crown fr u c ta n
c o n te n t in
1992............................................................................ 15
5
P rim e r sequences, s o u rce s, chromosomal lo c a tio n s
and a l l e l ism a n a ly s e s ................................. ; ............................24
6
R e s t r ic t io n s i t e v a r ia t io n in PCR p ro d u c ts ..................25
7
D e s c r ip tio n s , chromosomal lo c a t io n , and numbers o f
mapped p ro g e n ie s assignm ents f o r m arkers used to
lo c a te q u a n t it a t iv e t r a i t lo c i in th e doubled
h a p lo id progeny o f D ic k to o x M orex. D e f in it io n s o f
m arker p r e fix e s are p ro v id e d in th e t e x t ...................... 40
8
R egression c o e f f ic ie n t s o f mR and BCD265b lo c i
w ith w in te rh a rd in e s s p a ra m e te rs..........................................55
'j
9
S ig n if ic a n t le v e ls o f a s s o c ia tio n s among
m arkers and w in te rh a rd in e s s p a ra m e te rs ............... ..58
viii
LIST OF FIGURES
F ig u re
1
Page
HPLC chromatograms o f crown fr u c ta n c o n te n t
f o r D ic k to o and Morex c u l t i v a r s . .......................................
9
2
DNA sequence o f PSTl-340 c lo n e s ..........................................23
3
A m p lif ic a t io n o f b a r le y DNA from D ic k to o and Morex
w ith e ig h t p rim e r s e ts on 1% agarose g e l ...................... 26
4
D ig e s tio n o f PCR p ro d u c ts w ith r e s t r i c t i o n
endonucleases w ith fo u r-b a s e r e c o g n itio n sequences
were e le c tro p h o re s e d on a 7% p o ly a c ry la m id e g e l ___ 27
5
S e g re g a tio n o f DxM doubled h a p lo id l i n e s ....................... 28
6
A m p lif ic a t io n o f w h e a t-b a rle y chromosome a d d itio n
l i n e s ................................................................................................... 29
7
Map c o n tr u c tio n in th e D ic k to o x Morex c r o s s ................44
8
The d i s t r i b u t i o n o f f i e l d s u r v iv a l measured a t
C o r v a llis , OR in 1991 f o r D ic k to o x Morex l i n e s ___ 45
9
The d i s t r i b u t i o n o f f i e l d s u r v iv a l measured a t
Bozeman, MT in 1992 f o r D ic k to o x Morex l i n e s ___ _.46
10
The d i s t r i b u t i o n o f LT50 f o r D ic k to o x Morex l i n e s . .47
11
The d i s t r i b u t i o n o f heading d a te under 24 h l i g h t
f o r D ic k to o x Morex l i n e s ........................................................48
12
The d i s t r i b u t i o n o f heading d a te under green house
c o n d itio n (16 h l i g h t ) f o r D ic k to o x Morex l i n e s . . . 49
13
The d i s t r i b u t i o n o f fr u c ta n c o n te n t in 1992 f o r
D ic k to o x Morex l i n e s ..........................
14
50
The d i s t r i b u t i o n o f p r o s tr a te gro w th h a b it f o r
D ic k to o x Morex l i n e s ................................................................. 51
ix
ABSTRACT
The poor e f f ic ie n c y o f s e le c tio n f o r im proved c o ld to le ra n c e in
w in t e r b a r le y has been a t t r ib u t e d to a c o m b in a tio n o f complex
in h e r ita n c e and d i f f i c u l t i e s in t r a i t assessm ent. S everal p h y s io lo g ic a l
param eters have been used as s e le c tio n c r i t e r i a f o r c o ld to le r a n c e .
However, d e f i n i t i v e a n a ly s is o f th e M endelian fa c to r s c o n t r o llin g these
param eters has n o t y e t been p e rfo rm e d . The o b je c tiv e s o f t h i s re search
were to g e n e t ic a lly in v e s tig a te th e r e la t io n s h ip s among c h a ra c te rs
a s s o c ia te d w ith w in t e r s u r v iv a l and to i d e n t i f y th e chromosomal
lo c a tio n s o f genes c o n t r o llin g those t r a i t s .
One hundred doubled h a p lo id lin e s d e riv e d from Fi p la n ts from a
cro s s between D ic k to o ( w in te r ) x Morex ( s p r in g ) were used t o stu d y th e
g e n e tic s o f f i e l d s u r v iv a l, LT50, fr u c ta n c o n te n t, flo w e r in g d a te and
p r o s t r a t e grow th h a b it . A medium d e n s ity lin k a g e map was c o n s tru c te d
from th e se doubled h a p lo id lin e s u s in g DMA, p r o te in isoenzym e, and
m o rp h o lo g ic a l m a rke rs. T h is map was th e n u t i l i z e d to i d e n t i f y th e
lo c a tio n o f q u a n t it a t iv e t r a i t lo c i (QTL) a s s o c ia te d w ith th e se t r a i t s .
S e q u e n c e -ta g g e d -s ite s (STS) were developed f o r polym erase c h a in r e a c tio n
as DNA (PCR) m arke rs.
S ig n if ic a n t c o r r e la t io n s between crown fr u c ta n c o n te n t and w in te r
h a rd in e s s param eters were d e te c te d . These r e s u lt s suggested t h a t
f r u c ta n c o n te n t may be one f a c t o r c o n t r ib u t in g to c o ld to le r a n c e .
However, s e le c tio n based on h ig h fr u c ta n c o n te n t d id n o t p o s i t i v e l y
im prove o th e r w in te rh a rd in e s s a s s o c ia te d t r a i t s .
The STS-PCR system f a c i l i t a t e d map c o n s tr u c tio n in t h i s c ro s s . A
s e t o f 77 DNA m arkers were re s o lv e d in t o e ig h t lin k a g e groups and
d is t r ib u t e d t o seven chromosomes.
Large QTL e f f e c t s f o r fr u c ta n c o n te n t
and a l l w in t e r h a rd in e s s param eters e xce p t p r o s tr a te g ro w th h a b it were
d e te c te d in a re g io n o f a p p ro x im a te ly 20 centiM organ s on th e lo n g arm o f
chromosome 7. C a re fu l a n a ly s is o f lin e s c a r r y in g re c o m b in a tio n
b re a k p o in t w it h in t h i s 20cM in t e r v a l suggested t h a t a t le a s t 2 genes
a f f e c t in g measured param eters were lo c a te d w it h in t h i s in t e r v a l .
T h is
in t e r v a l a n a ly s is suggested t h a t th e a s s o c ia tio n among a t le a s t some o f
th e se t r a i t s is due to lin k a g e r a th e r th a n p le io t r o p y .
S m a lle r QTL
e f f e c t s m o d ify in g c h a ra c te rs measured in t h i s stu d y were d e te c te d on
chromosome 2 and chromosome 5.
e
I
CHAPTER I
INTRODUCTION
I t s lim it e d a b i l i t y to s u rv iv e c o ld s tre s s l i m i t s th e p ro d u c tio n o f
w in te r
b a r le y ,
a cro p
w ith
tremendous
p o t e n t ia l
fo r
p r e v a ilin g w in t e r wheat m o nocu lture o f N o rth A m erica.
have been made to
und erstan d th e g e n e tic
w in t e r h a rd in e s s in c e r e a ls .
c o ld
to le r a n c e
id e n t i f i e d .
is
one o f
However,
d iv e r s if y in g
th e
S e vera l a tte m p ts
and p h y s io lo g ic a l
c o n tr o l
of
The a s s o c ia tio n o f fr u c ta n a ccu m u la tio n w ith
th e
p h y s io lo g ic a l
a c c u ra te e s tim a te s
param eters
th a t
o f th e g e n e tic
has
been
dependence o f
w in t e r h a rd in e s s on fr u c ta n c o n te n t have n o t been r e p o rte d .
The complex g e n e tic b a s is o f c o ld to le ra n c e has t r a d i t i o n a l l y been
s tu d ie d th ro u g h te c h n iq u e s o f q u a n t it a t iv e a n a ly s is .
The developm ent o f
m o le c u la r m arker te c h n o lo g ie s p e rm itte d th e r a p id c o n s tr u c tio n o f g e n e tic
maps and e v a lu a tio n
o f th e g e n e tic c o n tr o l
o f q u a n t it a t iv e
tr a its .
In
b a r le y , isozym es, seed s to ra g e p r o te in s , and DNA m arkers have been used in
lin k a g e a n a ly s is e x p e rim e n ts .
The r e s u lt in g lin k a g e maps have p e rm itte d
th e d e s c r ip tio n o f th e g e n e tic c o n tr o l o f q u a n t it a t iv e c h a ra c te rs .
T r a d i t i o n a l ly , DNA m arkers were id e n t if ie d u sin g S outhern b l o t t in g
t o d e te c t r e s t r i c t i o n
fra g m e n t le n g th
polym orphism s
(R FLP s).
A r a p id ,
n o n - r a d io a c tiv e , and p r a c t ic a l te c h n iq u e , polym erase c h a in r e a c tio n (PCR)
has been
Used to
te c h n iq u e
r e q u ire s
a m p lif ic a t io n
d e te c t
s h o rt
RFLPs in
many genome mapping
sequence p rim e rs
o f t a r g e t DNA fra g m e n ts .
p r o je c ts .
w hich prom ote th e
The
s e le c tiv e
The S e quen ce-T agged -site
(STS)
approach helped span th e gap between RFLP-based mapping p r o je c ts and PCR
te c h n o lo g y .
2
Doubled h a p lo id te c h n iq u e s can p ro v id e im m ortal g e n e tic re fe re n c e
p o p u la tio n s
fr e e
o f n o n -a d d itiv e
s e g re g a tin g g e n e ra tio n
ana lyses
ty p e s
in
o f gene a c tio n
t h a t c o m p lic a te
autogamous s p e c ie s .
They are a ls o
id e a l ly s u ite d f o r lin k a g e map c o n s tr u c tio n and QTL m apping.
A doubled
h a p lo id p o p u la tio n from a s p rin g x w in t e r b a rle y was used in t h is s tu d y .
Our o b je c tiv e s
were
i)
to
g e n e t ic a lly
s tu d y
fr u c ta n a c c u m u la tio n and w in te r h a rd in e s s ; i i )
te c h n o lo g y ; i i i )
th e
r e la t io n s h ip
between
to deve lo p th e STS-PCR map
t o g e n e ra te th e g e n e tic map o f th e c ro s s and lo c a te th e
QTL c o n t r o ll in g w in te r h a rd in e s s and i t s
a s s o c ia te d c h a ra c te rs .
3
CHAPTER 2
GENETICS OF FRUCTAN CONTENTS AND WINTERHARDINESS IN
A WINTER X SPRING BARLEY
In tr o d u c tio n
S everal s tu d ie s on th e r e la t io n s h ip between fr u c ta n a ccu m u la tio n and
w in t e r h a rd in e s s have been re p o rte d in many p la n t sp e cie s in c lu d in g b a rle y
( P o n tis and d e l CampiH o , 1985; C h a tte rto n e t a l , 1988; C h a tte rto n e t a l ,
1989;
P o n tis ,
C h a tte rto n
et
1989;
al
Suzuki
(1989)
and
Nass,
dem onstrated
1987;
th a t
L iv in g s to n
te m p e ra tu re
et
a l,
had
1989).
a profound
e f f e c t upon th e a c c u m u la tio n o f t o t a l n o n s tr u c tu ra l c a rb o h y d ra te s in many
g ra ss s p e c ie s .
Exposure o f many c e re a ls and grasses to low te m p e ra tu re s
induced
a c c u m u la tio n
la rg e
C a m p illo ,
1985).
of
In b a r le y ,
fr u c ta n
L iv in g s to n
and
sucrose
e t al
( P o n tis
and
Del
(1989) dem onstrated t h a t
fr u c ta n c o n te n t was in c re a s e d in c o ld a c c lim a te d p la n ts and t h a t s to re d
fr u c ta n s
were h y d ro ly z e d when th e
te m p e ra tu re s .
more
Long c h a in fru c ta n s
c lo s e ly
( L iv in g s to n
a s s o c ia te d
1991,
w ith
Suzuki
p la n ts
r e in itia te d
g row th
a t normal
(h ig h DP fr u c ta n s ) were found t o
c o ld
and Nass,
h a rd in e s s
198 7).
than
A lth o u g h
lo w
th e
DP
be
fru c ta n s
presence
of
fr u c ta n s in p la n t appeared to be advantageous f o r w in t e r h a rd in e s s
( P o in t is and Del C a m p illo , 1985), no d i r e c t evidence o f c r y o p r o te c tio n by
fr u c ta n s has been p re se n te d ( P o llo c k , 1986).
The g e n e tic and p h y s io lo g ic a l bases o f w in te rh a rd in e s s are p o o rly
u n d e rsto o d .
e s tim a te
( O lie n ,
a t w hich
P h y s io lo g ic a l measurements w hich have been commonly used to
c o ld
h a rd in e s s
in
c e re a ls
in c lu d e
lo n g -te rm
fie ld
s u r v iv a l
1978) and c o n t r o lle d fre e z e t e s t s t o d e te rm in e LT50 (te m p e ra tu re
50% o f th e
p la n t
p o p u la tio n
is
k ille d )
(Pomeroy and F ow ler,
4
1973).
W hile
e lu c id a te d ,
th e
g e n e tic
c o n tr o l
of
w in te rh a rd in e s s
w in t e r and s p rin g grow th h a b it
in
genes
s tr o n g ly
a ffe c te d
v e r n a liz a t io n
not
yet
been
b a r le y were found to
c o n t r o lle d by th e a c tio n o f a lt e r n a t iv e a lle le s
These
has
be
a t th re e u n lin k e d l o c i .
re q u ire m e n t,
respon se, and m a tu r ity (Takashashi and Yasuda, 1969).
p h o to p e rio d
E p is ta s is a t these
th re e lo c i made i t d i f f i c u l t to s tu d y gene a c tio n d i r e c t l y .
Takahashi and
Yasuda (1969) found t h a t th e g e n e tic c o n tr o l o f grow th h a b it co u ld more
e a s ily be s tu d ie d i f p la n ts were grown in c o n t r o lle d e n viro nm ents under 24
hour l i g h t i n g .
D o ll e t a l . (1989) re p o rte d t h a t v e r n a liz a t io n re q u ire m e n t
is a s s o c ia te d w ith c o ld to le r a n c e .
In t h i s e xp e rim e n t we s tu d ie d th e g e n e tic c o n tr o l o f crown fr u c ta n
a c c u m u la tio n in a s e t o f doubled h a p lo id lin e s w hich d e riv e d from a sim p le
cro s s between a c o ld - s u s c e p tib le s p rin g b a r le y and a hardy w in t e r b a r le y .
Our
o b je c tiv e s
in
t h is
s tu d y
were
i)
to
p ro v id e
th e
f ir s t
g e n e tic
in v e s t ig a t io n o f fr u c ta n a c c u m u la tio n , w in t e r h a rd in e s s and r e la te d t r a i t s
in a p o p u la tio n o f lin e s d e riv e d from a w in t e r x s p rin g b a r le y cross i i )
t o e v a lu a te th e r e la t io n s h ip s between p h y s io lo g ic a l c h a ra c te rs a s s o c ia te d
w ith w in t e r s u r v iv a l in t h i s p o p u la tio n o f T ines and i i i )
t o d e te rm in e how
much o f th e a p p a re n t a s s o c ia tio n between c h a ra c te rs was due to p le io tr o p y .
M a te ria ls and Methods
P la n t M a te ria l
One hundred doubled
h a p lo id
(DH)
lin e s
were developed u sin g th e
Hordeum bulbosum te c h n iq u e , as d e s c rib e d by Chen and Hayes (1 9 8 9 ), from F1
p la n ts o f th e c ro s s o f D ic k to o x Morex.
D ic k to o (C l 5529) is a s ix -ro w e d
5
w in t e r fe e d b a r le y o f p o o rly c h a ra c te riz e d a n c e s try w hich was re le a s e d by
th e Nebraska A g r ic u lt u r a l Experim ent S ta tio n in 1952 .
Morex (CI15773) is
a s ix -ro w e d s p rin g m a ltin g b a r le y re le a s e d by th e M innesota A g r ic u lt u r a l
E xperim ent S ta tio n in 1978 (Rasmussen, 1978).
T r a i t Assessment
We used
C o r v a llis ,
th re e
measures
Oregon, f i e l d
of
c o ld
s u r v iv a l
to le r a n c e :
fie ld
a t Bozeman1 Montana,
s u r v iv a l
and LT50.
at
F ie ld
s u r v iv a l a t C o r v a llis was measured by v is u a ll y a ssessing p e rc e n t s u r v iv a l
f o llo w in g th e w in te r o f 1990-1991.
Each DH li n e was re p re s e n te d by an
u n r e p lic a te d p lo t c o n s is tin g o f two 1 .5 m row s.
F ie ld s u r v iv a l a t Bozeman
was measured as th e d iff e r e n c e between i n i t i a l
(O c to b e r, 1991) and p o s t­
c o ld
s tre s s
(M arch,
1992)
p la n t
stands
in
a random ized com plete b lo c k
e xp e rim e n t c o n ta in in g th re e r e p lic a t io n s . Mean s u r v iv a l (MeanSUR) was th e
mean o f th e f i e l d
s u r v iv a l e s tim a te s a t both lo c a tio n s . The LT50 o f each
DH l i n e was de te rm in e d u s in g p la n t m a te ria l hardened a t 2°C f o r 5 weeks
w ith a 10 h li g h t / 1 4 h d a rk p h o to p e rio d re g im e .
Four te m p e ra tu re s (0 , -4 ,
- 8 , -12°C) w ith te n p la n ts a t each te m p e ra tu re were used t o d e te rm in e th e
LT50 o f each DH l i n e .
1990 t o F e b rua ry 1991.
A to ta l
o f th re e r e p lic a t e s was ru n from O ctober
P la n t m a te ria l was prepared f o r f r e e z in g , and LT50
v a lu e s were computed, as d e s c rib e d by K o la r e t a l . (1 9 9 1 ).
Heading d a te was e s tim a te d u s in g th e heading d a te o f u n v e rn a liz e d
p la n t m a te ria l
under greenhouse c o n d itio n s
of
18°C d a y /n ig h t
and 24 h
lig h t.
The c o n te n t o f fr u c ta n s w ith a degree o f p o ly m e riz a tio n (DP) > 5 was
d e te rm in e d u s in g crowns from f i e l d
hardened p la n ts .
F iv e p la n ts o f each
genotype were dug from f i e l d p lo ts a t C o r v a llis , OR in Ja n u a ry , 1992.
In
6
1991,
o n ly
th e
measurement
at
f ifte e n
le a s t
C o r v a llis ,
and most
OR in
1991 were
a n a ly s is .
P la n ts were washed, trim m ed,
fro z e n
liq u id
in
n itr o g e n .
t is s u e was ground in l i q u i d
fr u c ta n e x t r a c t io n .
hardy
based on
sampled
fie ld
fo r
s u r v iv a l
crown
fr u c ta n
and th e crowns were im m e d ia te ly
T issu e was ly o p h iI i zed f o r
15 days.
Crown
n itro g e n and 50 mg per sample were used f o r
F iv e ml o f d i s t i l l e d w a te r Were added to each 50 mg
sam ple; samples were shaken and then in c u b a te d f o r 15 min in a 90°C w a te r
b a th .
The s u p e rn a ta n t was poured th ro u g h an A m b e rlite MB-3A io n exchange
r e s in column and th e e lu a te volume a d ju s te d t o 5 ml w ith w a t e r . ^ T h is ste p
was re p e a te d tw ic e .
The column was th e n washed w ith 5 ml o f 90°C w a te r,
and th e w a te r e x tr a c ts were d r ie d a t 60°C f o r 6 to 8 h.
D rie d samples were
d is s o lv e d in I ml o f GO0C w a te r and c e n tr ifu g e d a t 12,000 rpm f o r 10 m in.
The
s u p e rn a ta n t
in je c t io n
was
f ilt e r e d
th ro u g h
a 0.4 5
m icron
filte r
p r io r
to
in t o th e HPLC, w hich was equipped w ith a BioRad carbo-C guard
column and a BioRad HPX-42 C c a rb o h y d ra te column.
F ru cta n c o n te n t (> DP
5) was expressed as mg g "1 on a d ry w e ig h t b a s is (m g/gdw).
RFLP and C o r r e la tio n A n a ly s is
S eventy-seven m arkers were used in
th is
e x p e rim e n t.
Genomic and
cDNA c lo n e s were s u p p lie d by C o rn e ll U n iv e r s it y , th e N o rth Am erican B a rle y
Genome P r o je c t o r were among those d e s c rib e d in Shin e t a l . (1 9 9 1 ).
m arkers
were
C hapter 3 in
developed
t h is
as d e s c rib e d
t h e s is ) ^
B r ie fly ,
by Tragoonrung
fo r
et
RFLP a n a ly s is ,
al
( in
PCR
p re s s ,
p la n t DNA was
prepa red from fre s h t is s u e u sin g th e te c h n iq u e o f D e lla p o r ta e t a l .(1 9 8 4 ).
DNA samples (10 ^ g ) were d ig e s te d w ith a p p ro p ria te r e s t r i c t i o n endonuclea­
ses, se p a ra te d on 0.8% agarose g e ls in T r is - B o r a te b u f f e r and tr a n s fe r r e d
to charged n y lo n membranes u s in g th e a lk a lin e t r a n s f e r p ro to c o l o f Reed
7
and Mann (1 9 8 5 ).
Cloned DNA in s e r t s
agarose and la b e le d
F ilt e r s
were c u t
by random p rim in g
were p r e h y b rid iz e d ,
from
low m e ltin g
( F einbe rg and V o g e ls te in
h y b rid iz e d
o v e r n ig h t,
washed w ith
s trin g e n c y o f 0.1 XSSC, 0.1% SDS, 65 C, and exposed t o
Isozymes were assayed as d e s c rib e d
p o in t
by N ie ls e n
film
a fin a l
as needed.
and Johansen
s to ra g e p r o te in s were assayed as d e s c rib e d by Blake e t a l
1984).
(1986)
and
(1 9 8 2 ).
The
m o rp h o lo g ic a l m arkers were scored u s in g a ste re o m icro sco p e o r w ith a hand
le n s .
R e s u lts and D is c u s s io n
F ru cta n chromatograms o f D ic k to o and Morex c u l t i v a r s h a rve ste d under
both h a rd e n in g and n o n -h a rd e n in g c o n d itio n s are shown in F ig u re I .
non-hardened crown t is s u e was used, f r u c ta n was n o t d e te c te d
D ic k to o o r M orex.
A f t e r h a rde ning in th e f i e l d
c o n ta in e d
mg
fru c ta n /g d w ,
In
1991,
137
fru c ta n /g d w .
t r a n s fe r r e d
to
a grow th
w h ile
b a rle y
Morex
p la n ts
chamber
and
hardened
in e it h e r
in 1992, D ic k to o crowns
crowns
grown
When
c o n ta in e d
i n . th e
at
66.2
greenhouse
2°C f o r
fiv e
mg
were
weeks.
F ru cta n c o n te n ts o f D ic k to o and Morex were 90 .9 and 15.7 mg fru c ta n /g d w ,
r e s p e c t iv e ly .
When compared to
fr u c ta n
s ta n d a rd s ,
th e
m a jo r
fr u c ta n
sp e cie s w hich d if f e r e d in c o n c e n tra tio n between D ic k to o and Morex were DP5
o r g r e a te r .
Lower DP (DP3-DP5) fr u c ta n s were d e te c te d
were a c c lim a te d
(1992)
c o n ta in e d
p la n t s .
in
th e
le s s
grow th
chamber
DP3-DP5 fr u c ta n
(1 9 9 1 ).
th a n
F ie ld
grow th
in p la n ts which
hardened p la n ts
chamber a c c lim a te d
S im ila r r e s u lte d were re p o rte d by C h a tte rto n e t a l . (1989) and
P o llo c k (1 9 8 6 ).
The range and mean o f crown fr u c ta n c o n te n t,
and heading d a te are shown in Table I .
LT50, f i e l d
s u r v iv a l,
S p e c ific s on th e d i s t r i b u t i o n o f
8
th e se
tr a its
s u b m itte d ).
were
re p o rte d
in
our
companion
paper
(Hayes
et
a l,
F ru cta n c o n te n t o f te s te d doubled h a p lo id lin e s in 1991 was
le s s th a n t h a t o f 1992 (T a b le I ) . T h is may be due to d iffe r e n c e s in grow th
stages o f th e p la n ts
a t h a rv e s t,
o r to e n viro n m e n ta l
v a r ia t io n .
P la n t
tis s u e s c o lle c t e d from th e f i e l d were la r g e r and had been dorm ant lo n g e r
th a n th e p la n ts grown in th e greenhouse and a c c lim a te d in grow th chambers.
L e v itt
(1980)
re p o rte d
th a t
th e
degree o f w in te r
h a rd in e s s
o f p la n ts
depended upon developm ental stage and e n viro n m e n ta l c o n d itio n s .
fie ld
s u r v iv a l a t C o r v a llis , OR (ORSUR) was h ig h e r than t h a t a t Bozeman,
MT (MTSUR).
H a lf o f th e doubled h a p lo id lin e s
s u ffe re d com plete w i n t e r k i l l .
C o r v a llis
D ic k to o
and
and 62% s u r v iv a l
Morex were
screened a t Bozeman, MT
F ie ld s u r v iv a l o f Morex were 10%, and 0% a t
C o r v a llis , OR and Bozeman, MT, r e s p e c t iv e ly .
at
Average
at
- 5 . 5°C and
Bozeman.
62
days,
D ic k to o showed 82% s u r v iv a l
LT50 and heading da te
and
-3.9°C
and
fo r
45 days,
r e s p e c t iv e ly .
T ab le I . Mean, minimum,
w in t e r h a rd in e s s .
T r a it s
Fructan91
Fructan92
LT50
Heading d a te
ORSUR
MTSUR
and maximum va lu e s o f p h y s io lo g ic a l
Min
0.00
58.60
-1 1 .0 0
27.00
1.00
0.00
,
Mean
Max
36.00
97.80
-6 .0 7
47.18
53.73
25.08
103.00
237.40
-2 .7 0
97.00
tr a its
fo r
100.00
8 5.00
Fructan91 = fr u c ta n c o n te n ts measured in 1991; F ructan92 = fru c ta n
c o n te n ts measured in 1992; ORSUR = f i e l d s u r v iv a l a t C o r v a llis , OR in
1991; MTSUR = f i e l d s u r v iv a l a t Bozeman, MT in 1992.
9
o
a
■d- 'T
i
'M
iJ j
'M
T
-d"
r-
>M
0:1
fv.
B
A
C4J
C
D
F ig u re I .
HPLC chromatograms o f fr u c ta n c o n te n ts f o r D ic k to o and Morex
c u l t i v a r s . A) F ru cta n chromatogram o f non-hardened D ic k to o t is s u e . B)
F ru cta n chromatogram o f non-hardened Morex t is s u e . C) F ru cta n chromatogram
o f hardened D ic k to o . D) F ru cta n chromatogram o f hardened Morex t is s u e .
10
C o r r e la tio n s
param eters
among
a s s o c ia te d
crown
w ith
fr u c ta n
w in te r
c o n te n t
h a rd in e s s
are
and
p h y s io lo g ic a l
shown
in
Table
2.
S ig n if ic a n t c o r r e la t io n s were observed between crown f r u c ta n c o n te n t and
each o f th e o th e r measured param eters in th e 1992 e x p e rim e n t.
In 1991,
o n ly th e f i f t e e n most and le a s t hardy lin e s were sampled f o r crown fr u c ta n
c o n te n t.
As would be e xpe cted , th e reduced sample s iz e r e la t iv e to th e
1992 e xp e rim e n t r e s u lte d in reduced e x p e rim e n ta l s e n s i t i v i t y .
T ab le 2.
C o r r e la tio n c o e f f ic ie n t s
1992 and p h y s io lo g ic a l p a ram eters.
T r a it s
LT50
Heading d a te
ORSUR
MTSUR
MeanSUR
between fr u c ta n c o n te n t in
Fructan91
Fructan92
0.29"=
0.24"=
0.63**
0.57**
0.60**
0 .27*
0.47**
0.47**
0.41**
0.50**
1991 and
* * S ig n if ic a n t le v e l a t 1%
* S ig n if ic a n t le v e l a t 5%
ns N o n - s ig n ific a n t .
ORSUR = f i e l d s u r v iv a l a t C o r v a llis , OR in 1991; MTSUR = f i e l d s u r v iv a l a t
Bozeman, MT in 1992; MeanSUR = mean f i e l d s u r v iv a l from both lo c a tio n s .
F ie ld s u r v iv a l is among th e most e r ro r- p r o n e measurements commonly
made
by
p la n t
d i s t r ib u t i o n s
C o r v a llis ,
or
c o r r e la t io n
s c ie n t i s t s .
We compared
had s e le c tio n
u s in g
th e ir
o f ta ils
o v e r a ll
th e
s im ila r it y
mean va lu e
v a lu e between crown fr u c ta n
(T a b le
p e rfo rm
a t a ile d - d is t r ib u t io n
1989) one must be c a r e fu l
of
3 ).
The sim p le
c o n te n t and f i e l d
s u r v iv a l was
between f r u c ta n c o n te n t in 1991 and 1992 was 0 .4 6 .
to
t a ils
been perform ed a t e it h e r Bozeman,
h ig h e r u s in g d a ta from C o r v a llis than t h a t a t Bozeman.
g o in g
of
a n a ly s is
The c o r r e la t io n
O b v io u s ly , i f one is
(Lander and B o ts t e in,
in o b ta in in g th e d i s t r i b u t i o n
upon w hich th e
11
t a ils
are s e le c te d .
The c o r r e la t io n between fr u c ta n c o n te n t and LT50Was much lo w e r than
t h a t re p o rte d
by L iv in g s to n
at
a l.
(1 9 8 9 ).
T h is may r e s u lt
from th e
d iff e r e n c e
in sample s iz e o r reduced dependence o f LT50 on crown fr u c ta n
c o n te n t in
progeny from t h i s
c ro s s .
S ig n if ic a n t p o s it iv e
c o r r e la t io n s
were found between LT50 and heading d a te , LT50 and MEANSUR, and heading d a te
and MEANSUR (0 .4 3 , 0 .4 5 , 0 .5 , r e s p e c t iv e ly ) .
T ab le 3.
Percentage o f lin e s in common among p h y s io lo g ic a l t r a i t s
and 25% s e le c tio n i n t e n s it ie s .
T r a it s
LT50
Heading
da te
F ructan92
15% D ic k to o ty p e
Oregon s u r v iv a l
Bozeman s u r v iv a l
Heading da te
LT50
40
33
56
100
33
33
100
56
47
40
60
36
15% Morex Type
Oregon s u r v iv a l
Bozeman s u r v iv a l
Heading d a te
LT50
20
ND
20
100
33
ND
100
20
13
ND
'33
26
25% D ic k to o ty p e '
Oregon s u r v iv a l
Bozeman s u r v iv a l
Heading d a te
LT50
44
52
56
100
60
52
100
56
40
48
48
36
25% Morex ty p e
Oregon s u r v iv a l
Bozeman s u r v iv a l
Heading d a te
LT50
40
ND
48
100
32
ND
100
48
20
ND
36
28
.
ND= Data n o t d e te rm in e d .
Fructan92 = fr u c ta n c o n te n ts measured in 1992.
a t 15
12
To d e te rm in e how e f f e c t i v e l y s e le c tio n f o r any o f th e se c h a ra c te rs
would r e s u lt in c o r r e la te d s e le c tio n f o r a n o th e r o f th e se c h a ra c te rs , we
c a lc u la te d
th e
percen tag e
(Tragoonrung e t a
l 1990) .
of
common
lin e s
among
d is tr ib u tio n
t a ils
The percen tag e o f li n e in common f o r p a irs o f
c h a ra c te rs u s in g t a i l s c o m p ris in g e it h e r 15% and 25% o f each d i s t r ib u t i o n
are shown in T able 3.
I f s e le c tio n were perform ed s o le ly on th e b a s is o f
h ig h fr u c ta n c o n te n t,
a p p ro x im a te ly 50% o f th e lin e s s e le c te d would show
good s u r v iv a l and delayed heading d a te .
T h i r t y - s ix p e rc e n t o f lin e s w ith
h ig h fr u c ta n c o n te n t showed low LT50.
I t has been suggested t h a t fr u c ta n s may a c t as t is s u e c ry o p r o te c ta n t
by m o d ify in g ic e c r y s ta l fo rm a tio n (O lie n and L e s te r, 198 5).
been
suggested
indepe nden t
th a t
de la ye d
fu n c tio n s
in
s u c c e s s fu l o v e r w in te r in g .
tr a its * a n d
a n a ly s is
s p rin g
a t t a in in g
th e
th e
and
u ltim a te
low
LT50 va lu e s
phenotype
needed
have
fo r
To d e te rm in e th e a s s o c ia tio n o f w in te rh a rd in e s s
g e n e tic m a rke rs,
w ith
heading
I t has a ls o
a s e t o f m arkers was used f o r c o r r e la t io n
f o u r p h y s io lo g ic a l
c h a ra c te rs .
s ig n if ic a n t c o r r e la t io n s f o r a l l th e t r a i t s
Four m arkers
(T a b le 4 ) .
showed
These fo u r m arkers
map to th e lo n g arm o f b a r le y chromosome 7 and com prise a f a i r l y t i g h t l y
lin k e d
set o f lo c i.
S ix o th e r m arkers showed s ig n if ic a n t c o r r e la t io n s
w ith fr u c ta n c o n te n t and a co m b in a tio n o f s ig n if ic a n t c o r r e la t io n s w ith
th e
o th e r measured
c h a ra c te rs .
T h is
suggests
th a t
one gene o r gene
c lu s t e r on th e lo n g arm o f b a rle y chromosome 7 c o o r d in a te ly m o d ifie s crown
fr u c ta n c o n te n t,
r e s u lt
from
LT50, w in t e r s u r v iv a l,
e it h e r
lin k a g e
or
a n o th e r lo c u s in th e v i c i n i t y
and flo w e r in g d a te .
p le io t r o p y .
T h is co u ld
On chromosome 5 th e re
is
o f th e gene encoding D h o rd e in which has
s ig n if ic a n t im pact on both crown fr u c ta n c o n te n t and s u r v iv a l a t C o r v a llis
13
Oregon, b u t w hich showed l i t t l e
in t e r a c t io n w ith o th e r c h a ra c te rs .
On th e
s a t e l l i t e o f chromosome 7 (KSU32) th e re appears to be a gene which has an
e f f e c t on crown fr u c ta n c o n te n t and heading d a te .
These r e s u lt s
suggest t h a t
a gene
( o r perhaps
a t ig h t ly
lin k e d
c lu s t e r o f genes) w ith m a jo r im pact on developm ent re s id e s on th e lo n g arm
o f b a r le y chromosome 7.
D ic k to o ,
r e s u lt s
a s s o c ia te d
w ith
in
a
T h is gene o r gene c lu s t e r ,
b a rle y
good w in te r
li n e
w ith
s u r v iv a l.
many
of
when d e riv e d , from
th e
The D ic k to o
c h a r a c t e r is t ic s
a lle le
r e s u lt s
in
de la ye d he a d in g , h ig h crown fr u c ta n c o n te n t, low LT50 and r e l a t i v e l y good
f ie ld
s u r v iv a l
a t e it h e r Bozeman o r C o r v a llis .
Two o th e r l o c i ,
one on
chromosome 5 and th e o th e r on th e s a t e l l i t e o f th e s h o r t arm o f chromosome
7, have an im pact on crown fr u c ta n c o n te n t.
a ls o
im p lic a te d
The chromosome 5 lo c u s is
in w in te r s u r v iv a l w h ile th e lo c u s on th e s a t e l l i t e
of
chromosome 7 is im p lic a te d in delayed flo w e r in g .
Some o f th e d i f f i c u l t y
w ith
im proved
la r g e s t
c o ld
to le ra n c e
f a c t o r r e s u lt in g
in
e xp e rie n ce d in d e v e lo p in g b a r le y germplasm
can be e x p la in e d
by th e se
th e e x c e lle n t f i e l d
s u r v iv a l
r e s u lt s .
p o t e n tia l
The
of
D ic k to o appears to be in h e r it e d as though i t were a s in g le M endeli an gene
on th e lo n g arm o f chromosome 7.
U n fo r tu n a te ly , t h i s one M endelian gene
appears t o r e s u lt in delayed flo w e rin g in a d d itio n to im proved s u r v iv a l,
low ered LT50 and in c re a s e d crown fr u c ta n c o n te n t.
decades sought e a r l i e r flo w e r in g ,
both
e a s ie r
and more e f f e c t iv e
w in t e r s u r v iv a l,
to
P la n t b re e d e rs have f o r
hardy b a rle y gen o typ e s.
s e le c t
fo r
flo w e r in g
Since i t
d a te than
is
fo r
in w ide cro sses u s in g D ic k to o as a p a re n t th e s e le c te d
progeny would l i k e l y n o t c a r r y th e D ic k to o a l l e l e f o r w in t e r s u r v iv a l a t
t h i s lo c u s due t o i t s
a s s o c ia tio n w ith dela ye d heading.
14
Two o f th e th re e lo c i
a ls o
shown
to
a ffe c t
found t o m o d ify crown f r u c ta n
flo w e r in g
h y p o th e s iz e a p h y s io lo g ic a l
d a te .
It
rough
awns,
and th e
s t r a ig h t fo r w a r d
to
Once a v a ila b le ,
p h y s io lo g ic a l
appear re a s o n a b le .
deve lop
seem
reason able
r e la t io n s h ip between fr u c ta n d e p o s itio n
reduced tu rn o v e r ) and delayed in flo re s c e n c e .
7 m a rke r,
would
c o n te n t were
fla n k in g
is o g e n ic
(o r
Using th e sim p le chromosome
m arker
lin e s
to
BCD265b,
w hich v a ry
fo r
it
should
t h is
be
re g io n .
a n a ly s is o f d iffe r e n c e s among lin e s would
L in e developm ent is
in p ro g re s s , and is o g e n ic p a irs
can be accessed from D r. P a tr ic k M Hayes when th e y are a v a ila b le .
T ab le 4. C o r r e la tio n c o e f f ic e in t s o f RFLP m arkers t h a t showed s ig n if ic a n t in te r a c tio n s w ith
fr u c ta n c o n te n t (F ru c ta n 9 2 ).
M arkers
Loc1
AWN
pKSU24
BCD265b
C3a
RACH
HorD '
ap340a
pKSU32
CS
F4
7L
7L
7L
7L
7L
5L
5L
7S
?
?
Fructan92
•f
I!
0 .5 0 m
0.23
0.2 5
0 .2 6 ee
0 .4 4
0.3 9
0 .4 8
LT50
-
0.43
0 . 03ns
0 .0 5 ns
0 .0 1 ns
0 .3 2 ns
0 .2 3 ":
0.05"=
Heading da te
0.65*
0.23
0 .0 2 ":
0 .0 1 ":
0.59**
0 .0 7 ":
0 .0 5 ":
MTSUR
ORSUR
MeanSUR
0.77
0.28
0 .0 7 ":
0 .0 4 ":
0 .3 2 ":
0 .0 2 ":
0 .2 1 ":
0.57**
0.60**
0.44**
0.78**
0.29**
0.20*
0.19*
0 .3 7 ":
0 .0 3 ":
0 .2 3 ":
0.72**
0.65**
0.66**
0.80**
0.32**
0 .1 6 ":
0 .1 4 ":
0 .3 6 " :.
0 .0 1 ":
0 .2 2 ":
T 1OL*'''----------------
* * = S t a t i s t i c a l l y s ig n if ic a n t le v e l a t 99%
* = S t a t i s t i c a l l y s ig n if ic a n t le v e l a t 95%
ns = S t a t i s t i c a l l y non s ig n if ic a n t
MTSUR = f i e l d s u r v iv a l a t Bozeman, MT in 1992; ORSUR = f i e l d
MeanSUR = mean f i e l d s u r v iv a l o f both lo c a tio n s .
s u r v iv a l a t C o r v a llis , OR in 1991'
H
Ul
16
CHAPTER 3
STS FACILITATED PCR FOR BARLEY GENOME MAPPING.
In tr o d u c tio n
C u rre n t genomic lin k a g e
fra g m e n t
le n g th
maps are g e n e r a lly
polym orphism s
(RFLPs)
based on r e s t r i c t i o n
( B o ts te in
et
a l.,
1980).
C o n ve n tio n a l d e te c tio n o f RFLPs by S outhern b lo t a n a ly s is (S o u th e rn , 1975)
is la b o r io u s and c o s t ly (Beckmann and S e lle r , 1983; Beckmann, 1988).
polym erase ch a in r e a c tio n (PCR) ( S a ik i e t a l . ,
1987)
p ro v id e s
p o p u la tio n s .
between
a r a p id ,
sa fe
The
1985; M u llis and Faloona,
and e f f i c i e n t method f o r
scre e n in g la rg e
U n lik e Southern b lo t a n a ly s is , PCR can d i r e c t l y d is tin g u is h
in s e r t io n / d e le t io n , o r
p o in t
m u ta tio n
polym orphism s can be d e te c te d by h y b r id iz in g
e v e n ts .
P o in t m u ta tio n
PCR p ro d u c ts w ith
a lle le -
s p e c if ic o lig o n u c le o tid e s (H ig u ch i e t a l . , 1988; Li e t a l . , 1988; K u rth e t
o
a l . , 1991), DNA sequencing o f PCR p ro d u c ts (Wong e t a l . , 198 7), cleavage
o f PCR p ro d u c ts w ith a r e s t r i c t i o n endonuclease ( S a ik i e t a l . , 1985), and
d e n a tu rin g g r a d ie n t gel e le c tr o p h o r e s is
a l . , 1990).
(Myers e t a l . ,
I n s e r t io n / d e le t io n polym orphism s can be an a lyze d by s iz in g o f
PCR p ro d u c ts v ia g e l e le c tr o p h o r e s is (H ig u c h i e t a ! . ,
1990).
A m a jo r l i m i t a t i o n
sequence
in fo r m a tio n
(W illia m s e t a l . ,
n u c le o tid e
1987; R ie d e l, e t
in
1991).
f o r PCR a n a ly s is
o rd e r
to
is
s y n th e s iz e
1988; Shin e t a l . ,
th e need f o r e x te n s iv e
th e
a p p ro p ria te
p rim e rs
W illia m s e t a l .(1 9 9 0 ) proposed u s in g a r b it r a r y
sequences as s in g le
p rim e rs f o r a m p lif ic a t io n
o f random DNA
fra g m e n ts (RAPD).
In
g e n e tic
th e
human genome mapping p r o je c t ,
map t o
a p h y s ic a l
map has
been
an a tte m p t
g r e a t ly
to
c o n v e rt
s im p lif ie d
th e
by u sin g
17
sequence-tagged
s it e s
(S TS ).
An
STS
is
a
s h o r t,
unique
sequence,
a m p lifie d by PCR, w hich id e n t i f i e s a known lo c a tio n on a chromosome (O lson
e t a l . , 1989).
To d a te , a l l STSs t h a t have been used in mapping p r o je c ts
have been d e riv e d from w e ll- c h a r a c te r iz e d DNA probes o r sequences (C ole e t
a l . , 199 1).
th a t
DzO v id io e t a l.( 1 9 9 0 ) and W eining and La n g rid g e (1991) showed
PCR can be used to
d e te c t g e n e tic
polym orphism s
in
c e re a ls
w ith
p rim e r sequences d e riv e d from th e sequence o f a - y - g lia d in gene and a <*amylase gene, r e s p e c t iv e ly .
Here, we propose two approaches to o b ta in STSs f o r b a rle y genome
m apping.
One approach
c o n s tr u c t p rim e rs .
is
to
u t iliz e
sequence d a ta
fro m
Genebank to
A n o th e r is to sequence anonymous DNA clo n e s used in
genome mapping p r o je c ts .
P o in t m u ta tio n polym orphism s w it h in
a m p lifie d
fra g m e n ts were d e te c te d on p o ly a c ry la m id e g e ls o f d ig e s te d PCR p ro d u c ts
w ith fo u r-b a s e c u t t e r s .
I n s e r t io n / d e le t io n even ts were observed d i r e c t l y
by g e l e le c tr o p h o r e s is .
M a te ria l and Methods
G e n e tic S tocks
Two doubled
h a p lo id
p o p u la tio n s
were
gen erate d
from
b a rle y
cv.
D ic k to o x Morex (DxM), a w in te r x s p rin g c ro s s , and fro m S teptoe x cv.
Morex (SxM ), a s p rin g x s p rin g cro ss by th e N orth Am erican B a rle y Genome
Mapping P r o je c t
(NABGMP).
For th e DxM c ro s s , 30 dou ble d h a p lo id lin e s
were used f o r s e g re g a tio n a n a ly s is .
One hundred and f i f t y doubled h a p lo id
p ro g e n ie s were used f o r s e g re g a tio n a n a ly s is in th e SxM c ro s s .
w h e a t-b a rle y
a d d itio n
(c v .
lin e s
Chinese
d e s c rib e d
S p rin g -c v .
by
Isla m
et
chromosomal assignm ent o f polym orphism s.
Betzes
a l.
c u ltiv a r s )
(1981)
was
A set o f
chromosomal
u t iliz e d
fo r
18
DNA I s o la t io n
Genomic DNA was prepared
(1 9 8 7 ), m o d ifie d as f o llo w s :
u s in g
th e
procedure
o f Ausubel
et
a l.
10 g fre s h w e ig h t o f young l e a f tis s u e was
ground to a f in e powder under l i q u i d n itr o g e n and in c u b a te d a t 55 C in 20
ml o f e x tr a c tio n b u f f e r (100 mM T ris -H C l pH 8 .5 , 100 mM EDTA, 250 mM NaCl,
0.5% SDS, and 100 ug/m l p ro te in a s e K) f o r 2 h rs . T h e ,ly s a te was e x tra c te d
w ith
20 ml
isoam yl
p h e n o l: c h lo ro fo rm
a lc o h o l) ,
p r e c ip it a t io n .
fo llo w e d
s o lu tio n
by
(50% p h e n o l, .49% c h lo ro fo rm ,
c h lo ro fo rm
e x t r a c t io n ,
and
1%
eth a n o l
DNA was c e n tr ifu g e d a t 5000 rpm f o r 15 m in . , suspended in
2 ml o f s t e r i l e w a te r, and tr e a te d w ith 20 ug DNase fr e e RNase f o r 2 h r .
DNA was e x tra c te d w ith p h e n o l: c h lo ro fo rm , and c h lo ro fo rm , a d ju s te d to a
c o n c e n tra tio n
o f 0 .3 M sodium a c e ta te ,
and e th a n o l
p r e c ip it a t e d .
The
p u r if ie d DNA was d is s o lv e d in s t e r i l e w a te r and s to re d a t -20 C u n t i l use.
DNA Sequencing
Four DNA c lo n e s ,
random ly
s e le c te d
fo r
P s tl-3 1 9 ,
sequencing
P s tl- 3 2 7 ,
from
P s tl-3 3 7 ,
a P s tI
and P s tl-3 4 0 , were
b a rle y
p ro v id e d by D r. Nora L a p ita n (C olorado S ta te U n iv . ) .
genomic l i b r a r y ,
S in g le -s tra n d e d DNA
(ssDNA) te m p la te s were g e n erate d u s in g PCR as d e s c rib e d by H iguch i and
Ochman
(1 9 8 9 ).
PCR
p rim e rs ,
5'
TACGACTCACTATAGGGC
3'
and
5'
CGCCAAGCTATTTAGGTG 3 ' , were used to a m p lify th e in s e rte d DNA fragm ents in
th e pGEM v e c to r . One o f th e p rim e rs was p h o s p h o ry la te d p r i o r to PCR w ith
T4 DNA k in a s e . A f t e r PCR a m p lif ic a t io n ,
was
tr e a te d
s p e c ific a lly
w ith
lambda
d ig e s ts
o n ly
exonucle ase,
th e
DNA
th e d o u b le -s tra n d e d DNA (dsDNA)
a
5'
s tra n d
to
3'
nuclease
s y n th e s iz e d
pho sphoryl ate d p rim e r le a v in g th e com plem entary s tra n d (ssDNA).
from
which
th e
The ssDNA
was p u r if ie d u s in g Prep-A-Gene (BIO-RAD), and sequenced by th e te c h n iq u e
19
of
Sanger
p r o to c o l
et
a l.,
1977
u s in g
Sequenase
f o llo w in g
th e
m a n u fa c tu re r's
(USB).
P rim e r S y n th e s is
O lig o n u c le o tid e p rim e rs were s y n th e s iz e d by sta n d a rd p h o sp h o ra m id ite
c h e m is try on a A p p lie d Biosystem s 391 DNA s y n th e s iz e r .
from th e s o lid
h y d ro x id e
at
A f t e r h y d r o ly s is
s u p p o rt and removal o f p r o te c tin g groups in 30% ammonium
55 C f o r
15 h r . ,
th e
d is s o lv e d in 200 u l o f s t e r i l e w a te r.
DMAs were d r ie d
under vacuum,
and
DNA y ie ld was a p p ro x im a te ly 800 ug
p e r s y n th e s is .
PCR A m p lif ic a t io n
PCR a m p lific a t io n s were perform ed in a 100 u l r e a c tio n c o n ta in in g
2 .5 u n its Taq polym erase (P e rk in Elmer C e tu s ), l x PCR b u f f e r (50 mM K C l,
10 mM T ris -H C l
pH 8 .3 ,
and 1.5 mM MgCl2) ,
200 uM o f each dNTP, 0 .3 uM
p rim e rs , and 25 ng o f genomic DNA te m p la te s .
w ith m in e ra l o i l
The r e a c tio n was o v e r la id
and s u b je c te d to one c y c le o f 95 C f o r 5 m in . , fo llo w e d
by 32 c y c le s o f 94 C f o r I m in ., 55 C f o r I m in ., and 72 C f o r 90 sec.
D e te c tio n o f Polymorphisms
PCR p ro d u c ts
were
d e le t io n polym orphism s.
run
on
1% agarose
g e ls
to
d e te c t
in s e r t io n /
To d e te c t base s u b s t it u t io n polym orphism s, 20 ul
o f a m p lifie d frag m en ts were d ig e s te d w ith f o u r base c u t t e r s : A l u l, H a e III,
H h a I,
H in fI,
endonucleases,
r e a c tio n
M spI,
exce p t
R s a I,
and
R saI,
T a q I.
Two
were d i r e c t l y
a f t e r a m p lif ic a t io n .
u n its
added to
of
th e
20 u l
r e s tr ic t io n
of
th e
PCR
For R s a I, 20 u l o f th e PCR p ro d u c t were
p r e c ip it a t e d w ith th re e volumes o f 95% e th a n o l and th e p e l l e t was washed
w ith 70% e th a n o l p r i o r to d ig e s t .
D ig e ste d frag m en ts were e le c tro p h o re s e d
20
on 7% p o ly a c ry la m id e g e ls a t 250 V o lt f o r 100 m in.
R e s u lts
STS P ro d u c tio n
E ig h t PCR p rim e r s e ts and t h e i r sources are shown in T able 5.
p rim e r s e ts
were designed
from
p u b lis h e d
Four
sequences o f a - h o r d o th io n in ,
a lc o h o l dehydrogenase 2 (ADH-2), B l-h o rd e in , and th e Bam H I-SstI fragm ent
o f pMSU21.
The o th e r f o u r ,
P s tl- 3 1 9 ,
were o b ta in e d from sequencing
genomic l i b r a r y .
P s tl- 3 2 7 , P s tl- 3 3 7 ,
and P s tl-3 4 0 ,
s h o rt p o r tio n s o f DNA c lo n e s from a P s tl
F ig u re 2 shows »the DNA sequence o f s h o r t p o r tio n s a t
b oth ends o f th e genomic clo n e s o b ta in e d by th e method d e s c rib e d .
th e se sequences,
PCR p rim e rs were designed to
From
be about 20 bases lo n g ,
c o n ta in 50% GC and h a rb o r no in v e rte d re p e a t sequences.
PCR a m p lif ic a t io n and polym orphism d e te c tio n
Each p rim e r s e t was used to
D ic k to o
and Morex b a r le y
(F ig u re
a m p lify sequences from t o t a l
3 ).
Two p rim e r
s e ts
d if f e r e n t ia t e d
between th e two c u l t i v a r s based on agarose g e l e le c tr o p h o r e s is .
DNA
fra g m e n ts
polym orphism .
bp fra g m e n t
P a re n ta l
from
pMSU21
p rim e rs
id e n t if ie d
an
DNA o f
A m p lifie d
in s e r t io n / d e le t io n
A 670 bp fra g m e n t is a m p lifie d from D ic k to o DNA, and a 450
is
a m p lifie d
and progeny
from Morex DNA when th e se
banding
p a tte rn s
are
shown
in
p rim e rs
F ig u re
are used.
5A.
T h is
polym orphism was p r e v io u s ly determ ined to be a l l e l i c t o t h a t id e n t if ie d by
Southern b lo t a n a ly s is w ith th e probe pMSU21 (S hin e t a l .
1990).
B l-
h o rd e in p rim e rs a m p lifie d two frag m en ts in D ic k to o (a p p ro x . 960 and 860
bp)
and one fra g m e n t in
Morex (a p p ro x .
860 b p ).
The PCR and p r o te in
polym orphism s (d e te rm in e d by SDS-PAGE) dem onstrated p e r f e c t c o s e g re g a tio n
(T a b le 6 ) .
21
PCR p ro d u c ts a m p lifie d w ith th e o th e r s ix p rim e r s e ts d id n o t show
s iz e polym orphism s.
These p ro d u c ts were d ig e s te d w ith
c u t te r s and e le c tro p h o re s e d on 7% p o ly a c ry la m id e g e ls .
polym orphism s
h o r d o th io n in ,
p rim e r s e ts
were
d e te c te d
in
PCR
a lc o h o l dehydrogenase 2,
(F ig u re 4 ) .
p ro d u c ts
P s tl-3 2 7 ,
seven fo u r-b a s e
R e s tr ic t io n s it e
a m p lifie d
P s tl- 3 3 7 ,
from
<*-
and P s tl-3 4 0
No polym orphism was d e te c te d w ith th e P s tl-3 1 9
p rim e rs . S e g re g a tio n based on PCR a m p lif ic a t io n from P s tl-3 2 7 and P s tl-3 3 7
p rim e rs agreed p e r f e c t ly w ith t h a t de te rm in e d by S outhern b lo t a n a ly s is o f
doubled h a p lo id lin e s from th e DxM c ro s s (T a b le 6 ) .
P a re n ta l and progeny
banding p a tte rn s f o r th e a n a ly s is based on th e P st-337 p rim e rs are shown
in F ig u re SB.
The p ro d u c t o f ADH-2 p rim e r a m p lif ic a t io n , however, d id n o t
show a l l e l ism w ith any o f f i v e RFLP polym orphism s i d e n t i f i e d th ro u g h th e
use o f th e ADH-2 c lo n e in Southern b lo t a n a ly s is o f a s e r ie s o f doubled
h a p lo id lin e s
(p e rs . comm, A. K le in h o fs , NABGMP, 1991).
Chromosomal L o c a tio n
Chromosomal lo c a tio n s o f STSs were id e n t if ie d
by lin k a g e a n a ly s is
w ith p r e v io u s ly mapped m arkers in doubled h a p lo id p ro g e n ie s and by PCR
a m p lif ic a t io n o f w h e a t-b a rle y a d d itio n lin e s (T a b le 5 and F ig u re 6 ) .
The
re c o m b in a tio n ana lyses were perform ed u s in g MAPMAKER (Land er and B o ts t e in,
1989).
The P s tl-3 2 7
and a-
h o rd o th io n in
a m p lif ic a t io n
p ro d u c ts
were
e v a lu a te d in both th e SxM ( p o p u la tio n s iz e = 150) and DxM (p o p u la tio n s iz e
= 30) c ro s s e s .
They were lo c a te d on chromosome 2 and 5 r e s p e c tiv e ly in
both
I d e n t ic a l
c ro s s e s .
a n a ly s is
and
chromosomes were lo c a te d
PCR a m p lif ic a t io n
of
DNA from
u s in g
re c o m b in a tio n
th e w h e a t-b a rle y
a d d itio n
lin e s .
The polym orphism i d e n t if ie d
by th e ADH-2 based p rim e rs mapped to
22
b a r le y chromosome 7 by both re c o m b in a tio n a n a ly s is and PCR a m p lific a t io n
o f w h e a t-b a rle y chromosome a d d itio n lin e s .
C u r io u s ly , . a lth o u g h th e probe
ADH-2 h y b r id iz e s to s e v e ra l p o lym o rp h ic bands from genomic d ig e s ts , none
o f th e RFLP polym orphism s cosegregated w ith th e PCR polym orphism .
E f f ic ie n c y o f PCR A n a ly s is
Four p r e v io u s ly unmapped clo n e s were s e le c te d from a P s t - I l i b r a r y
p re s e le c te d
to
e xclu d e
re p e a t
sequences
and
o r g a n e lla r
sequences.
Sequence d a ta was o b ta in e d from th e te rm in a l 200 bp from each end o f each
c lo n e and p rim e rs were s y n th e s iz e d .
Seven r e s t r i c t i o n endonucleases w ith
f o u r base p a ir r e s t r i c t i o n s it e s were u t i l i z e d w ith th e f o u r PCR p ro d u cts
from th e b a r le y c u l t i v a r s Morex and D ic k to o .
s it e s
were su rveyed ,
g e n o typ e s.
and a t o t a l
A to ta l
o f 79 r e s t r i c t i o n
o f 16 were absent in
one o f th e two
T h is p ro v id e s a c o n s e rv a tiv e e s tim a to r o f sequence d iv e rg e n ce
between th e s e two genotypes o f 16/316 bases, about 5%.
S ince th e clo n e s
were n o t p re s e le c te d on th e b a s is o f polym orphism s, we have no reason to
exp e ct
b ia s
in
th is
e s tim a te
of
base
p a ir
d iff e r e n t ia t io n
homologous s in g le copy sequences o f D ic k to o and Morex b a r le y .
between
23
* 11A —» *—B "«
A C G T A C GT
F ig u re 2. DNA sequence o f P s tI-3 4 0 c lo n e . S h o rt p o r tio n s o f both ends (A
and B) o f th e clo n e s were used to d e sig n PCR p rim e rs . The arrow s in d ic a te
th e p o r tio n s o f sequence from which p rim e rs were d e sig n e d .
T a b le 5
Prim er sequences, sources, chromosomal lo c a t io n s and a l l el ism analyses.
Prim er
set
Prim er
sequences
aMSUZl
5'GGTCTTTCATGTACCTACC 3 '
5'CGAGCTCCTGTCGAGG 3 '
Shin e t a l . ,
B l-H o rd e in
5 ' CCACCATGAAGACCTTCCTC 3 '
5 ' TCGCAGGATCCTGTACAACG 3 '
Forde e t a l . ,
e -H o rd o th io n in
5 ' CTGGGGTTGGTTCTGG 3 '
5 ' GGCAGCAACATGGCATTC 3 '
R o d rig u e z-P a le n zu e la
e t a l . , 1988
5 (ND)
Alcohol
dehydrogenaseZ
5 ' GGGGAGATATCGACCAAAGT 3 '
5 ' CACGCCCTCGCCAACGCTCTCCA 3 '
T ric k e t a l . ,
7 U nlinked2*
P s tI-3 1 9
5 ' AGCTGAGCAAGCTTCTTTGG 3 '
.5'AACATGCTGGGCAACTCCCA 3 '
Genomic l i b r a r y
P s t I- 3 2 7
5 ' GGTACGAACATGGAGGTACT 3 '
5'ATCCAGTTCTTGTGCACCTG I '
Genomic l i b r a r y
2 ( 3 0 /3 0 )
P s t I- 3 3 7
5'ATCCAGTTCTTGTGCACCTG 3 '
5'AGCTACGTGGATCACACCAC I '
Genomic l i b r a r y
7 (3 0 /3 0 )
P s tI-3 4 0
5 ' TAGCATCGGTAATCTCTCGC 3 '
5 ' CCCTTTATATACACTGCCGA 3 '
Genomic l i b r a r y
5 (ND)
Primer
source
Chromosomal
Io c a tio n (A lle lis m )
1991
1985
1988
’ Data p r e v io u s ly r e p o r te d (Shin e t a l . , 1990)
7
2Southern b l o t d a ta communicated by D r. A. K le in h b fs
-Nb polymorphisms were i d e n t i f i e d e i t h e r by Southern b l o t a n a ly s is or PCR
ND = d a ta not determ ined
2 1(1 0 0 /1 0 0 )
5 (7 0 /7 0 )
Table 6.
Restriction Site Variation in PCR Products.
Prim er
sets
DNA
source
PCR a m p li f ie d
fragm ent s iz e s
P s tI-3 1 9
D icktoo
Morex
1100 bp
1100 bp
D icktoo
Morex
P s t I- 3 3 7
P s tI-3 4 0
P s t I -327
a -h o r d o th io n in
•
Alcohol
dehydrogenase?
R e s t r i c t i o n s it e s
HhaI
H in fI
A lu I
H a e lII
4
4
3
3
I
1000 bp
1000 bp
I
2
2
3
D icktoo
Morex
1200 bp
1200 bp
2
4
D icktoo
Morex
1100 bp
1100 bp
D icktoo
Morex
1050 bp
1050 bp
D icktoo
Morex
630 bp
630 bp
ND = d a ta not d eterm ined.
.
MspI
RsaI
I
I
I
I
I
I
3
3
I
I
3
2
{
O
ND
ND
I
I
3
3
I
3
2
4
3
4
ND
ND
4
5
5
5
O
I
O
O
5
5
I
I
O
O
I
I
I
I
2
2
I
I
I
I
O
O
ND
ND
3
2
3
3
2
3
O
O
2
2
O
I
I
I
ND
ND
'
r
TaqI
26
M l
2
3 4
5 6
7 8
9 10 11 12 13 14 15 16 M
Fig u re 3 .
A m p l i f i c a t i o n o f b a r le y DNA from D icktoo and Morex w ith e ig h t
p rim e r sets on a 1% agarose g e l . DNA tem p lates in the odd lanes are
D ic k to o , in the even lanes are Morex. A m p lifie d products w ith P s t I- 3 1 9
prim ers are in lanes I and 2, those w ith P s t I- 3 2 7 prim ers a re in lane 3
and 4, those w ith P s t I- 3 3 7 prim ers are in la n e 5 and 6, those w ith P s t I 340 prim ers are in lan e 7 and 8, those w ith pMSU21 prim ers are in lane 9
and 10, those w ith B l-h o r d e in primers are in lane 11 and 12, those w ith a h o rd o th io n in
prim ers
are
in
lane
13 and 14,
those w ith
alcohol
dehydrogenase 2 prim ers are in lane 15 and 16. DNA s iz e markers are shown
in lan e M. The s iz e s o f the marker bands are given in base p a i r s .
27
Fig u re 4. D ig e s tio n o f PCR products w ith r e s t r i c t i o n endonucleases w ith
fo u r -b a s e
r e c o g n it io n
sequences
were
e le c tro p h o re s e d
on
a
7%
p o ly a c ry la m id e g e l . H i n f I d i g e s t io n o f a m p li f ie d products w ith P s t I- 3 2 7
prim ers are in lan e I (D ic k to o ) and lan e 2 (M o r e x ). H i n f I d i g e s t io n o f
a m p li f ie d products w ith P s t I- 3 3 7 prim ers are in lane 3 (D ic k to o ) and lane
4 (M o r e x ). H a e I I I d i g e s t io n o f a m p lif ie d products w ith P s t I - 3 4 0 primers
are in la n e 5 (D ic k to o ) and lan e 6 (M o r e x ). TaqI d i g e s t io n o f a m p lif ie d
products w ith a -h o r d o th io n in primers are in lane 7 (D ic k to o ) and lane 8
(M o r e x ). RsaI d i g e s t io n o f a m p lif ie d products w ith a lcohol dehydrogenase
2 prim ers are in lan e 9 (D ic k to o ) and la n e 10 (M o r e x ). DNA s iz e markers
a re shown in lan e M w ith the s iz e o f the marker bands given in base p a i r s .
200»
150»
94»
F ig u re 5A, B. S egregation o f DxM doubled h a p lo id l i n e s . DNA s iz e markers
are shown in la n e M w ith th e s iz e o f marker bands given in base p a i r s . The
te m p la te DNA was from D icktoo in lan e I , Morex in la n e 2 and doubled
h a p lo id s in lan e 3 to la n e 12. A A m p lifie d products w ith pMSU21 primers
showed i n s e r t i o n / d e l e t i o n polymorphisms on a 1% agarose g e l . B H i n f I
d i g e s t io n o f a m p li f ie d products w ith P s t I - 3 3 7 primers were e le c tro p h o re s ed
on a 7% p o ly a c ry la m id e g e l .
29
F ig u re 6A, B. A m p l i f i c a t i o n o f w h e a t-b a r le y chromosome a d d i t i o n l i n e s was
from b a r le y cv Betzes in lan e I , Chinese s prin g wheat in la n e 2, b a r le y
chromosome I ,
2, 3, 4, 6, 7 a d d it io n l i n e s are in lan es 3 to 8,
r e s p e c t i v e l y . DNA s iz e markers are shown in lane M w ith a given s iz e o f
marker bands in base p a i r s . A A m p l i f i c a t i o n o f w h e a t- b a r le y chromosome
a d d i t i o n l i n e s w ith P s t I- 3 2 7 primers e le c tro p h o re s e d on a 1% agarose g e l .
B A m p l i f i c a t i o n o f w h e a t- b a r le y chromosome a d d itio n l i n e s w ith P s t I- 3 3 7
prim ers e le c tro p h o re s e d on a 1% agarose g e l .
30
Discussion
Three o f the fo u r randomly s e le c te d s in g le copy clones from which we
obta ine d p rim er sequences i d e n t i f i e d a l l e l i c v a r i a t i o n between Morex and
D icktoo b a r le y .
v a r ie s
F u r th e r ,
between Morex
is o la te d
s in g le
it
appears as though about one base in twenty
and D ic k to o .
copy genomic
A pproxim ately
clones
tested
37% o f
a g a in s t
the
Morex
randomly
and Dicktoo
i d e n t i f i e d polymorphisms when s ix s ix -b a s e r e s t r i c t i o n endonucleases were
u tiliz e d .
When i n d i v i d u a l
r e s t r i c t i o n s i t e s were e v a lu a te d , we a c t u a l l y
observed a frequency o f I s i t e
in 5 l o s t by m utation
(39 m u ta tio n s /1 1 8 8
bases e v a l u a t e d ) ,
o r 3.3% (d a ta not shown).
3.3%)
s i m i l a r and suggest t h a t polymorphisms between w i n t e r
and
seem f a i r l y
s p rin g
b a r le y
c u ltiv a rs
are
fre q u e n t
These fr e q u e n c ie s
enough
to
p e rm it
(5% vs.
e ffe c tiv e
mapping using e i t h e r RFLP or PCR based approaches.
F ive o f th e s i x PCR-based e v a lu a t io n systems in which a l l el ism was
determ ined
fo r
Southern
b lo t
based
lin k a g e s to p r e v io u s ly mapped m arkers.
polymorphisms
provided
expected
The s i x t h , ADH-2, provided a c l e a r
polymorphism but no lin k a g e to polymorphisms i d e n t i f i e d by Southern b l o t
a n a ly s is .
On one hand,
it
is c l e a r t h a t th e ADH-2 prim ers used in t h i s
study p ro v id e a useful g e n e t ic m arker.
On th e o th e r hand,
it
is l i k e l y
t h a t th e locus a m p li f ie d by these prim ers has no r e l a t i o n s h i p to alcohol
dehydrogenase.
This
F u r th e r study could help e x p la in t h i s anomaly.
study demonstrates
a n a ly s is f o r genome mapping.
an a l t e r n a t i v e
method t o
Southern
b lo t
However, th e need o f p rim e r sequences can be
a l i m i t i n g f a c t o r in PCR-based genome mapping.
To overcome t h i s problem,
th e RAPD approach was introduc e d ( W illia m s e t a l . , 1 9 9 0 ).
D7O vid io e t a l .
(1 9 90 ) and Weining and Langridge (1 991) d e r iv e d PCR prim ers from published
31
DNA sequences.
Comparable to STS proposed in the human genome p r o j e c t
(Olson e t a l . , 1 9 8 9 ) ,
s e le c te d
I extended th e STS idea by e v a lu a t in g fo u r randomly
DNA clones
these fo u r clones
from a genomic l i b r a r y .
id e n tifie d v a ria tio n
PCR products o f th r e e
between Morex and D ic k to o .
of
PCR-
based sequencing methods ( G ylle n s te n and E r l i c h , 1988; In n is e t a l . , 1988;
Higuchi
and Ochman, 1989; Kusukawa e t a l . ,
1990) make production o f STS
from s e le c te d DNA clones p r a c t i c a l .
The p roduction o f medium d e n s it y genomic maps using RFLPs has proven
to be f e a s i b l e in many crop spec ie s.
m a n ip u la tio n
a n a l y s is .
sequences
demands the
In t h i s
t e s te d
s eg re g a tio n
Using these maps f o r e f f i c i e n t QTL
use o f technology
s im p le r than
experim ent we found t h a t
could
by PCR.
be e a s i l y
b lo t
seven o f th e e ig h t marker
m anipulated
As s i g n i f i c a n t
Southern
QTL l o c i
to
p e rm it
e v a lu a tio n
are
id e n tifie d
of
in crops,
c o n v e rtin g mapped RFLP markers which f l a n k a g ro n o m ica lly im p o rta n t l o c i to
PCR based d e t e c t io n systems w i l l pro v id e a u s e r - f r i e n d l y technology to the
u l t i m a t e users o f genome maps, th e p l a n t b re ed e rs .
Although th e v a r i a t i o n o u ts id e th e STS re g io n cannot be d e te cted as
in
Southern
s u ffic ie n t
b lo t
a n a l y s is ,
it
e x is ts
v a ria tio n
w ith in
systems (L i e t a l . ,
1 9 8 9 ).
has
been
demonstrated
a m p li f ie d
fragments
th a t
in
th e r e
is
mammalian
1988; L i t t and L u ty , 1989; T a u tz , 1989; Weber and May,
In p l a n t systems,
our study supports a s i m i l a r c o n clu s io n .
In
c o n t r a s t to S k o ln ic k and W allace ( 1 9 8 8 ) , t h i s study demonstrates t h a t PCR
polymorphisms a re m a in ly due to th e absence or presence o f r e s t r i c t i o n
endonuclease s i t e s r a t h e r than the fragm ent le n g th d i f f e r e n c e s .
Chromosomal
a n a ly s is
and
use
lo c a tio n s
of
th e
were
id e n tifie d
w h e a t- b a r le y
using
a d d it io n
both
recom bination
lin e s .
Chromosomal
32
lo c a t io n s
id e n tifie d
in
progeny from two crosses
(D ic k to o
x Morex and
S teptoe x Morex) corresponded w e ll and were confirmed by PCR a n a ly s is o f
DNA from th e w h e a t- b a r le y chromosome a d d it io n l i n e s .
Although r e l a t i v e costs might be debated, the ease o f PCR a n a ly s is ,
the
c la rity
of
re s tric tio n
p a tte r n
of
PCR products
and
the
ease
of
d i s t r i b u t i o n o f p rim e r sequence data (as opposed to maintenance o f c lo nes)
a ll
argue
in
chromosomal
sTable
fa v o r o f PCR as a to o l
lo c a tio n
of
crosses
if
over
C le a rly ,
the
a polymorphism must be w e l l - c h a r a c t e r i z e d
and
it
is
to
in genome a n a l y s is .
be
g e n e ra lly
u s e fu l.
A ll
seven
polymorphisms e v a lu a te d in t h i s study appear sTable and appear to map to
s in g le l o c i .
One o f th e prim er sets (ADH-2) a m p lif ie d a product from an
unexpected lo c u s .
The clone from which our sequence d a ta d e riv e d c l e a r l y
h y b r id iz e d t o sequences a t several d is p ers e d l o c i .
lo c i
of
th is
s o rt,
d is p e rs e d ,
m u ltig e n e
cand id a te s f o r RCR based g e n e tic a n a l y s is .
We would suggest t h a t
fa m ilie s ,
may
be
u n s u ita b le
33
CHAPTER 4
MAPPING OF TRAITS ASSOCIATED WITH WINTERHARDINESS
IN A WINTER x SPRING BARLEY CROSS
I n t r o d u c tio n
The p r o p o r tio n o f w i n t e r b a r le y p la n ts s u r v iv in g to produce g r a in w in te r h a r d in e s s
- is determined by many i n t e r a c t i n g f a c t o r s .
thought to range from low tem perature t o le r a n c e to b i o t i c
ta n c e .
Included
to le ra n c e ,
among these
v e rn a liz a tio n
c o n trib u tin g
re q u ire m e n t,
fa c to rs
are
photoperiod
These are
s tre s s r e s i s ­
low tem perature
response,
p r o te in
m etabolism , carbohydrate s t a t u s , and membrane l i p i d com position (Thomashow
1 9 9 0 ).
The g e n e t ic and p h y s io lo g ic a l
p o o r ly
understood.
In
th is
report
bases o f most o f these fa c to r s are
we
addressed
w in te r h a r d in e s s by s y s t e m a t i c a l l y measuring i t s
th e
g e n e t ic
basis
of
components in a doubled
h a p lo id p o p u la tio n o f b a r le y and re g re s s in g these phenotypic data sets on
m o le c u la r marker genotypes t o i d e n t i f y q u a n t i t a t i v e t r a i t l o c i
Long-term
fie ld
s u r v iv a l
is
th e
(Q TL).
best measure o f w in te rh a rd in e s s
( O lie n 1 9 7 8 ), but o b ta in in g long term performance d a ta a re expensive and
d iffic u lt.
High l e v e l s o f genotype x environment i n t e r a c t i o n and enormous
measurement e r r o r in c re a s e th e d i f f i c u l t y o f c o r r e c t d a ta i n t e r p r e t a t i o n .
Furtherm ore,
e ffe c tiv e
th e
i n fr e q u e n t
d is c rim in a tio n
g e n e ra lly in e ffe c t iv e
occurrence
among
of
genotypes
a te s t
makes
w in te r
th a t
fie ld -b a s e d
perm its
s e le c t io n
(M c In ty r e e t a l . 1 9 8 8 ).
Crown f r u c t a n c o n te n t and c o n t r o l l e d f r e e z e t e s t s t o determ ine LT50
( t h e te m p e ratu re a t which 50% o f th e p o p u la tio n is k i l l e d ) a re reasonably
sim ple measures which show s i g n i f i c a n t c o r r e l a t i o n s w ith f i e l d
s u r v iv a l
34
(S tu s h n o ff e t
a l.
ba sis
to le ra n c e
o f cold
c u m u l a t i v e ly ,
th e
th re e
1 9 8 4 ).
Thomashow ( 1 9 9 0 ) ,
in
h ig h e r p l a n t s ,
in re v ie w in g th e m o le c u la r
c ite d
over 20 r e p o r ts
th a t,
assign c old to le r a n c e genes to every chromosome in each o f
genomes
of
wheat.
Chromosomes
5A and
5D
a re
most
o ft e n
im p l ic a t e d ; Sutka and Snape (1 989) and Roberts (1 990) pro v id ed com pelling
evidence f o r th e importance o f chromosome BA.
V e rn a liz a tio n
but t r a i t
and cold t o le r a n c e a re considered t o be a s s o c ia te d ,
a s s o c ia tio n
in
b a r le y
( D o ll
et
a l.
1989)
and wheat
1990) has been a t t r i b u t e d t o lin k a g e r a t h e r than p l e i o t r o p y .
of
growth
h a b it
in to
its
components
of
v e rn a liz a tio n
photo p erio d s e n s i t i v i t y , and m a t u r i t y i s c h a lle n g in g .
(Roberts
S ep a ra tio n
re q u ire m e n t,
Three u n lin k e d l o c i
have been i d e n t i f i e d which behave e p i s t a t i c a l I y to de te rm in e w i n t e r vs.
s p rin g
h a b it
phenotype
in
can
b a r le y
(Takahashi
be m o d ifie d
s e p a r a tio n o f these e f f e c t s
m a tu rity
genes
(Roberts
et
r e p o r te d
th a t
photo period
and Yasuda 1 9 7 1 ).
by both
v e rn a liz a tio n
The w i n t e r
and
h a b it
p h o to p e rio d ,
and
can be f u r t h e r c om plicated by th e a c tio n o f
a l.
1 9 8 8 ).
response,
Barnham and
as
d is tin c t
Rasmusson
from
(1981)
m a tu rity ,
is
q u a n tita tiv e ly in h e rite d .
P r o s t r a t e o r r o s e t t e growth h a b i t has been shown t o be c o r r e l a t e d
w ith c o ld hardiness
Low tem p e ratu re s
MacDonald 1 9 8 4 ).
in wheat (Salmon 1971; T a y lo r 1983; Chaudhry 1 9 8 6 ).
induced re d u c tio n
in r o s e t t e growth h a b i t
In our b a r le y p a r e n t s ,
(Roberts and
th e w i n t e r c u l t i v a r
showed p r o s t r a t e growth w h ile th e s p rin g c u l t i v a r
(Morex)
(D ic k to o )
showed a much
more e r e c t growth p a t t e r n .
C arbohydrates,
c ry o p ro te c tiv e
ro le
and p a r t i c u l a r l y
in
c e r e a ls .
fru c ta n s ,
S everal
are r e p o r te d
to
p la y
a
mechanisms have been proposed
35
(O lie n and L e s te r 1985) to e x p la in t h i s
(1989)
found
a
marked
e ffe c t
of
a s s o c ia t io n .
carbohydrate
L iv in g s to n e t a l .
le v e l
on
the
f r e e z in g
t o le r a n c e and sugar composition o f b a r le y crown t i s s u e in a s e t o f b a r le y
genotypes which included our w i n t e r p a ren t ( D i c k t o o ) .
no d i r e c t
re la tio n s h ip
between
s p e c ific
However, th e r e was
com positional
makeup o f
to ta l
c lo n in g
and
carbohydrate and f r e e z in g t o l e r a n c e .
S u b s ta n tia l
in te re s t
has
developed
in
th e
c h a r a c t e r i z a t i o n o f genes re g u la te d o r induced by s p e c i f i c s tr e s s e s .
d a te ,
th r e e
genes
r e g u la te d
by
cold
in
( C a t t i v e l l i and B a r t e l s , 1990; Dunn e t a l . ,
provided by D r.
id e n tify
e v a lu a te d
1 9 9 1 ).
have
been
rep o rte d
Two clones were k in d ly
One o f th e two clones
(A086) was found to
a polymorphism between Morex and D ic k to o .
Although one clone
in
C a ttiv e lli.
b a r le y
To
these
experim ents
was
not
re a d ily
o b ta in e d ,
published
sequence d a ta p e r m itte d us to re c o v e r homologous sequences from several o f
these using PCR.
in th e c ro s s .
to lo c i
No polymorphism was d e te c te d from PCR using t h i s marker
The clone A086 was examined t o determ ine whether i t mapped
a s s o c ia te d w ith cold t o le r a n c e o r i t s components.
Doubled h a p lo id s pro v id e g e n e t ic r e fe r e n c e p o p u la tio n s f r e e o f the
n o n - a d d i t i v e types o f gene a c tio n t h a t com p lic a te s e g re g a tin g g e n e ra tio n
analyses in autogamous s p e c ie s .
map
c o n s tr u c tio n
w in te r h a r d in e s s ,
and
which
analyses and th w a rte d
QTL
have
They a re a ls o i d e a l l y s u it e d f o r lin k a g e
mapping.
The
of
b a r le y
con ve n tio n a l
g e n e tic
are e x c e l l e n t t a r g e t s
f o r QTL
posed c h a lle n g e s
s e le c tio n
e ffo rts ,
mapping in doubled h a p lo id p o p u la tio n s .
components
fo r
36
M a t e r i a l s and Methods
Germolasm
One hundred doubled h a p lo id (DH) l i n e s were developed by the H o rd e u m
b u l b o s u m te c h n iq u e ,
o f th e
cross
as described by Chen and Hayes ( 1 9 8 9 ) , from F1 p la n ts
o f D icktoo
x Morex.
D icktoo
is
a six -ro w e d
w in te r
feed
b a r le y o f unknown a n c e s try and mixed d e s c r i p t i o n re le a s e d by th e Nebraska
A g ric u ltu ra l
Experiment S t a t i o n
in
1952.
Morex
is
m a ltin g b a r le y re le a s e d by th e Minnesota A g r i c u l t u r a l
a s ix-row ed
spring
Experiment S ta tio n
in 1978.
T r a i t Assessment
We
measured
to le ra n c e :
fie ld
th r e e
s u r v iv a l
Bozeman, Montana, and LT50.
v is u a lly
tra its
at
which
are
C o rv a llis ,
F i e ld s u r v iv a l
assessing p e rce n t s u r v iv a l
d ire c t
Oregon,
measures
fie ld
of
s u r v iv a l
cold
at
a t C o r v a l l i s was measured by
fo llo w in g
the w i n t e r o f
1990-1991.
Each DH l i n e was re p res e n te d by an u n r e p lic a te d p l o t c o n s is tin g o f two 1 .5
m rows.
F i e l d s u r v iv a l a t Bozeman was measured as the d i f f e r e n c e between
in itia l
(O c to b e r, 1991) and p o s t-c o ld s tr e s s (March, 1992) p l a n t stands in
a th ric e
r e p l i c a t e d exp e rim en t.
using p l a n t m a t e r ia l
The LT50 o f each DH l i n e was determined
hardened a t 2°C f o r 5 weeks w ith a 10 h l i g h t / 1 4 h
d a rk photo period regim e.
Four tem peratu res
(0 ,
-4 ,
-8 ,
-12°C) w ith ten
p la n ts a t each te m p e ratu re were used t o determ ine th e LT50Of each DH l i n e .
A to ta l
o f th r e e r e p l i c a t e s were run from October 1990 t o February 1991.
P la n t m a t e r ia l
was prepared f o r f r e e z i n g ,
and LT50 value s were computed,
as d e s c rib e d by K o la r e t a l . (1 9 9 1 ).
Two e s tim a te s o f heading d a te were made.
Heading d a te was measured
37
using
u n v e r n a liz e d
p la n t
m a te r ia l
te m peratu re o f 18°C and 24 h l i g h t .
in
the
greenhouse
at
a
constant
The heading date under normal green
house c o n d itio n s was a ls o measured' a t the same time as th e heading date
under continuous l i g h t .
Normal green house c o n d itio n s a t C o r v a l l i s were
16 h l i g h t , w ith tem peratures o f 20°C d u rin g the day,
and 18°C during the
n ig h t.
Growth h a b i t d a ta were taken in th e f i e l d a t C o r v a l l i s , OR 1992.
s c a le from one to e ig h t was used.
and e ig h t
A
One in d ic a te d extreme p r o s t r a t e h a b it
in d ic a te d e r e c t growth.
These d a ta were taken b e fo re digging
p l a n t s f o r f r u c t a n a n a l y s is .
The crown c o n te n t o f fr u c ta n s w ith a degree o f p o ly m e r iz a tio n (DP)
> 5 was determ ined using f i e l d
hardened p l a n t m a t e r i a l .
F iv e p la n ts o f
each genotype were dug from f i e l d p l o t s a t C o r v a l l i s , OR in January, 1992.
P la n ts were washed,
liq u id
n itro g e n .
ground
in
liq u id
e x tra c tio n .
trimmed,
and th e crowns were im m ediately froze n
T issue was T y o p h iliz e d
n itr o g e n
fo r
and 50 mg pe r
15 days.
in
Crown tis s u e was
sample were used f o r
fr u c ta n
F iv e ml o f d i s t i l l e d w a te r were added to each 50 mg sample;
samples were shaken and then incubated f o r 15 min in a 90°C w a te r bath. The
s u p e rn a ta n t
was
poured
column and remixed
in
through
5 ml
an A m b e r lite
w a te r;
th is
MB-3A ion
exchange
step was re p e a te d
r e s in
tw ic e .
The
column was then washed w ith 5 ml o f 90°C w a t e r , and th e w a te r e x t r a c t s were
d r i e d a t 60°C f o r 6 t o 8 h.
w a te r
and c e n t r if u g e d
filte re d
which
using
was
at
D rie d samples were d is s o lv e d in I ml o f GO0C
12,000 rpm f o r
a 0 .4 5 micron f i l t e r
equipped
carb o h y d ra te columns.
w ith
BioRad
p rio r
carbo-C
10 min.
to
The supernatant was
in je c tio n
guard
and
in to
BioRad
the HPLC,
HPX-42
C
Fructan c o n te n t (> DP 5) is expressed as mg g*1 on
38
a dry w eight basis (mg/gdw).
Map C o n s tru c tio n and QTL A n a lys is
Markers,
Table 7.
d e s c rip tio n s ,
m orphological
sup p lie d by C o rn e ll
Heun e t a l .
and
isozyme
b a r le y cDNA,
clone
m arkers,
and oat cDNA c lo n e s ,
re s p e c tiv e ly .
I t h a c a , NY
developed
by
re s p e c tiv e ly .
Clones
14853-1902.
th e
p re fix
desig n a te s
North
chain
to :
U n iv e rs ity ,
known
re a c tio n .
Dr.
The ABC p r e f i x designates a
American
B a rle y
Requests
Bozeman, MT
320
fo r
Leon
Genome
P ro je c t.
Dr. Andy K le in h o fs , 271
Pullman, WA 991 6 4-64 2 0.
DNA sequence
Tom B lake,
Requests f o r
Dr. Mark S o r r e l l s , 252 Emerson H a l l ,
Johnson H a l l , Washington S ta te U n i v e r s i t y ,
d ire c te d
in
The p r e f i x e s "m" and " i "
Requests f o r these clones should be d i r e c t e d to :
polymerase
lis te d
( 1 9 9 0 ) , where the p r e f i x e s WG, BCD, and CDO d e sig n a te wheat
C o rn e ll U n i v e r s i t y ,
"ap"
are
U n i v e r s i t y were named f o llo w in g th e nomenclature o f
these clones should be d i r e c t e d t o :
cDNA
assignments
The fo llo w in g conventions were used.
d e s ig n a te
genomic,
and chromosome
polymorphism
v is u a l iz e d
p r im e r ' sequences
Johnson
H a ll,
The
should
Montana
by
be
S ta te
59715.
B r i e f l y , f o r RFLP a n a l y s is , p l a n t DNA was prepared from fre s h t i s s u e
using th e p r o te in a s e k procedure.
a p p r o p r ia t e
re s tric tio n
DNA samples (10 ^g) were d ig e s te d w ith
endonucleases,
separated
on
agarose
g els
and
t r a n s f e r r e d t o charged nylon membranes using th e a l k a l i n e p ro to c o l o f Reed
and Mann
(1 9 85 ).
agarose
and la b e le d
F ilte rs
were
Cloned
DNA i n s e r t s
were cut
by random prim ing
p r e h y b r id iz e d ,
h y b r id iz e d
s tr in g e n c y o f 0 .1 XSSC, 0.1% SDS, 65 C,
from low m e l t i n g - p o i n t
( Feinberg
o v e r n ig h t ,
and V o g e ls te in
washed w ith
and exposed to f i l m
1 9 8 4 ).
a fin a l
as needed.
PCR markers were based on published sequences o r on p a r t i a l sequencing o f
39
th e t e r m in i
s y n th es is
o f i n fo r m a tiv e RFLP c lo n e s .
procedures
were described
Isozymes were assayed as described
Sequencing and o l i g o n u c le o t id e
by Tragoonrung e t
by N ie ls e n
a l.
(in
and Johansen
s torage p r o te in s were assayed as described by Blake e t a l .
p r e s s ).
(1986)
(1 9 8 2 ).
and
The
m orphological markers were scored under a stereomicroscope o r w ith a hand
le n s .
Map c o n s tr u c tio n and QTL a n a ly s is were performed using MAPMAKER and
MAPMAKER/QTL programs (Lander and B o t s t e i n,
1 9 8 9 ).
The Sequence-Tagged-Site approach (Olsen e t a l . ,
g e n e ra te
prim ers
homologous
to
the
sequences
1991) was used to
published
fo r
r e g u la te d genes A086, T59 and BLT4 ( C a t t i v e l l i and B a r t e l s ,
a l.,
1 9 9 1 ).
s e le c te d .
From th e
s iz e d
fragm ent le n g th
th e
sequence
data
p rim er
cold
1990; Dunn e t
sequences
were
A m p l i f i c a t i o n regimes were s e le c te d to promote a m p l i f i c a t i o n o f
a p p ro p ria te ly
from
published
the
products which were then e v a lu a te d
polymorphisms o r s iz e
c u ltiv a rs
D ic kto o
and
fo r
re s tric tio n
polymorphisms using te m plate DNA
Morex.
These
were
then
u tiliz e d
as
m o le c u la r m arkers.
Results and Discussion
Map C o n s tru c tio n
The 77 markers were re so lv ed i n t o e ig h t lin k a g e groups (Ta b le 7 and
F ig u re 7 ) .
Two u n lin k e d groups were mapped to b a r le y chromosome I .
o f these was anchored t o chromosome I
o th e r
by
a U b iq u itin
chromosome
I
by
th e
lo c u s ,
North
both
American
(K le in h o fs and K i l l i a n , i n p r e s s ! .
aWGllOS
and
aPST240B
of
and aMSU21,
One
by th e presence o f His3 and the
which
were
B a rle y
p r e v io u s ly
Genome
mapped to
Mapping
P r o je c t
The chromosome 2 map was anchored by
spaning
a re g io n
of
213 .1
cM.
S ix
markers were found, on chromosome 3 l i n k e d w ith th e known marker iEST4.
40
These markers spanned a d is ta n c e o f 1 10 .6 cM.
by marker aG1u2 and a H rth ,
b a r le y chromosome 4.
Chromosome 4 was anchored
both o f which had p r e v io u s ly been mapped to
These markers span a regio n o f 152.3 cM lon g .
Known
lo c a t i o n seed storage p r o te in markers on chromosome 5 were d e te cted in a
lin k a g e group o f 13 m arkers.
o f 180.3 cM.
markers
The map o f chromosome 5 covered a d is ta n c e
Chromosome 7 spanned a re g io n 1 8 9 .8 cM lo n g .
mapped to
chromosome 7 are
previous mapping p r o j e c t s
know n-location
(Shin e t a l . ,
S ix o f th e 13
markers
e ith e r
from
1990; K le in h o fs and K i l l i a n ,
p r e s s ) o r by PCR o f w h e a t-b a r le y chromosome a d d itio n l i n e s .
One lin k a g e
group c o n s is tin g o f 7 markers is ambiguous f o r chromosome l o c a t i o n .
unambiguously p r e v io u s ly mapped markers mapped w i t h i n t h i s group.
th e
markers
mapped
w ith in
th is
group
is
a
product
of
in
No
One o f
a m p lific a tio n
d i r e c t e d by prim ers w ith homology to th e b a r le y n i t r a t e reductase ( N a r l )
lo c u s ,
known to
r e s id e
on b a r le y
chromosome 6.
U n f o r t u n a t e ly ,
th r e e
a m p l i f i c a t i o n products which upon d ig e s t io n provided r e s t r i c t i o n fragment
le n g th polymorphisms were produced under th e d i r e c t i o n o f these prim ers .
Two o f
them were
lin k e d
w ith
markers
on chromosome 2.
The t h i r d
is
assumed t o r e s id e on chromosome 6 , but we suggest t h a t t h i s may be a weak
a s s e rtio n .
T able 7.
D e s c r ip t io n s , chromosome l o c a t i o n , and numbers o f mapped
progenies assignments f o r markers used to lo c a t e q u a n t i t a t i v e t r a i t l o c i
in th e doubled h a p lo id progeny o f D icktoo X Morex. D e f i n i t i o n s o f marker
p r e f i x e s are provided in th e t e x t .
Marker
D e s c r ip tio n
a N a rla
aHIS3
WG118S
D2A
CS
STS-PCR
STS-PCR
Wheat genomic RFLP
B a rle y cDNA RFLP
B a rle y cDNA RFLP
Chromosome
Numbers
of
l i n e s mapped
I
I
I
I
I
100
100
30
30
30
41
Table 7.
Marker
aUbiq
apT59b
C3b
aWGHOS
WG645
aPST327
aMSU21
PST59
A086
CD0588
aNar7HinF
aWGi78
aNAR7S
aPST340b
AlOA
A12
D7A
1EST4
F8
F4
aGlu2
WSEP
TB19-20
WG1026b
BCD265a
PST316
D7B
HorB
HorC
aG lul
iG P Il
WG789
WG118L
AlOB
aWGHOL
HorD
aPST340a
iPGD
BCD265C
aHRTH
D2B
aWG669
pKSU44
aNar7L
LMWP
pKSU24S
OPAll
(c o n tin u e d )
D e s c r ip tio n
Chromosome
STS-PCR
STS-PCR
B a rle y cDNA RFLP
STS-PCR
Wheat genomic RFLP
STS-PCR
STS-PCR
B a rle y genomic RFLP
B a rle y cDNA RFLP
Oat cDNA RFLP
STS-PCR
STS-PCR
STS-PCR
STS-PCR
B a rle y cDNA RFLP
B a rle y cDNA RFLP
B a rle y cDNA RFLP
Esterase 4
B a rle y cDNA RFLP
B a rle y cDNA RFLP
STS-PCR
H2O -S o lu b le endosperm p r o t e i n
STS-PCR
Wheat genomic RFLP
B a rle y cDNA RFLP
B a rle y genomic RFLP
B a rle y cDNA RPLP
B-hordein
C -hordein
STS-PCR
Glucosephosphate isomerase I
Wheat genomic RFLP
Wheat genomic RFLP
B a rle y cDNA RFLP
STS-PCR
D -hordein
STS-PCR
Phosphogluconate dehydrogenase 2
B a rle y cDNA RFLP
STS-PCR
B a rle y cDNA RFLP
STS-PCR
B a rle y cDNA RFLP
STS-PCR
Low m o le c u la r w eight p r o t e i n
B a rle y cDNA RFLP
B a rle y cDNA RFLP
Numbers
of
l i n e s mapped
I
I
I
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
6'
6
6
6
6
6
6_
.
100
30
30
100
100
100
100
30
30
100
100
100
100
30
30
30
30
100
30
30
100
100
100 .
100
100
100
30
100
100
100
100
30
30
30
100
100
100
100
100
100
30
100
30
100
100
100
30
42
Table 7.
(c o n tin u e d )
Marker
D e s c r ip tio n
pKSU32
pTA71
apT59a
aADH
BCD298
mS
WG364b
PST321
PKSU24
C3a
mR
BCD265b
aPST337
B a rley cDNA RFLP
B a rle y genomic RFLP
STS-PCR
STS-PCR
B a rle y cDNA RFLP
R a c h il l a h a i r len gth
Wheat genomic RFLP
B a rle y genomic RFLP
B a rle y cDNA RFLP
B a rle y cDNA RFLP
Awn roughness
B a rle y cDNA RFLP
STS-PCR
Chromosome
Numbers
of
l i n e s mapped
7
7
7
7
7
7
7
7
7
30
100
30
100
100
100
100
30
100
30
100
100
100
I
I
I
7
Cold To le ran c e and QTL A n a lys is
Each
measure
of
cold
phenotypes (F ig u re s 8 ,
9,
t o le r a n c e
and 1 0 ) .
gave
a d is tin c t
F i e l d s u r v iv a l
consequence o f a unique s e t o f hardening, growth,
d is trib u tio n
of
is thought to be the
and s tr e s s c o n d itio n s
and th e d i s t i n c t p o p u la tio n frequency d i s t r i b u t i o n s f o r Oregon and Montana
fie ld
s u rv iv a l
100% s u r v iv a l
r e f l e c t these d i f f e r e n c e s .
At C o r v a l l i s ,
and o n ly two showed complete m o r t a l i t y .
e i g h t l i n e s had
A t Bozeman,
l i n e s s u f f e r e d complete m o r t a l i t y and th e h ig h e s t s u r v iv a l was 85%.
50
The
hardening and f r e e z i n g s tre s s e s used to determ ine LT50Ie d t o a more normal
d i s t r i b u t i o n o f t r a i t performance: LT50 values ranged from - 2 .7 to - 1 1 .0
0C.
The tr a n s g r e s s iv e
s p rin g
paren t,
Morex,
segregants
may have
fo r
cold t o le r a n c e
c o n tr ib u te d
f a v o r a b le
in d ic a te
a lle le s
t h a t the
or
th a t
e p i s t a s i s may be an im p o rta n t d e te rm in a n t o f t r a i t e x p re s s io n .
Large QTL e f f e c t s f o r a l l measures o f cold t o l e r a n c e were found on
chromosome 7.
For f i e l d
s u r v iv a l
in Oregon, th e QTL e f f e c t was d e te cted
in th e i n t e r v a l o f mR-BCD265b w ith th e l o g - l i k e l i h o o d o f 8 .5 6 and account
43
f o r 37.8% o f th e v a r i a t i o n .
exceeding
th e
For f i e l d
lo g -lik e lih o o d
of
2 .0
s u r v iv a l
in Montana, QTL e f f e c t s
th re s h o ld
were
d e te c te d
in
two
i n t e r v a l s : WG364b-PST321, and pKSU24-C3a. The l a t e r i n t e r v a l was covered
up to mR-BCD265b i n t e r v a l .
s u r v iv a l
A n a ly s is
o f the QTL f o r mean o f the f i e l d
in th e two lo c a tio n s d e te cted the QTL e f f e c t s a t two i n t e r v a l s :
WG364b-PST321, mR-BCD265b.
f o r WG364b-PST321,
L o g - l i k e l i h o o d o f the two i n t e r v a l s were 6 .0 7
and 19.23 f o r mR-BCD265b.
d e te c te d in th e f o llo w in g i n t e r v a l s :
For LT50, LODs > 3 .0 were
apT59a-a'ADH, and mR-BCD265b.
Because t h i s re p re s e n ts a f i r s t a tte m p t to map QTL a ss ociated w ith
cold
to le ra n c e
in
b a r le y ,
previous
it
is
d iffic u lt
p e r s p e c tiv e
of
re p o rts .
d is to rtio n ,
in fa v o r o f th e w i n t e r p a r e n t ,
to ,p u t
We re p o rte d
our
fin d in g s
s ig n ific a n t
in
the
s egregation
in the v i c i n i t y o f Rrn2 in the
F2 progeny o f D icktoo X Morex Fi p la n ts s e l f - p o l l i n a t e d under low tempera­
t u r e ( Schon e t a l .
v i c i n i t y o f mR.
1 9 9 1 ).
No s eg re g a tio n d i s t o r t i o n was d e te c te d in the
In th e case o f Oregon f i e l d s u r v iv a l and L-T50, modest QTL
e f f e c t s were d e te c te d in th e v i c i n i t y o f Rrn2.
In
th e
fa c e
of
p o p u la tio n
frequency d i s t r i b u t i o n s
such as those
shown in F igures 2a, 2b, and 2c, pre vious i n v e s t i g a t o r s have r e s o rte d to
c la s s i c a l
q u a n tita tiv e
h e rita b ility .
procedures
The r e s u l t s
example,
Rhode
s u r v iv a l
ranging
and
e s tim a te
g e n e t ic
have not been p a r t i c u l a r l y
Pulham
from
to
(1960)
-8% to
85%.
r e p o r te d
The
varia n ce s
s a tis fy in g .
h e rita b ilitie s
a v a ila b ility
of
fo r
and
For
fie ld
a p p ro p ria te
c y t o g e n e tic stocks in wheat has allow ed f o r th e measurement o f chromosome
e ffe c ts ,
lo c i
and both Roberts
(1990) and Sutka and Snape (1 9 8 9 ) r e p o r t t h a t
on wheat chromosome 5 are a s s o c ia te d w ith cold t o l e r a n c e .
chromosome 7 is homoeologous w ith wheat chromosome 5 ( Is la m e t a l ,
B a rley
1 9 8 1 ).
44
-
iKir11
12.1
AlCA
HorB
13.7
2 7 .6
..
1H1S3
..
KollfiS1 D2A
6.7
— HorC
3 .7
3.4
CS
A12
■7
D7A
3 0 .9
VG7E9
F8
»p759b
pKSL'32
1 0 .3
H G llB L
p7A71
1 0 .3 .
11.8
AlOB
1 1 .9
iV G llO L
F<
C3b
ap759l
7 .9 I
2 9 .6
'■
IS P l
2 3 .7
6 .7
6 .9
» G lu l
7 .8 I
26.1
i£S74
i l lb l q
10.1
a ADH
1 5 .9
2 6 .6
Chromosome I
BCD298
Chromosome 3
HorD
20.0
7 .8
*PS1430«
VG364b
2 6 .5
2 7 .8
IPGD
- aVGUOS
9.8 _
V564S
BCD265C
1 4 .0
37.5
16.5
18 .7
CS*
21.0
I
-■ VSP
T A086
18.1
■■ 1KAR7HF
1 5 .6
"
IVG178
"
INAR7S
2 9 .5
32.3
. . «PS73«0b
Chromosome 2
BCD265b
T 1VG669, D2
9 .9
■■ COOS88
mR
Chromosome S
■■ PST69
12.0
2 7 .8
PKSU24
4 .9
•HRTH
■ - »G1u2
*KSU21
9 .4
1 2 .9
2 1 .7
~ 1PS7327
-
PS1321
7 .6
16 19 20
2.6
W.7 ..
PKSU44
3 8 .9
HG1026b
5 .0
BCC265*
. . *p337
3 0 .6
"
2 7 .0
"
PS1316, D7B
INAR7L
3 8 .8
Chromosome «
LMWP
1 9 .6
PKSU24S
12.1
O P A ll
Chromosome
6
Figure 7. Map c o n s t r u c t io n o f Dicktoo x Morex cross
Chromosome 7
45
Field survival at Corvallis,
OR (%)
Figure 8. The d is trib u tio n of fie ld survival measured at C orvallis, OR in
1991 fo r Dicktoo x Morex cross.
46
50 --
in 40 -•••
I
30 - -
z 20 +•
PiMrmii RffAitfmi AAi A iikifiii APk ifffAA AA AifAi Aufii Am AiAm
D
10
20
30
40
50
60
70
MT (%)
80
Field survival at Bozeman,
Figure 9. The d is trib u tio n of fie ld survival measured at Bozeman, MT in
1992 fo r Dicktoo x Morex lines.
47
-1 0
—11
LT50 ( C)
Figure 10. The d is trib u tio n of LT50 fo r Dicktoo x Morex lines
48
25 30
40
50
60
70
80
Heading Date under 2 4 h light (days)
Figure 11. The d is trib u tio n of heading date under 24 h lig h t for
Dicktoo x Morex lines.
49
Heading date under 16 h light (days)
Figure 12. The d is trib u tio n of heading date under greenhouse condition
(16 h lig h t) fo r Dicktoo x Morex lines.
50
76V)
60
80
100 120 1 40 1 6 0 180
Frucian content (m g /g d w )
200
220
240
Figure 13. The distribution of fructan content in 1992 for Dicktoo x Morex lines.
51
20 ......
1
2
3
4
5
6
7
8
9
10
Prostrate growth habit
Figure 14. The d is trib u tio n of prostrate growth habit fo r
Dicktoo x Morex line s.
52
Heading Date
V e rn a liz a tio n
in te ra c t
response,
m a tu rity ,
to determ ine w i n t e r vs.
and photoperiod
response genes
sp rin g growth h a b i t .
Follow ing the
example o f Takahashi and Yasuda (1 971) we chose heading d a te under 24 h
lig h t
as
a
c h a r a c te r
t r a n s g r e s s iv e
s eg re g a tio n
under 24 l i g h t
measures
growth
of
ass oc iate d
can
cold
h a b it
to le ra n c e ,
(F ig u r e 11 and 1 2 ) .
growth
and d iscontinuo us
be a t t r i b u t e d
a lle le s ,
d is p la y s t y p i c a l
w ith
or
to
both
tra it
h a b it.
expression
m u ltig e n ic
p a rents
is
s ig n ific a n t
o f heading date
c o n tro l.
may have
expression
The
the
As w ith
c o n tr ib u te d
re s u lt
of
w in te r
e p is ta s is
The accession o f D icktoo t h a t we used as a p a ren t
w i n t e r growth h a b it under f i e l d c o n d itio n s
throughout th e f a l l
the
and w i n t e r and mid-season m a t u r i t y
-
- p ro s tra te
but under the
de sc rib e d t e s t c o n d itio n s i t is o n ly 16 days l a t e r to head than Morex.
It
i s a lr e a d y c l e a r from a s e r ie s o f ongoing experiments in v o lv in g v arious
perm utations o f v e r n a l i z a t i o n , p h o to p e rio d , and ambient te m p e ra tu re , t h a t
th e w i n t e r phenotype observed under f i e l d
c o n d itio n s
i s a t t r i b u t a b l e to
photo period s e n s i t i v i t y r a t h e r than t o a v e r n a l i z a t i o n
e xam ination
of
v e rn a liz a tio n
v a ria tio n
may
s ev e ral
accessions
of
responses from none to
be
a ttrib u ta b le
to
D icktoo
complete
th e
re q u ire m e n t.
r e v e a le d
(d a ta
not
h e te r o g e n e ity
a
range
shown).
of
the
An
of
This
in itia l
germplasm, as documented in the v a r i e t y r e le a s e d e s c r i p t i o n .
The skewness o f
th e
growth h a b i t
frequency histogram
toward the
s p rin g phenotype would lend some support t o the e p i s t a t i c model proposed
by Takahashi
and Yasuda
(1 9 71 ).
According
to
th is
model,
w in te r
vs.
s p rin g growth h a b i t i s determ ined by th e e p i s t a t i c i n t e r a c t i o n o f 3 l o c i :
Sh on chromosome 4;
sh2 on chromosome 7;
and sh3 on chromosome 5.
The
53
a l l e l i c c o n s t i t u t i o n ShShsh2sh2sh3sh3 is re q u ire d f o r expression o f w i n t e r
h a b i t and a l l o th e r a l l e l i c combinations g iv e s prin g growth h a b i t .
While
th e r e is reasonable f i t to a tw o -lo c u s , 3 :1 model (Chi square = 2 .6 1 ; p >
.1 0 0 )
based
D icktoo vs.
on
th e
grouping
of
a ll
phenotypes
those l a t e r than the w i n t e r p a r e n t,
e a rlie r
maturing
than
such a grouping ignores
th e presence o f t r a n s g r e s s iv e segregants on both sides o f th e d i s t r i b u ­
tio n .
Based on th e l e v e l
o f genome s a t u r a t io n t h a t we have achieved to
d a t e , i t would appear t h a t Dicktoo and Morex may c a r r y c o n t r a s t in g a l l e l e s
a t o n ly one o f th e Sh l o c i .
not
d e te c te d
on
re s p e c tiv e ly ).
S i g n i f i c a n t QTL e f f e c t s f o r growth h a b it were
chromosome
4
and
5
(s ite
of
th e
and
Sh3
lo c i,
Large QTL e f f e c t s (LOD > 19) were d e te c te d on chromosome
7, again in the mR-BCD265b i n t e r v a l .
Takahashi and Yasuda (1971) re p o rte d
13.1% re com bination between mR and th e Sh2 lo c u s .
and BCD265b i s
Sh
th e
re g io n
showing th e
la rg e s t
The re g io n between mR
QTL e f f e c t .
Small
QTL
e f f e c t s were d e te c te d in chromosome 2 in th e aNAR7HF-aWG178 i n t e r v a l w ith
th e LOD v a lu e 2 . 4 8 .
fe ll
in
th e
In th e greenhouse, QTL f o r heading d a te t r a i t
same i n t e r v a l
as heading d a te
at
24 h r
lig h t.
als o
The lo g -
l i k e l i h o o d f o r th e heading d a te QTL in mR-BCD265b was 1 1 .3 7 .
Fructan Content o f Crown Tissue
The p a r e n ta l
polymorphism f o r
fru c ta n
c o n te n t,
to g e t h e r w ith
the
wide range o f crown f r u c t a n c o ntent in t h e i r progeny ( F ig u r e 13) provides
a unique o p p o r tu n it y t o s im u ltan e o u s ly assess th e r o l e o f f r u c t a n content
and cold t o l e r a n c e .
The p o p u la tio n d i s t r i b u t i o n
and skewed toward th e Morex phenotype.
6 3 .8
mg/gdw.
The t r a n s g r e s s iv e
h ig h e r than D ic kto o
(137 mg/gdw),
is a p p ro x im a te ly normal
Morex had a f r u c t a n
segregants w ith
fru c ta n
c onte nt o f
s u b s ta n tia lly
in d i c a t e s t h a t both p a re n ts may have
54
c o n tr ib u te d a l l e l e s f o r f r u c t a n accum ulation.
The QTL a n a ly s is re v e a le d s u b s t a n t ia l
LOD p lo t s
fo llo w
s u rv iv a l,
a p p ro xim a te ly
the
LT50, and growth h a b i t .
same
e f f e c t s on chromosome 7 The
p a tte r n
as
those
fo r
fie ld
L o g - l i k e l i h o o d o f th e QTL in the mR-
BCD265b was 5 .0 5 and accounted f o r 20.8% o f the v a r i a t i o n .
P r o s t r a t e Growth H a b it
The d i s t r i b u t i o n
shown in F ig u re 14.
o f p ro s tra te
growth h a b it
in t h i s
p o p u la tio n
Morex is e r e c t w h ile D icktoo is p r o s t r a t e .
tr a n s g r e s s iv e s eg re g a tio n toward D ic k to o , th e w i n t e r b a r l e y .
o f th is
tra it
pKSU24-C3a.
and 2 . 3 6 ,
fe ll
i n t o th r e e i n t e r v a l s ;
ap327-aMSU21,
is
There was
QTL e f f e c t s
ap340a-iPGD, and
L o g - l i k e l i h o o d values o f th e f i r s t two i n t e r v a l s were 2 .3 1
re s p e c tiv e ly .
L o g - l i k e l i h o o d o f th e pKSU24-C3a (Chromosome 7)
was 3 . 3 7 .
From our d a ta , th e mR-BCD265b i n t e r v a l on th e long arm o f chromosome
7 c o n tr ib u te d to QTL e f f e c t s f o r a l l
h a b it.
th e
th e t r a i t s
except p r o s t r a t e growth
M u l t i p l e r e g re s s io n a n a ly s is was performed in t h i s
R2 values
are
shown in Table 8 .
As expected,
growth h a b i t i s th e s m a lle s t among th e t r a i t s .
th e
in te rv a l
and
R2 o f p r o s t r a t e
F i e ld s u r v i v a l a t Bozeman,
MT gave h ig h e s t R2.
The d i f f e r e n c e o f R2 values f o r f i e l d s u r v iv a l between
th e
may
two
lo c a tio n s
c o n d itio n s
and
th e
re s u lt
p r e c is io n
from
of
th e
th e
d iffe re n c e s
e xperim ent.
in
Three
cold
re p lic a tio n s
conducted a t Bozeman may have increased th e p r e c is io n o f f i e l d
measurements.
s u r v iv a l
O b v io u s ly , th e accuracy o f QTL a n a ly s is i s dependent upon
th e accuracy w ith which th e t r a i t s
th e
s e v e r it y
value s
of
two f l a n k i n g
h ig h e r
a s s o c ia tio n
w ith
markers
fru ctan
can be measured.
o f th e
c o n te n t,
in te rv a l,
and f i e l d
C onsidering the R2
th e mR locus
s u rv iv a l
than
has
has
55
BCD265b locus does.
The r e v e r s e is t r u e f o r heading d a te a t . 24 h l i g h t .
Since d i f f e r e n t R2 values between th e two markers were d e te c te d f o r heading
date under 24 h l i g h t and f i e l d
8 ),
means o f the
groups
of
th e
ra tio n
o f the
recombinant
s u r v iv a l
a t Oregon were d e te c te d
two t r a i t s
in d iv id u a ls
were analyzed
in
the
d i f f e r e n c e s between th e two means were observed.
(Ta b le
from the
in te rv a l.
two
Two
fo ld
This suggested t h a t the
r e l a t i o n s h i p between f i e l d s u r v iv a l a t Oregon and heading d a te under 24 h
l i g h t was due to l i n k a g e ,
not p l e i o t r o p y .
c a r r y in g
in
recombinant
type
th is
More markers and i n d iv id u a l
in te rv a l
are
needed
to
re s o lv e
the
q ue stion o f p l e i o t r o p i c e f f e c t .
T a b le 8 .
Regression
c o e ffic ie n ts
w in te r h a r d in e s s parameters
T ra its
mR
Fructan
Oregon s u r v iv a l
Bozeman s u r v iv a l
LT50
Heading d a te 16 h l i g h t
Heading d a te 24 h l i g h t
P r o s t r a t e growth h a b it
0.21
0.31
0.51
0 .2 0
0 .3 2
0 .2 9
0 .1 3
of
th e
power w ith
which
and
BCD265b
BCD265b
(1 9 92 )
m iddle
and
found t h a t
8%
from
a p p ro x im a te ly equal
marker l o c i
th e
ta ils
th e
if
(1 986) and Lander and B o ts te in
we d e te c te d
2% o f th e
ta il
of
w ith
0 .2 3
0 .3 3
0 .6 8
0 .2 5
0.4 1
0 .4 2
0 .1 3
QTL v a r ia n c e
e ffe c ts
s e l e c t i v e genotyping was s m a lle r than w ith random sam pling.
Wyler
lo c i
mR and BCD265b
0 .1 5
0 .1 8
0 .5 6
. 0 .2 0
0 .3 3
0 .3 8
0 .0 4
As was suggested by Lebowitz e t al
(1 9 89 ),
mR
w ith
W e lle r and
i n d i v i d u a l s were s e le c te d a t th e
th e
d is trib u tio n ,
t o t h a t obta ine d w ith
then
random sam pling.
power
was
Since many
used in t h i s map were based on s e l e c t i o n o f phenotypes from
of
th e
fie ld
s u r v iv a l
d is trib u tio n
at
C o rv a llis ,
OR,
we
56
c o n s tru c te d a c o r r e l a t i o n m a tr ix to e s tim a te th e mean e f f e c t s
markers on phenotypes.
technique
to
H i s t o r i c a l l y , t h i s was the most commonly u t i l i z e d
map genes
(L in c o ln and Lander,
o f mapped
c o n tro llin g
q u a n tita tiv e ly
measured
phenotypes
1 9 9 0 ).
Of th e 77 markers mapped in t h i s e xperim ent, 30 markers were mapped
using 30
in d iv id u a ls
p o p u la tio n .
lin k a g e
w h ile
th e
remainder were mapped using
the e n t i r e
Sample s iz e can have a d ra m atic impact on th e accuracy o f a
map,
and th e
p r e c is io n
o f our recom bination
e s tim a te s
markers t h e r e f o r e is v a r i a b l e w i t h i n th e lin k a g e map.
between
F u rth e r,
in te rv a l
a n a ly s is approaches to QTL i d e n t i f i c a t i o n u t i l i z e d a ta o n ly when genotypic
d a ta i s a v a i l a b l e on both sides o f th e i n t e r v a l .
I f a marker in which a
complete d a ta s e t was a v a i l a b l e was a d ja c e n t t o a marker c o n ta in in g only 30
genotype
d a ta p o in ts ,
then
th e
QTL a n a ly s is
would
u tiliz e
o n ly
the
30
phenotypic values corresponding to th e i n t e r s e c t i o n o f th e two genotype
d a ta se ts .
As an example,
seven from KSU24-c3a.
th is
occurred
in
the
in te rv a l
on chromosome
The accuracy o f th e QTL a n a ly s is as implemented by
Mapmaker-QTL i s q u e s tio n a b le in in s tan ce s o f t h i s ty p e .
Simple
c o rre la tio n
and
r e g re s s io n
a n a ly s is
p ro v id es
a
simple
approach to QTL a n a ly s is which u t i l i z e s a l l o f th e d a ta a v a i l a b l e f o r each
mapped m arker.
When T a b le 9 is c o n tra s te d w ith th e LoD score estim a te s o f
Mapmaker-QTL, a l l o f th e s i g n i f i c a n t i n t e r a c t i o n s i d e n t i f i e d by MapmakerQTL a re s i m i l a r l y
id e n tifie d
by sim ple c o r r e l a t i o n
a n a ly s is .
However,
Mapmaker-QTL f a i l e d t o i d e n t i f y i n t e r a c t i o n s d e te c te d through th e use o f
c o rre la tio n
a n a l y s is .
When d a ta s e ts a re le s s than p e r f e c t ,
a n a ly s is may p ro v id e more in fo r m a tio n than i n t e r v a l
c o rre la tio n
a n a l y s is .
A s e p a ra te q u e stio n concerns th e use o f s e l e c t i v e genotyping as a
57
c o s t - e f f e c t i v e approach f o r th e i d e n t i f i c a t i o n
o f QTL.
address
to
th is
id e n tifie d
q ue stion
w ith
th is
d a ta s e t
is
using complete d a ta s e ts would l i k e w is e
ask
The best way to
w hether
have been
the
QTL
id e n tifie d
w ith s e l e c t i v e ge notyping.
When c o r r e l a t i o n
a n a ly s is
o f marker mR,
KSU24,
and BCD265b were
conducted on th e 30 l i n e s i d e n t i f i e d by s e l e c t i v e genotyping, s i g n i f i c a n t
c o r r e l a t i o n s were found f o r a l l t r a i t s ,
and the c o r r e l a t i o n s were o f the
same magnitude as were those i d e n t i f i e d using 100 progeny.
s ta tis tic a l
The l e v e l s o f
s i g n i f i c a n c e f o r LT50 and mR, and LT50 and BCD265b were reduced
t o 5% in s te a d o f 1%.
le s s com plete,
but i t
More complete d a ta s e ts are always t o be p r e f e r r e d to
appears in t h i s case t h a t a more complete d a ta s e t
would r e s u l t in th e conclusions we o b ta in e d .
T a b le 9 .
S i g n i f i c a n t l e v e l s o f a s s o c ia tio n a among markers and w in te rh a rd in e s s param eters.
Markers
Fructan
Oregon
s u r v iv a l
Bozeman
s u r v iv a l
P rostrate
growth
Heading
d a te (1 6 )
Heading
d a te (2 4 )
LT50
CS
aMSU21
aPST327
PST59
CD0588
aWGHOS
aNAR7HnF
aWG178
aPST340b
F8
F4
aGlu2
BCD265a
WG1026b
D7B
WSP
HorD
BCD265c
aPST340a
iPGD
LMWP
aNAR7L
OPAll
pTA71
WG364b
BCD298
BCD265b
pKSU24
pKSU32
C3a
*
NS
NS
NS
*
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
**
NS .
**
NS
NS
*
NS
NS
NS
NS
*
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
*
NS
NS
NS
NS
NS
NS
NS
NS
NS
*
NS
NA
NS
NS
NS
*
NS
*
NS
NS
NS
NS
NS
NS
NS
**
**
*
**
NS
NS
NS
NS
*
NS
NS
NS
NS
NS
NS
NS
NS
*
NS
NS
NS
*
NS
NS
NS
**
*
**
NS
*
NS
*
*
*
**
*
**
NS
NS
NS
NS
NS
*
**
**
**
**
NS
**
**
NS
NS
NS
NS
NS
*
**
NS
**
NS
**
NS
NS
NS
NS
NS
*
**
NS
**
**
NS
NS
NS
NS
NS
NS
**
NS
NS
NS '
NS
*
*
**
NS
NS
NS
NS
NS
NS
NS
*
NS
NS
NS
NS
NS
NS
NS
*
NS
NS
NS
**
**
**
**
-
NS
NS
NS
**
**
**
**
NS
NS
NS
NS
NS
NS
*
NS
**
NS
NS
**
**
NS
*
T able 9 (c o n tin u e d )
Markers
Fructan
Oregon
s u r v iv a l
Bozeman
s u r v iv a l
P ro strate
growth
Heading
d a te (1 6 )
Heading
d a te (2 4 )
mS
mR
WGI 026a
PST321
*
**
**
**
**
**
**
**
**
NS
**
**
NS
NS
NS
NS
*
NS
NS
NS
NS
* NS
*
*
**
**>
**
*
**
**
**
*
**
Cl
BCD175
NS -
NS
**
LT50
NS
**
**
NS
*
NS
NS = S t a t i s t i c a l l y non s i g n i f i c a n t .
*
= S t a t i s t i c a l l y s i g n i f i c a n t a t 95% l e v e l .
* * = S t a t i s t i c a l l y s i g n i f i c a n t a t 99% l e v e l .
CJI
CO
.
I
^
60
CHAPTER 5
SUMMARY
This study provides the f i r s t reasonably complete d a ta s e t designed
to p e rm it e v a lu a tio n o f the g e n e tic c o n tr o l
w i n t e r hardiness
in b a r le y .
o f p h y s io lo g ic a l
aspects o f
The dependence o f cold t o le r a n c e on crown
f r u c t a n c o n te n t, heading d a te , growth h a b it and LT50Was i n v e s t i g a t e d .
lo c a tio n
The
o f genes m odifying expression o f each o f these c h a ra c te rs was
id e n tifie d ,
and
th e
magnitude
of
th e
e ffe c ts
of
a lle lic
v a ria tio n
e s tim a te d .
Crown f r u c t a n c o n te n t appeared to be a ss o c iate d w ith w i n t e r s u r v iv a l
and cold t o l e r a n c e .
d ire c tly
Growth h a b it and heading d a te appeared to be le s s
a s s o c ia te d w ith w i n t e r
s u rv iv a l,
although li n k a g e
among genes
c o n t r o l l i n g each o f these c h a r a c te r s may have made s e l e c t i o n f o r useful
recombinants d i f f i c u l t .
A n a ly s is o f th e p e rc e n t o f l i n e s
demonstrated th e p o t e n t i a l
d iffic u ltie s
use o f t a i l e d d i s t r i b u t i o n a n a l y s is .
th e b est e s t im a t o r o f w in te r h a r d in e s s ,
in t a i l s
of tr a it
d is trib u tio n s
which can be g e nerated from the
Long term w i n t e r s u r v i v a l
and one o r two e xp e rim e n ts ' d a ta
can le a d t o a poor assignment o f l i n e s t o d i s t r i b u t i o n t a i l s .
w h ile
th e
Bozeman w i n t e r o f
which showed r e l a t i v e l y
is s t i l l
1992 provided good s e p a r a tio n
F u r th e r ,
among l i n e s
good w i n t e r s u r v i v a l , h a l f o f th e l i n e s
experim ent showed no s u r v i v a l .
M u l t i p l e y e a r and l o c a t i o n
in th e
experiments
would a id in th e development o f a c c u ra te phenotypic d e s c r i p t i o n o f these
lin e s .
T h is p r o j e c t d id pro v id e support f o r th e c o n te n tio n t h a t recombinant
61
inbred
lin e s ,
e s p e c ia lly
doubled
h a p lo id s ,
are
u n iq u e ly
s u ite d
to
q u a n t i t a t i v e t r a i t a n a ly s is when the t r a i t under a n a ly s is is d i f f i c u l t to
measure.
Only recombinant inbreds pro v id e mapping resources which p e rm it
th e development o f r e p l i c a t e d experiments a t m u l t i p l e lo c a t io n s and over
m u ltip le years.
Many RFLP maps have been c o n stru cted
a n a l y s is .
In t h i s
p ro je c t,
s o le ly
using
Southern
we used polymerase chain r e a c t i o n
f a c i l i t a t e map c o n s tr u c tio n in t h i s c ro s s .
b lo t
(PCR) to
The prim er sequences f o r PCR
were generated from s e q u e n c e -ta g g e d -s ite s o f the clones from the b a r le y
l i t e r a t u r e and from th e b a r le y genome mapping p r o j e c t .
We found t h a t the
frequency w ith which we d e te c te d polymorphisms using PCR was s i m i l a r to
Southern b l o t a n a l y s is .
Seventy-seven markers
lin k a g e groups.
chromosomal
in
th is
study were r e s o lv e d
to
e ig h t
Seven lin k a g e groups were known from p r i o r mapping o f the
lo c a tio n
b a r le y chromosome
w ill
used
6
o f m arkers.
One lin k a g e
) may be i n a c c u r a t e l y l o c a t e d .
group
(th a t
assigned to
F u r th e r use o f markers
c l a r i f y t h i s problem.
QTL e f f e c t s o f a l l w i n t e r - h a r d i n e s s -a s s o c ia te d param eters (e x c e p tin g
p r o s t r a t e growth h a b i t )
s in g le
20cM i n t e r v a l
measured in
th is
study were found t o map t o
on th e long arm o f chromosome 7.
a
The mR-BCD265b
i n t e r v a l on chromosome 7 was th e l o c a t i o n o f genes m od ify in g these t r a i t s .
When recombinants w i t h i n t h i s i n t e r v a l were c a r e f u l l y e v a lu a te d , we found
t h a t a gene m odifying s u r v iv a l
a t Oregon and a gene m o d ify in g flo w e r in g
d a te under 24 hours' i l l u m i n a t i o n occupied l o c a t io n s a t d i f f e r e n t ends o f
th e i n t e r v a l .
This prov ides to our knowledge th e f i r s t t e s t o f lin k a g e
versus p l e i o t r o p y f o r QTL r e s i d i n g in a common i n t e r v a l .
I t a ls o suggests
62
th a t
recombinants
s u r v iv a l
can
be s e le c te d
which
break th e
a s s o c ia tio n
between
and l a t e fl o w e r in g .
Three l o c i were found to modify crown fr u c ta n c o n te n t.
The l a r g e s t
e f f e c t mapped to the i n t e r v a l on the long arm o f chromosome 7, w h ile two
l o c i w ith le s s dra m atic e f f e c t s were mapped to the s h o rt arm o f chromosome
7 and th e
long
arm o f chromosome "5.
The f r u c ta n
QTL on chromosome 5
appeared to cosegregate w ith w i n t e r s u r v iv a l measured a t Oregon in 1991.
The chromosome 7 f r u c t a n QTL showed no d e t e c t a b l e i n t e r a c t i o n w ith w i n t e r
s u r v iv a l a t e i t h e r l o c a t i o n .
among these
lin e s
tru ly a c r itic a l
A to tal
m o d ifie d
accounted
More a c c u ra te measurement o f w i n t e r s u r v iv a l
may help determ ine whether crown f r u c t a n
content
is
f a c t o r in d e te rm in in g w in te r h a r d in e s s .
o f th r e e mapped i n t e r v a l s were found to c o n ta in QTL which
growth
fo r
h a b it.
1 5 .2
and
In te rv a ls
1 5 .7
p e rcent
on
of
chromosome
th e
2
and
v a ria tio n
chromosome
fo r
th is
5
tra it.
M ajor e f f e c t s on heading d a te were lo c a te d to th e chromosome 7 i n t e r v a l
and to an i n t e r v a l on chromosome 2 which accounted f o r a p p ro xim a te ly 13%
o f th e v a r i a t i o n f o r th e c h a r a c te r .
These
accompanied
s e le c tio n
d a ta
by
is
suggest
e a rlie r
perform ed.
th a t
improvement
heading
They
and
more
a ls o
in
erect
w in te r
growth
a cc entuate
th e
s u r v iv a l
if
may
be
a p p r o p r ia te
importance
of
a p p r o p r ia t e s e l e c t i o n o f p arents and a c c u ra te a n a ly s is o f th e phenotypic
v a lu e o f l i n e s when perform ing a QTL exp e rim en t.
63
REFERENCES CITED
A l l a r d , R.W. 1988. G enetic changes ass o c iate d w ith th e e v o lu t io n o f
adaptedness in c u l t i v a t e d p la n ts and t h e i r w ild p r o g e n ito r s . J . o f
H e r e d it y 7 9 :2 2 5 -2 3 8 .
Barnham, R .W ., and Rasmusson, D.C. 1981. In h e r it a n c e
. response in b a r le y . Crop Sci 2 1 :4 5 4 -4 5 6 .
of
photoperiod
Beckmann,
J .S ., S e lle r,
M.
1983.
R e s tric tio n
fragment
le n g th
polymorphisms in g e n e tic improvement: m ethodologies, mapping and
c o s ts . Theor Appl Genet 6 7 : 3 5 - 4 3 .
Beckmann, J . S . , 1988. O lig o n u c le o tid e polymorphisms: A new to o l f o r
genomic g e n e t ic s . Bio/Technology 6 :1 0 6 1 -1 0 6 3 .
B la k e ,
T . K . , U l l r i c h , S . E . , and N i I an, R.A. 1982. Mapping o f the Hor-3
locus encoding D hordein in b a r le y . Theor Appl Genet 6 3 : 3 6 7 -3 7 1 .
B o t s t e i n , D . , W h ite , R . L . , S k o ln ic k , M ., D a v is , R.W. 1980. C ons tru c tio n o f
a
g e n e t ic
map
in
man using . r e s t r i c t i o n
fragm ent
le n g th
polymorphisms. Am J Hum Genet 3 2 : 3 1 4 -3 3 1 .
C a ttiv e lli,
L .,
and
B a rte ls ,
D.
1990.
M o le c u la r
c h a r a c t e r i z a t i o n o f c o l d - r e g u la t e d genes in b a r l e y .
9 3 :1 5 0 4 -1 5 1 0 .
c lo n in g
and
P la n t physiol
C h a t t e r t o n , N . J . , H a r r is o n , P . A . , T h o r n le y , W .R., and B e n n e tt, J .H . 1988.
New methods o f a n a ly z in g f r u c t a n in cool season p l a n t s . P la n t
Physiol 8 6 : 1 2 .
C h a t t e r t o n , N . J . , H a r r is o n , P . A . , B e n n e tt, J . H . , and Asay, iK.H. 1989.
Charbohydrate p a r t i t i o n i n g in 185 accessions o f gramineae grown
under warm and cold te m p e ra tu re s . J P la n t Physiol 1 3 4 :1 6 9 -1 7 9 .
Chen,
F . , and Hayes, P.M. 1989. A comparison o f H o r d e u m b u l b o s u m m ediated h a p lo id p roduction e f f i c i e n c y in b a r le y using i n v i t r o
f l o r e t and t i l l e r c u l t u r e . Theor AppT Genet 7 7 : 7 0 1 -7 0 4 .
C o le ,
C . G . , G o o d fe llow, P . N . , Bobrow, M ., and B e n tle y , D.R. 1991.
G en era tion o f novel sequence tagged s i t e s (STSs) from d i s c r e t e
chromosomal re g io n using Alu-PCR. Genomics 1 0 :8 1 6 -8 2 6 .
D o ll,
H . , Hahr, V . , and Sogaard, B. 1989. R e la t io n s h ip between v e r n a l ­
i z a t i o n re quirem ent and w i n t e r hardiness in doubled h aploids o f
b a r l e y . Euphytica 4 2 :2 0 9 -2 1 3 .
DzO v id io ,
R . , T a n z a r e l l a, O .A .,
and Proceddu, E.
1990.
Rapid and
e f f i c i e n t d e t e c t io n o f g e n e t ic polymorphisms in wheat through
a m p l i f i c a t i o n by polymerase chain r e a c t i o n . P la n t Mol Biol 1 5:16 9 171.
J
64
Dunn,
M. A . , Hughes, M .A ., Zhang, L . , Pearce, R . S . , Q u ig le y , A . S . , and
Jack, P .L . 1991. N u c le o tid e sequence and m o le c u la r a n a ly s is o f the
low te m peratu re induced c e re a l gene, BLT4. Mol Gen Genet 2 2 9 :3 8 9 394.
Fe in b erg , A . P . , and Vogel s t e i n , B. 1984. A technique f o r r a d i o l a b e l l i n g
DNA r e s t r i c t i o n endonuclease fragments to high s p e c i f i c a c t i v i t y .
Anal Biochem 137: 266 -2 6 7.
Forde,
B .G ., Heyworth, A . , PywelT, J . , M i f l i n , B .J . 1985. N u c le o tid e
sequence o f a B l-h o r d e in gene and th e i d e n t i f i c a t i o n o f p o s s ib le
upstream r e g u l a t o r y elements in endosperm storage p r o t e i n genes from
b a r le y , wheat and maize. N u c le ic Acids Res 1 3 :7 3 2 7 -7 3 3 9 .
Fow ler, D . B . , and Gusta, L .V . 1979. S e le c tio n f o r w in te r h a r d in e s s in
wheat. I . I d e n t i f i c a t i o n o f ge noty pic v a r i a b i l i t y . Crop Sci 19: 769-
Heun, M ., Kennedy, A . E . , Anderson J . A . , L a p ita n , N . L . V . , S o r r e l l s , M .E .,
and T a n k s le y , S.D. 1991. C o n s tru c tio n o f a r e s t r i c t i o n fragment
le n g th polymorphism map in b a r le y .
Genome 3 4 : 4 3 7 -4 4 7 .
G y lle n s t e n , U . B . , and E r l i c h , H.A. 1988. G eneration o f s in g le stranded DNA
by th e polymerase chain r e a c t i o n and i t s a p p l i c a t i o n to d i r e c t
sequencing o f th e HLA-DQalpha lo c u s . Proc N atl Acad Sci USA 8 5 :7 6 5 2 7656.
H ig u c h i, R . , Von B e ro ld in g e n , C . H . , Sensabaugh, G . F . , and E r l i c h ,
1988. DNA ty p in g from s in g le h a i r s . Nature 3 3 2 :5 4 3 -5 4 6 .
H.A.
H ig u c h i, G ., and Ochmah, H. 1989. P roduction o f s in g le stranded DNA
te m p la te s by exonuclease d ig e s t io n fo llo w in g th e polymerase chain
r e a c t i o n . N u c le ic Aci Res 17:58 6 5.
In n is ,
M .A .,
Myambo, K . B . , G e lfa n d , D . H . ,
Brow, M.A.D.
1988. DNA
sequencing w ith Thermus a q u a tic s DNA polymerase,
and d i r e c t
sequencing o f PCR a m p li f ie d DNA. Proc N atl Acad Sci USA 8 5 :9 4 3 6 9440.
Islam A . K . R . M . , Sheperd, K.W ., and Sparrow, D .H .B . 1981. I s o l a t i o n and
c h a r a c t e r i z a t i o n o f euplasmic w h e a t-b a r le y chromosome a d d itio n
l i n e s . H e r e d it y 4 6 :1 6 1 -1 7 4 { H o r d e u m v u l g a r e ) . Genome 3 4 : 4 3 7 -4 4 7 .
K o la r , S . , Hayes, P .M ., Chen, T . , and Linderman, R. 1991. Genotypic
v a r i a t i o n f o r c o ld t o le r a n c e in w i n t e r and f a c u l t a t i v e b a r le y . Crop
Sci 3 9 : 1 1 4 9 -1 1 5 2 .
Kusukawa, N-.T., Uem ori. T . , Asada, K . , and K a to , I . 1990. Rapid and
r e l i a b l e p ro to c o l f o r d i r e c t sequencing o f m a t e r i a l a m p li f ie d by
PCR. B iotechnique 6 6 : 2 - 6 .
Lander, E . S . , and B o t s t e i n , D. 1989. Mapping Mendelian f a c t o r s u n d e rlyin g
65
, q u a n tita tiv e t r a i t s
using RFLP lin k a g e maps. G enetics 1 2 1 :1 8 5 -1 9 9 .
Lander, E . S . , and Green, P. 1991. Counting a lg o rith m s f o r l i n k a g e : c o r r e c ­
t i o n to Morton and C o l l i n s . Ann Hum Genet 5 5 : 3 3 - 3 8 .
L i , H . , G y lle n s t e n , U . B . , C u i, X . , S a i k i , R . K ., E r l i c h , H . A . , and Arnheim,
N. 1988. A m p l i f i c a t i o n and a n a ly s is o f DNA sequences in s in g le human
sperm and d i p l o i d c e l l s . Nature 3 3 5 :4 1 4 -4 1 7 .
L i t t , M ., and L u ty , J .A . 1989. A h y p e r v a r ia b le m i c r o s a t e l l i t e r e v e a le d by
in v i t r o a m p l i f i c a t i o n o f a d i n u c le o t i d e re p e a t w i t h i n the c a rd ia c
muscle a c t i n gene. Am J Hum Genet 4 4 : 3 9 7 -4 0 1 .
L iu ,
B.H. and Knapp, S .J .
1990. GMENDEL: A program f o r Mendelian
s e g re g a tio n and lin k a g e a n a ly s is o f i n d i v i d u a l o r m u l t i p l e progeny
p o p u la tio n s using l o g - l i k e l i h o o d r a t i o s . J H e r e d it y 8 1 :4 0 7 .
L iv in g s to n , D . P . , O l ie n , C .R ., and Freed, R.D. 1989. Sugar composition and
f r e e z i n g t o le r a n c e in b a r le y crowns a t v a ry in g c arb ohydrate l e v e l s .
Crop Sci 2 9 :1 2 6 6 -1 2 7 0 .
L iv in g s to n , D.P . 1991. N o n s tru c tu ra l carbohydrate accum ulation in w i n t e r
o a t crowns b e fo re and du rin g cold hardening. Crop Sci 3 1 :7 5 1 -7 5 5 .
L i v i t t , J . 1980. Responses o f p la n ts
Academic Press, New York.
to
environm ental
stre ss .
2nd ed.
Mayers,
R .M ., M a n i a t i s , I . ,
and Lerman, L .S .
1987. D e te c tio n
l o c a l i z a t i o n o f s in g le base changes by d e n a tu rin g g r a d ie n t
e le c t r o p h o r e s is . Meth Enzymol 1 5 5 :5 0 1 -5 2 7 .
and
gel
M c In ty r e , B . L . , Chen, T . H . H . , and M e d e rick, M.F. 1988. P h y s io lo g ic a l
t r a i t s a s s o c ia te d w ith s u r v iv a l o f w i n t e r wheat and w i n t e r t r i t i c a l e
in A l b e r t a . Can J P la n t Sci 6 8 : 3 6 1 -3 6 6 .
Mull i s , K . B . , and Faloona, F. 1987. S p e c i f i c s y n th es is o f DNA in v i t r o v i a
a
polymerase chain r e a c t i o n . Meth Enzymol 1 5 5 :3 3 5 -3 5 0 .
N i e ls e n , G ., and Johansen, H.B. 1986. Proposal f o r th e i d e n t i f i c a t i o n o f
b a r le y v a r i e t i e s based on th e genotypes f o r 2 hordein and 39
isoenzyme l o c i o f 47 r e fe re n c e v a r i e t i e s . Euphytica 3 5 :7 1 7 -7 2 8 .
O lie n ,
C . R . , and L e s t e r , G.E. 1985. F reeze-induced
carbohydrates o f r y e . Crop Sci 2 5 :2 8 8 -2 9 0 .
changes
in
s o lu b le
O l ie n , C.R. 1978. A n a ly s is o f f r e e z in g s tr e s s and p l a n t response. In P.H.
Li and A. Sakai ( e d . ) P la n t c o ld hardiness and f r e e z i n g s t r e s s ,
mechanisms and crop i m p l i c a t i o n . Academic Press, New York.
Olson, M ., Hood, L . , C a n to r, C . , and D o s t s t e i n, D. 1989. A common language
f o r p h y sic a l mapping o f th e human genome. Science 2 54 :1 4 3 4 -1 4 3 5 .
4
66
P o llo c k , C .J . 1986. Fructans and th e metabolism o f sucrose
p l a n t s . New Phytol 1 0 4 :1 -2 4 .
in v a s c u la r
Pomeroy, M . K . , and Fow ler, D.B. 1973. Use o f l e t h a l dose tem perature
e s tim a te s as in d ic e s o f f r o s t t o le r a n c e f o r wheat cold a cclim ated
under n a tu r a l and c o n t r o l l e d
environm ent. Can J P la n t Sci 5 3 :4 8 9 494.
P o n t i s , H.G.
1989. Fructan in cold s t r e s s . J P la n t Physiol
1 3 4 :1 4 8 -1 5 0 .
P o n t is , H . G . , and CampiH o , E.D. 1985. F ru c ta n s . pp 2 0 5 -2 0 7 . In : Dey,
P .M ., and Dixon, R.A. (eds) B iochem istry o f s torage chabohydrate in
green p l a n t s . Academic Press, New York.
Reed, K. C. , and Mann, D.A. 1985. Rapid t r a n s f e r o f DNA from agarose g e ls
to nylon membranes. N u c le ic Acids Res 1 3 :7 2 0 7 -7 2 2 1 .
R e i d e l , G . E . , Swanberg, S . L . , Kuranda, K . D . , M a rq u e tte , K . , LaPan, P . ,
Bledsoe, P . , K e n n e d y ,.A ., and L in , B.Y. 1990. D e na turing gradein gel
e le c t r o p h o r e s is i d e n t i f i e s genomic DNA polymorphism w ith high
frequency in m aize. Theor Appl Genet 8 0 : 1 - 1 0 .
Rhode,
C . R . , and Pu!ham, C .F . 1960. H e r i t a b i l i t y e s tim a te s o f w i n t e r
hardiness in w i n t e r b a r le y determ ined by th e standard u n i t method o f
re g r e s s io n a n a l y s is . Agron J 5 2 :5 8 4 -5 8 6 .
R oberts, E . H . , Summerfie l d , R . J . , Cooper, J . P . , and E l l i s , R.H. 1988.
Environmental c o n tr o l o f flo w e r in g in b a r le y . I . Photoperiod l i m i t s
t o lo n g -d a y responses, photoperiod i n s e n s i t i v e phases, and e f f e c t s
o f low te m p e ratu re and s h o rt day v e r n a l i z a t i o n . 1988. Ann Botany
1 2 7 -1 4 4.
R oberts, D.W.A. 1990. I d e n t i f i c a t i o n o f l o c i on chromosome BA o f wheat
in v o lv e d in c o n tr o l o f c old h a rd in e s s , v e r n a l i z a t i o n , l e a f le n g th ,
r o s e t t e growth h a b i t , and h e ig h t o f hardened p l a n t s . Genome 3 3 :2 4 7 259.
R o d r ig u e z -P a le n z u e la , P . , P i n t o r - T o r o , J . , ' Carbonero, P . , and G a rc ia Olmedo,
F.
1988.
N u c le o tid e
sequence and endo s p erm -s p e cific
e xpre s sion o f th e s t r u t u r e a l gene f o r th e t o x i n a - h o r d o th io n in in
b a r le y (Hordeum v u lg a re L ) . Gene 7 0 : 2 7 1 -2 8 1 .
S a ik i,
R . K . , S c a r f , S . , Faloona, F . , Mull i s , K . B . , Horn, G . T . , E r l i c h ,
H .A .T and Arnheim, N. 1985. Enzymatic a m p l i f i c a t i o n o f b e ta -g lo b in
genomic sequences and r e s t r i c t i o n s i t e a n a ly s is f o r d iagnosis o f
s i c k l e - c e l l anemia. Science 2 3 0 :1 3 5 0 -1 3 5 4 .
S a ik i,
R . K . , Bugawan, T . L . , Horn, G.T.., Mull i s , and E r l i c h , H.A. 1986.
A n a ly s is o f e n z y m a t ic a lly a m p li f ie d , - g l o b i n and HLA-DQn DNA w ith
a l l e l e - s p e c i f i c o lig o n u c le o t i d e probes. Nature 3 2 4 :1 6 3 -1 6 4 .
J
67
Sanger, F . , N ic k le n , S . , and Coulson, A.R. 1977. DNA sequencing w ith chain
t e r m in a t in g i n h i b i t o r s . Proc N a tl Acad Sci USA 7 4 :5 4 6 3 -5 4 6 7 .
Schdn, C . C . , Hayes, P .M ., B lake, T . K . , and Knapp, S .J . 1991. Gametophytic
s e l e c t i o n in a w i n t e r x s p rin g b a r le y cross. Genome 3 4 :9 1 8 -9 2 2 .
S hin, J . S . , Chao, S . , Corpuz, L . , and B la ke , T .K . 1990. A p a r t i a l map o f
th e
b a r le y
genome
i n c o r p o r a t in g
re s tric tio n
fragment
le n g th
polymorphism, polymerase chain r e a c t i o n , isozyme, and morphological
marker l o c i . Genome 3 3 :8 0 3 -8 1 0 .
S k o ln ic k , M . H . , and W allance, R.B. 1988. Simultaneous a n a ly s is o f m u l t i p l e
polymorphic l o c i using a m p li f ie d sequence polymorphism (ASPs).
Genomics 2 :2 7 3 -2 7 9 .
. S tu s h n o f f , C . , Fow ler, B ., and B r u e le - B a b e l, A. 1984. Breeding and
s e l e c t i o n f o r r e s is t a n c e to low te m p e ra tu re . I n : P.B. Vose, S.G.
B l i x t ( e d s . ) Crop b re ed in g , a contemporary b a s is . Pergamon Press,
O x fo r d .
S utk a, J . and Snape, J.W. 1989. Location o f a gene f o r f r o s t r e s is ta n c e on
chromosome 5A o f wheat. Euphytica 4 2 : 4 1 -4 4 .
S u zu k i, M. , and Nass, H.G. 1988. Fructan in w i n t e r w heat, t r i t i c a l e , and
f a l l ry e c u l t i v a r s o f v a ry in g c old h a rd in e s s . Can J Bot 6 6 :1 7 2 3 1728.
T a k a h a s h i, R. and Yasuda, S. 1969. G enetics o f e a r l i n e s s and growth h a b i t
in b a r le y . I n . B a r le y G enetics I I . (R. N i l a n , e d . ) Proc o f th e Second
I n t l B a rle y Genet Symp. Pullman, WA, USA.
T a u tz , D. 1989. H y p e r v a r i a b i l i t y o f sim ple sequences as a general sources
f o r polymorphic DNA m arkers. N u c le ic Acids Res 1 7 :6 4 6 3 -6 4 7 1 .
Thomsashow, M.F. 19,90. M o le c u la r g e n e tic s o f cold a c c lim a t io n in h ig h e r
p la n ts Adv Genet 2 8 : 9 9 -1 3 1 .
Tragoonrung, S . , Hayes, P . M , , and B ro ic h , S . l .
1990. N e a r - i n f r a r e d
r e f l e c t a n c e e s tim a te s o f g r a in p r o t e i n and m a lt e x t r a c t in h i l l and
row p l o t e v a lu a tio n s o f sp rin g m a ltin g b a r le y . Can J P la n t Sci
70:71 -7 8 .
Tragoonrung, S . , K anazin,
F a c i l i t a t e d PCR f o r
pre ss .
T ric k ,
V . , Hayes, P . M . , and B la k e , T .K . 1992. STS
b a r le y genome mapping. Theor Appl Genet In
M ., Dennis, E . S . ,
Edwards, K . J . K . , and Peacock, W.J. 1988.
M o le c u la r a n a ly s is o f th e a lcohol dehydrogenase gene f a m ily o f
b a r l e y . P la n t Mol B iol 1 1 :1 4 7 -1 6 0 .
Weber, J . L . , and May, P .E . 1989. Abundant c la s s o f human DNA polymorphisms
which can be typed using th e polymerase chain r e a c t i o n . Am J Hum
68
Genet 4 4 : 3 8 8 -3 9 6 .
Weining, S . , and Langridge, P. 1991. I d e n t i f i c a t i o n and mapping o f
polymorphisms
in . c e re a l
based
on
the
polymerase
chain
r e a c t i o n . Theor Appl Genet 8 2 : 2 0 9 -2 1 6 .
W e lle r,
J.I.,
and W yler,
A.
1992.
Power o f
d iffe re n t
sampling
s t r a t e g i e s to d e t e c t q u a n t i t a t i v e t r a i t l o c i v a r ia n c e e f f e c t s . Theor
Appl Genet 8 3 : 5 8 2 -5 8 8 .
W i l lia m s , J . G . K ; , K u b e lik , A . R . K . , L iv a k , J . L . , R a f a l s l a , J . A . , and
Tingey* S. V. 1990. DNA polymorphisms a m p lif ie d by a r b i t r a r y primers
are useful as g e n e t ic m arkers. N u c le ic Acids Res 1 8 :6 5 3 1 -6 5 3 5 .
Wong,
C . , Dowling, C . E . , S a i k i , R. K . , H ig u c h i, R. G. , E r l i c h , H. A. , and
K a za zia n , H. H. 1987. C h a r a t e r i z a t i o n o f b e ta -th a la s s a e m ia mutations
using d i r e c t genomic sequencing o f a m p lif ie d s in g le copy DNA. Nature
3 3 0 :3 8 4 -3 8 6 .