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Theor Appl Genet (1995) 90:74 0-74 5 9 Springer-Verlag 1995
R . M . D . K o e b n e r
Generat ion of P CR -based m arkers for the detect ion of rye chromat in
in a wheat background
Received: 4 Ma y 1994 / Accepted: 22 September 1994
A b s t r a c t O l i g o n u c l e o t i d e p r i m e r s w e r e d e v e l o p e d t o d e -
t e c t t h e p r e s e n c e o f f o u r r y e s e q u e n c e s u s i n g a P C R a s s a y.
T h e s e a s s a y s g i v e a r y e - s p e c i f i c s i g n a l f r o m w h e a t D N A
t e m p l a t e w h i c h c o n t a i n s v a r i o u s r y e c h r o m o s o m e s o r c h r o -
m o s o m e s e g m e n t s . T h e s e q u e n c e s i d e n t i f i e d w e r e a s s o -
c i a t e d w i t h t h e n u c l e o l a r o r g a n i s e r r e g i o n , t h e 5 S - R r n a - R 1
l o c u s , t h e t e l o m e r e , a n d a w i d e l y d i s p e r s e d , r y e - s p e c i f i c
r e p e t i t iv e e l e m e n t R i s - 1. T h e p r i m e r s a m p l i f i e d f r o m t h e
w e l l - e s t a b l i s h e d l o c i N o r - R 1 a n d 5 S - R r n a - R 1 o n r y e c h r o -
m o s o m e a r m 1 R S , a n d a l s o l o c a t e d a 5 s - R r n a l o c u s o n
c h r o m o s o m e 3 R . T h e t e l o m e r e - a s s o c i a t e d s e q u e n c e w a s
p r e s e n t o n e v e r y r y e c h r o m o s o m e , a n d w a s a l s o p r e s e n t ,
a t a l o w c o p y n u m b e r , i n b o th w h e a t a n d b a r l e y . T h e s e a s -
s a y s w i l l b e p a r t i c u l a r l y u s e f u l f o r i n t r o g r e s s i o n p r o -
g r a m m e s a i m e d a t r e d u c i n g t h e ry e c o n t e n t o f t he 1 B L . 1 R S
w h e a t - r y e t ra n s l o c a t io n . W h e n m u l t i p le x e d , t h e p r im e r s
w i l l e n a b l e a r a p id , s i m u l t a n e o u s a s s a y f o r a n u m b e r o f d i s -
t i n ct r y e l o c i, w h i c h c a n b e d e r i v e d f r o m a s m a l l p o r t i o n
o f m a t u r e e n d o s p e r m t is s u e.
K e y w o r d s P C R 9 W h e a t 9 R y e 9 N O R 9 5 S - r D N A
T e l o m e r e
Introduction
T h e d e v e l o p m e n t o f m o l e c u l a r m a r k e r s h a s o p e n e d u p n u -m e r o u s p o s s i b i l i t i e s f o r a p p l i c a t i o n t o p l a n t b r e e d i n g .
M u c h e f f o r t i s c u r re n t l y b e i n g e x p e n d e d o n t h e c r e a ti o n o f
R F L P - b a s e d g e n e t i c m a p s i n m a n y c r o p s p e c i e s , i n c l u d in g
w h e a t a n d r y e , bu t t h e u se o f R F L P t e c h n o l o g y i n a re a l
b r e e d i n g e n v i r o n m e n t i s re s t r ic t e d b o t h b y t h e c o m p l e x i t y
a n d t h e c o s t o f t h e p r o c e d u r e s , p a r t i c u l a r l y i n w h e a t a n d
b a r l e y, w h e r e n o n - r a d i o a c t i v e m e t h o d s h a v e n o t y e t b e e n
g e n e r a l l y e s t a b l i s h e d . I n c o n t r a s t , P C R o f f e r s a l e s s t e c h -
Comm unicated b y G. E. Hart
R. M. D. KoebnerCereals Research Department, John Innes Centre,Norwich Research Park, Colney NR4 7UJ, UK
n i c a l l y d e m a n d i n g a n d m o r e r a p i d m e t h o d o l o g y , a n d t h e
d i r e c t io n o f g e n e t ic m a p p i n g p r o g r a m m e s i s t h e r e f o r e
t e n d i n g t o b e fo c u s s e d o n t h e c o n v e r s i o n o f a n R F L P - b a s e d
t o a P C R - b a s e d a s s a y ( O l s o n e t a l . 1 9 8 9 ) .
T h e i n t r o d u c t io n o f ry e c h r o m a t i n i n to a w h e a t b a c k -
g r o u n d h a s b e e n a g o a l o f w h e a t c y t o g e n e t i c i s ts f o r m a n y
y e a r s , a s r y e o f f e r s a r a n g e o f a d a p t a t i o n n o t r e a d i l y a c -
c e s s i b l e w i t h i n t h e w h e a t g e n e p o o l ( Z e l l e r a n d H s a m
1 9 83 ) , a n d a w e a l t h o f c y t o g e n e t i c m a t e r i a l w h i c h a l l o w s
f o r t h e m a n i p u l a t i o n o f in d i v i d u a l r y e c h r o m o s o m e s i s
a v a i l a b l e ( li s te d i n S h e p h e r d a n d I s l a m 1 9 88 ) . T h e m a j o r
h u r d l e f o r i n t r o g r e s s i n g r y e g e n e s i n t o w h e a t i s t h e l o w
l e v el o f h o m o e o l o g o u s r e c o m b i n a t i o n w h i c h c a n b e
a c h i e v e d b e t w e e n w h e a t a n d ry e c h r o m o s o m e s ( K o e b n e r
a n d S h e p h e r d 1 9 8 5 , 1 9 8 6 ). E f f e c t i v e s e l e c t i o n o f i n f r e -q u e n t r e c o m b i n a n t s t h e r e f o r e n e c e s s i t a t e s m a r k e r s w h i c h
a r e b o th e f f i c i en t , p o l y m o r p h i c a n d p l e n t i fu l . B i o c h e m i c a l
( p r o te i n s a n d i s o z y m e s ) a n d m o r p h o l o g i c a l c h a r a c t e r s a r e
b o t h s u i t a b l e f o r t h is p u r p o s e , b u t f e w a r e a v a i l a b l e , a n d
t h e R F L P a s s a y, w h i l e p r o v i d i n g m a n y m a r k e r s , i s n o t r e a d -
i l y a d a p t a b l e t o l a r g e - s c a l e s c r e e n i n g . A p a r t i c u l a r a d v a n -
t a g e o f t h e P C R a s s a y i n t h i s c o n t e x t i s t h a t i t d o e s a l l o w
f o r t h e e f f ic i e n t s c r e e n i n g o f l a r g e p o p u l a t i o n s a n d , i n p r i n -
c i p le , i t c a n b e d e v e l o p e d f o r a n y t a r g e t e d p a r t o f t h e g e -
n o m e w h e r e n u c l e o t i d e - s e q u e n c e i n f o r m a t i o n i s e i t h e r
a v a i l a b l e o r c a n b e r e a d i l y o b t a i n e d f r o m R F L P p r o b e s .
T h e p r e s e n t s t u d y i s a i m e d a t th e d e v e l o p m e n t o f s u c h a s -
s a y s , s p e c i f i c a ll y i n r e l a ti o n t o t h e a g r o n o m i c a l l y i m p o r -t a n t w h e a t - r y e t r a n s l o c a t io n 1 B L . 1 R S , w h i c h a p p e a r s t o
g i v e a y i e l d a d v a n t a g e o v e r n o r m a l 1 B ca r r ie r s , b u t c o m -
m o n l y s u f f e r s f r o m a d o u g h - q u a l i t y d e f e c t ( D h a l i w a l e t al .
1 9 8 8 ) .
Mat er i a l s and m et hods
DN A was extracted from single seeds, using the CT AB method de-scribed in King et al. (1993), or from leaf tissue using phenol/chlo-roform (Sharp et al. 1988), from the following lines: wheat cvs 'Ch i-nese Spr ing ' , 'Gabo ' , 'F ron tana ' , 'Favor i t s ' , 'Pavon ' , 'Glennson ' ,'L icanka ' , 'M acvanka 1 ' , 'Kavk az ' and 'Dea n ' ( the la t ter f ive are
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known carriers of the wheat-rye translocat ion 1BL.IRS); rye cv'DS2'; disomic addition lines carrying rye cv 'Imperial' chromo-somes 1R, 2RL, 3R, 4R, 5R, 6R and 7R in wheat cv 'Chinese Spring '
(Driscoll and Sears 1971); the wheat-rye translocat ion 1DL.1RS(Shepherd 1973), and its derived wheat-rye recombinants 180, 1-93(Koebner and Shepherd 1986) and WR-1 (Rogowsky et al. 1991).The DNA was diluted to 25 ng/btl and 1 btl was used as template forPCR. For some assays, template DNA was obtained by first immers-
ing an Eppendorf tube containinga small piece (approximately 5 mg)of crushed endosperm in 20 btl of 1 xTE in a sonicat ing waterbathfor 10 min, followed by a 2-h incubation at 37~ After this the tubewas centrifuged and 1 gl of the resultant supernatant was taken as
template.Four pairs of oligonucleotides o be used as primers for PCR were
synthesised; these were directed at the loci Nor-R1 and 5S-Rrna-R1,at the telomere-associated 350 family (Appets and McIntyre 1985),and at the widely dispersed rye-specific repetitive sequence Ris-1(Moore et al. 1993). These will be referred to hereafter as, respec-tively, NOR, 5S, TEL and RIS. For NOR, the sequences of the inter-genic spacers between the ribosomal repeats of wheat (Barker et al.1988) and rye (Appels et al. 1986 b) were compared, and the regionof least homology (approximately 600 bp, starting 30 bp downstreamfrom the 3' end of the coding region of the 25S-RNA gene) of the ryesequence was used to design primers These correspond to bases
1383-1404, and 1768-1751 of the sequence given in Appels et al.(1986 b). For 5S, the 349-bp intergenic spacer sequence between the120-bp repeat units of the rye 'short lineage' (Reddy and Appels1989), which lie at bases 153-172 and 259-241, was used to designprimers. For the TEL assay, primers were designed from the consen-sus 380-bp sequence of the repeating unit of the 350 family [bases139-158 and 261-242, Appels et al. (1986 a)]. For RIS, the Ris-1element was sequenced (unpubl ished data) and this sequence of 497bp was used to design appropriate primers (posit ioned at bases 33-53
and 143-124).PCR condi tions were similar for each primer. The reactions were
carried out in a 25-}.tl volume, contain ing 25 ng of template, 0.4 Uof Taq polymerase, 2 nmol of dNTP and an optimized quantity ofprimer (0.1-1 pmol). The reaction buffer was made to 12% w/v su-crose, 0.2 mmol cresol red, as recommended by Hoppe et al. (1992),to allow direct loading of the PCR reaction into the agarose gel. The
PCR was carried out over 25-35 cycles consist ing of 15 s at 94~45 s at 65~ and 45 s at 72~ with a minimum transit ion time be-tween each temperature, and the run was completed with a 5-min fi-
741
nal extension at 72~ In later experiments, the extension step wasomitted to give a two-step cycle (15 s at 94~ and 45 s at 65~PCR products were separated on 1% or 2% agarose gels contain-ing 10 btg/ml of ethidium bromide, and visualised under UV light.
Results
NOR
The amplifi cation profile obtained from rye DNA tem-
plate consisted of four products, of approximate sizes
400 bp, 600 bp, 700 bp and 800 bp. Wheat DNA tem-
plate was not amplified. Three of the four (400 bp, 600
bp, 700 bp) rye-speci fic products were also ampl ified
from template where the entire rye chromosome 1R was
present, but not from template DNA of wheat lines car-
rying any other rye chromosome (Fig. 1A). The 800-bp
product was not produced from any of the addition line
templates. No amplification was observed from a range
of barley cultivars (data not shown). The expected am-
plific ation product size was 386 bp, and this was gener-
ally the most efficientl y ampli fied product of the profile;
however, inspection of the DNA sequence of the rye
interg enic spacer shows that the reverse primer sequenc e
recurs at bases 200 3- 198 6 and again, with only one base
mismatch at the 5 ' end, at bases 2136 -21 19. These two
primer sites generate additional amplification products
of sizes 621 bp and 754 bp, respectively. Anal ysis of the
wheat-rye recombinants showed that the three priming
sites present on chr omoso me 1R were also present on the
1DL. 1RS translocati on, and on the proximal rye re com-bina nt 180, but not in either of the two distal rye recom-
binan ts 1-93 or WR-1 (Fig. 2A).
Fig. 1 Amplifi cation profilesof wheat cv 'ChineseSpring'(W), rye cv. 'DS2' (R),and the Chinese Spring/Imperi-al rye disomic addition line set(I=IR, 2=2RL ..... 7=7R) usingprimers for A NOR, B 5S,C TEL, D RIS. M: kilobase lad-der molecular-weight marker
(sizes in bp marked on the leftof B). The same sequence oftemplate DNAs is used in eachfigure
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Fig. 2 Amplification profilesof wheat-rye translocation1DL. 1RS (a), thr ee o f its de-rived recombinants (b: 180, c:1-93, d: WR-1) and wheat cv'Gab o' (e) using primers for ANOR, B 5S, C TEL, D RIS
Tab le 1 Sequence of oligonu-cleotides used as primers for NORassaying rye Nor-1 (NOR), 5s- 5SRrna (5s), Telomere-associated TELsequence (TEL) and dispersed RISrepeat Ris-1 (RIS)
F: GCATGTAGCGACTAACTCATCG
F: TTTTTGCGTCTCGTGACAAG
F: CCAACGCCTATGAAAACGAT
F: TAATTTCTGCTTGCTCCATGC
R: CCCAGTTTTCCATGTCGC
R: ACCGGCCTAAAACGTATCG
R: GCCAACTCTCGCAAAGAAAC
R: ACTGGGGTGCACTGGATTAG
5S
T h e a m p l i f i c a t i o n p r o f i l e o b t a i n e d f r o m e i t h e r r y e D N A
t e m p l a t e, o r f r o m w h e a t D N A t e m p l a t e c o n t a i n in g t h e e n -
t i re rye chromosomes 1R or 3R , cons i s t ed of two produc t s
of appr oxim ate s i zes 100 bp and 600 bp (F ig . 1A) . In some
runs , a fur the r amp l i f i ca t ion produc t of app roxim ate s i ze
1000 bp was fa in t ly v is ib le . Whea t or ba r l ey t empla te was
not ampl i f i ed . The expec ted s i ze of ampl i f i ca t ion produc t
was 106 bp . The sam e rye-sp ec i f i c ampl i f i ca t ion produc t s
w e r e p r o d u c e d f r o m 1 D L . 1 R S a n d f r o m t h e w h e a t - r y e r e -
comb inant s 180 and 1-93, but not f rom WR -1 (F ig . 2B) .
T E L
At l eas t four d i s tinc t ampl i f i ca t io n produc t s wer e produced
f r o m t e m p l a t e s o f r y e o r w h e a t c o n t a i n i n g t h e t e l o m e r e o f
a n y o n e o f th e s e v e n r y e c h r o m o s o m e s . T h e s e w e r e o f a p -
prox imate s i zes 100 bp , 500 bp , 900 bp and 1300 bp . In
addi t ion the prof i l e a lways inc luded a sme ar of h ighe r -mo -
l e c u l a r - w e i g h t D N A ( F i g . 1 C ) . W h e a t a n d b a r l e y D N A
templa te pro duced a simi la r , bu t muc h fa in te r prof i l e when
t h e n u m b e r o f P C R c y c l e s w a s i n c r e a s e d t o 4 0, b u t w a s n o t
v i s ib ly ampl i f i ed a f t e r 35 cyc les . The expec ted PCR prod-
uc t s i ze was 123 bp . Th e rye prof i l e w as presen t in
1DL.1RS and the recombinant s 1-93 and WR-1, but not inthe proximal recombinant 180 (F ig . 2C) .
RIS
T h e R I S p r i m e r s a m p l i f i e d a s i n g l e p r o d u c t o f a p p r o x i -
m a t e l y 1 0 0 b p f r o m a ll t e m p l a te s c o n t a i n i n g r y e D N A , b u t
not f rom templa tes l acking rye DN A (F igs . 1 D and 2 D) .
The expe c ted PCR pro duc t s i ze was 111 bp . The as say a l so
worked sa t i s fac tor i ly when t empla te was obta ined d i rec t ly
f r o m a s m a l l s e c t io n o f m a t u r e e n d o s p e r m , w i t h o u t a n y
p r i o r D N A p u r i f i c a t i o n p r o c e d u r e , a s d e s c r i b e d a b o v e
(Fig, 3).
Fig. 3 RIS-generated amplification profiles obtained by primingtemplate derived from varieties without (tracks 1-4, 10), and with(tracks 5-9) the 1BL. 1RS translocation. * denotes template ob tainedby sonication/incubation. Tracks 1-4: cvs 'Chinese Spring', 'Pav on','Frontana', 'Favorits'; tracks 5-8: cvs 'Licanka', 'Macvanka 1','Kavkaz', 'Dean'; track 9: cv 'Glennson'; track 10: cv 'Gab&
T r i p l e x P C R f o r N O R , 5 S, a n d T E L
W h e n a ll t h re e p r i m e r p a i r s c o r r e s p o n d i n g t o N O R , 5 s a n dTE L were inc luded in the PCR reac t ion m ixture , a l l the ex-
p e c t e d P C R p r o d u c t s w e r e o b t a i n e d f r o m t e m p l a t e o f a
w h e a t ( c v ' L i c a n k a ' ) c o n t a i n i n g t h e w h e a t - r y e t r a n s l o c a -
t ion 1BL. 1RS (F ig. 4) . Howe ver the ba lance of pr imer con-
cent ra t ions to obta in th i s resu l t was de l i ca te . To obta in a
s imul taneou s a s say for al l th ree loc i, the concent ra t ions o f
the pr imers used were 0 .5 fM (NOR), 0 .07 fM (5S) and
0 . 05 fM ( T E L ) . W h e n a n y o n e o f t h e p r i m e r p a i rs w a s
present in exces s , ampl i f i ca t ion f rom a t l eas t one , and
som et imes f r om both , of the o ther ta rge t loc i was sup-
pres sed . The pr imer s equences w ere a l igned to unco ver any
b a s e - p a i r c o m p l e m e n t a r i t y b e t w e e n t h e v a ri o u s c o m b i n a -
t ions . Thi s ana lys i s showed tha t only be tween four and
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Fig. 4 Multiplex PCR amplificationprofiles from cv 'Licanka' tem-plate (carries wheat-rye translocation 1BL.1RS). N, 5S, T indicateposition of PC R products amplified by, respectively, primers NOR ,5S and TEL. Primers used in each reaction indicated by + at the baseof the figure. M: kilobase ladder molecular-weight marker
seven bases could be a l igned between any two of the pr imer
sequences, w hile four (TEL) or f ive (NOR, 5S) bases were
complementary when compar ing the ind iv idual fo rward
and reverse pr imer sequences . Thus i t was no t thought
l ike ly that p r imer complem enta t ion in ter fered wi th the ef -
f i c iency of the mul t ip lex react ions .
Discussion
The convers ion of the RFLP assay in to a PCR-based one
i s being wide ly pursued as a means o f improving the ap-
p l icab i li ty o f molecu lar markers as d iagnos t ic and se lec-
t ion tools . A necessary s tep in this process is a check that
the locus being assayed by the primers is ident ical to thatassayed by the RFLP probe. A num ber of ways of conf i rm-
ing this ident i ty are possible, including l inkage analysis
and hybr id i sa t ion between the PC R product and the RFLP
probe. In the present case, the avai labi l i ty of a number of
wheat - rye recom binants involv ing d i s t inc t segments o f t il e
shor t arm of rye chrom osome 1R al lows a s imple check .
These recombinants have been prev ious ly c lass i f i ed for
genotype a t N or , R 1 , 5S - R r na - R 1 and the t e lomere (Koeb-
ner et al. 1986; Ro gow sky et al. 1991), and these classifi -
ca t ions f i t ted exact ly w i th those ob ta ined by the presen t
PCR assay, both for the l ines i l lustrated in Fig. 2, and for
the remainder o f the 1RS recombinant l ines (data no t
shown) . The PCR product s cou ld now be used as p robes
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for in situ hybridisat ion s tudies to further confirm their
provenance, as the physical locat ion of the three loci on
chromosome 1R is well establ ished (Appels et al . 1980,
1981). The Ris-1 ele men t is kno wn to be widely dispersed
throughout the rye genome, except near the cen t romeres
and telomeres of the chromosomes (Moore et al . 1993),
and thus was expected to give a posi t ive s ignal for al l the
recombinant l ines .Whereas the or ig in of the mul t ip le PCR product s us ing
the NOR pr imers was due to repet i tion of the reverse pr imer
sequence at two other closely located posi t ions, the mult i -
ple bands in the 5S and TEL profi les have a different ori -
gin. The 5S-Rrna-R1 locus consis ts of mult iple repeats of
a 120-bp coding uni t separated by about 400 bp of non-
coding spacer DNA (Reddy and Appels 1989), in which
the primer anneal ing sequences l ie. The primary amplifi -
cat ion product is expected to be 106 bp in length, but for
any one forward priming si te, there wil l be reverse prim-
ing s i tes in each of the adjacent spacer segments , giving
rise to amplificat ion products o f lengths 610 bp and 1 120
bp (and higher), ful ly consis tent with the observed data
(Fig. 1B). The 610-bp product appears to be prefere nt ial ly
amp lified over the other two, and those larger than 1 120
bp (1630 bp, 2140 bp etc.) are probably too large to be am-
plified efficient ly under the react ion condit ions used. In an
at tempt to promote the product ion of the 106-bp product ,
the ex tens ion phase of the PCR cycle was omi t ted , bu t the
resul t ing profi le was s imilar to that achieved earl ier . I t re-
mains unclear why the 610-bp product i s p refer red over the
106-bp produ ct .
A similar s i tuat ion pertains with respect to the TE L PC R
profi le. The primary pr oduct length is 123 bp, but those of
approx imate lengths 500 bp, 900 bp and 1 300 bp were
usual ly dis t inguishable, as well as other even higher-mo-
lecu lar -weigh t p roduct s and a background h igh-molecu lar -
weight smear. These resul ts are readi ly expl icable by as-
suming that the 350 sequence o ccurs as blocks of mult iple
copies in a t andem array as sugges ted by Appels and M cIn-
tyre (1985). In this case, a series of PCR products with a
size periodic i ty of 380 bp (the length of the consensu s se-
quence) will be amplified, i .e., 123 bp, 503 bp, 883 bp,
1263 bp . . . . which matches the PCR profi le exact ly (Fig.
1C). Onc e again, the smallest PCR p roduct is not the most
ef f i c ien t ly ampl i f ied , and the prof i l e was no t mod i f ied by
omission of the extension s tep.
Sequences re la ted to the 350 fam i ly have been i so la tedin a range of grass species (Xin and Appels 1988; McNeil
et al . 1994), and the postulat ion is that sequence
'speci f i c i ty ' ref l ec t s dominance, ra ther than exclus ive
presence of that part icular sequence in a genome. The ob-
servat ion that h igher numbers o f PCR cycles p roduced a
signal from both barley and wheat template, and part icu-
larly that the PCR profi le was ident ical to that of rye, is
consis tent with the presence o f 350-l ike sequences in these
other species , but at a level magnitudes lower than is
present in rye. Comparison of the target s i tes for anneal-
ing of the TEL pr imers between the rye s i t es and those in
tetraploid wheat and other grass species [Fig. 7 in McNeil
et al. (1994)] shows just one mism atch in each of the two
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74 4
p r i m e r s e q u e n c e s i n th e t e t ra p l o i d w h e a t A B 1 3 5 0 s e -
q u e n c e , w i t h m o r e m i s m a t c h e s i n t h e o t h e r c o m p a r i s o n s .
T h e e x p e c t a t i o n w o u l d t h e r e f o r e b e t h a t o n l y a s u b s e t o f
t h e w h e a t 3 5 0 f a m i l y s e q u e n c e s w o u l d a m p l i f y f r o m t h e
T E L p r i m e r s . S i n c e t h e f r e q u e n c y o f t h e 3 5 0 f a m i l y i n
w h e a t i s b e lo w t h e h y b r i d i s a ti o n t h r e s h o l d ( M c N e i l e t al .
1 9 9 4 ) , t h e t a r g e t f o r t h e T E L p r i m e r s f r o m a w h e a t t e m -
p l a t e i s r e d u c e d t o a s u b s e t o f a s m a l l n u m b e r , w h i c h e x -
p l a i n s w h y o n l y h i g h e r n u m b e r s o f a m p l i f i c a t i o n c y c l e s
g e n e r a t e a v i s i b l e P C R p r o d u c t .
T h e P C R a s s a y f o r 5S-Rrna-R1 i d e n t i f i e d a s i t e o n c h r o -
m o s o m e 3 R , i n a d d i t i o n t o t h e o n e o n 1 R , t h e l o c a t i o n o f
t h e m a j o r r y e s i t e ( A p p e l s e t a l . 1 9 8 0 ). A 3 R s i t e h a s r e -
c e n t l y a l s o b e e n i d e n t i f i e d i n r y e u s i n g in situ h y b r i d i s a -
t i o n , a l t h o u g h n o t o n t h e c v ' I m p e r i a l ' 3 R p r e s e n t i n t h e
a d d i t i o n l i n e u s e d i n th i s s t u d y ( C u a d r a d o e t a l . 1 9 9 5 ). A s
q u a n t i t a t i v e v a r i a t i o n i n t h e n u m b e r o f r e p e a t u n i ts a t t h e
5S-Rrna l o c i h a s b e e n o b s e r v e d i n r y e ( A p p e l s e t a l. 1 9 8 9 ) ,
t h is a p p a r e n t a n o m a l y i s r e a d i l y e x p l i c a b l e a s a c o p y n u m -
b e r e f f e c t , w i th t h e h i g h e r s e n s i t iv i t y o f P C R e n a b l i n g t h e
i d e n t i f i c a t i o n o f a l o c u s a t w h i c h t h e c o p y n u m b e r i s b e -
l o w t h e l e v e l o f d e t e c t i o n w i t h t h e in situ h y b r i d i s a t i o n
t e c h n iq u e . A s i m i l a r P C R - b a s e d a s s a y h a s a l so l o c a t e d a
s it e on b a r le y c h r o m o s o m e 3 H a n d s h o w n p o l y m o r p h i s m
w i t h i n t h e s p e c i e s f o r c o p y n u m b e r ( K a n a z i n e t a l . 1 9 9 3 ) .
A f u r t h e r s i t e , 5S-Rrna-R2, o n c h r o m o s o m e 5 R w a s i d en -
t i f i e d b y R e d d y a n d A p p e l s ( 1 9 8 9 ) , b u t w a s n o t d e t e c t e d
i n t h e p r e s e n t s t u d y . T h i s i s p r o b a b l y b o t h b e c a u s e t h e s i t e
i s v ir t u a l ly n o n - e x i s t a n t i n r y e c v ' I m p e r i a l ' ( A p p e l s e t a l .
1 9 89 ) , a n d b e c a u s e e f f e c t i v e p r i m i n g f r o m t h e f e w r e m a i n -
i n g s i t e s m a y b e p r e v e n t e d b e c a u s e t h e o l i g o n u c l e o t i d e
p r i m e r s w e r e d e s i g n e d f r o m t h e s e q u e n c e o f t h e ' s h o r t '
v a r i a n t, w h i c h p r e f e r e n t i a l l y h y b r i d i s e s in situ t o t h e 1 R ,
r a t h e r t h a n t o t h e 5 R , s i t e ( R e d d y a n d A p p e l s 1 9 8 9 ) . A
c o m p a r i s o n o f th e n u c l e o t id e s e q u e n c e s o f t h e p r i m e r s i t es
b e t w e e n t h e l o n g a n d s h o r t v a r i a n t s s h o w s t h r e e m i s -
m a t c h e s i n th e f o r w a r d p r i m e r s e q u e n c e a n d f o u r in t h e re -
v e r s e , p r o b a b l y a s u f f i c i e n t d i f f e r e n c e t o i n h i b i t a d e q u a t e
p r i m e r a n n e a l i n g . A s i m i l a r s i t u a t i o n e x i s t s i n b a r l e y ,
w h e r e p r i m e r s c a n b e s e l e c t e d w h i c h a m p l i f y e x c l u s i v e l y
f r o m e i t h e r th e s i te w i t h th e ' l o n g ' s p a c e r ( 3 H ) , o r f r o m
t h e ' s h o r t ' s p a c e r ( 2 H ) ( K a n a z i n e t a l . 1 9 9 3 ) .
T h e s e P C R - b a s e d m a r k e r s a r e id e a l l y s u i te d t o p r o -
g r a m m e s a i m e d a t th e i n t r o g r e s s i o n o f r y e g e n e s i n t o
w h e a t . E v e n i n t he a b s e n c e o f th e Phi g e n e , w h i c h n o r -
m a l l y s u p p r e s s e s n o n - h o m o l o g o u s r e c o m b i n a t i o n i nw h e a t , t h e f r e q u e n c y o f w h e a t - r y e r e c o m b i n a t i o n i s l o w
( K o e b n e r a n d S h e p h e r d 1 9 85 , 1 9 8 6 ), a n d t h e r e f o r e r e c o m -
b i n a n t i n d i v i d u a l s a r e r a r e . T h e r e i s t h e r e f o r e a n e e d t o u s e
m a r k e r s w h i c h c a n b e r e a d i l y a p p l ie d t o l a rg e n u m b e r s o f
i n d i v i d u a l s a n d , i n p a r t i c u l a r , t o b e a b l e t o s c r e e n s e e d s
r a t h e r t h a n g r o w i n g p l a n t s . A l t h o u g h R F L P m a r k e r s a r e
e f f i c i e n t a t d i s t i n g u i s h i n g r y e f r o m w h e a t l o c i , t h e y a r e n o t
r e a d i l y a p p l i c a b l e t o t h i s s i t u a t i o n , b o t h b e c a u s e t h e a s s a y
r e q u i r e s m o r e D N A t h a n c a n b e e x t r a c t e d f r o m a s i n g l e
s e e d [ g e n e r a l l y a b o u t 1 ~ tg ( K i n g e t a l . 1 9 9 3 ) ] a n d b e c a u s e
t h e p r o c e d u r e i s t o o t i m e - c o n s u m i n g a n d e x p e n s i v e f o r
l a r g e p o p u la t i o n s . I n c o n t ra s t , t h e P C R c a n b e s c a l e d d o w n
t o a 1 0 - g l r e a c t i o n w h i c h r e q u i r e s m i n i m a l r e a g e n t s a n d ,
u s i n g t h e m u l t i p l e x a p p r o a c h , a s i n g l e r e a c t i o n c a n b e u s e d
t o a s s a y f o r m u l t i p l e l o c i s i m u l t a n e o u s ly . T h e D N A e x t r a c -
t io n s y s t e m u s e d i n t hi s s t u d y p r o d u c e d s u f f ic i e n t t e m p l a t e
f r o m a s i n g l e s e e d f o r 5 0 0 P C R r e a c t i o n s - c l e a r l y a l e s s
e f f ic i e n t, b u t m o r e r a p i d , s y s t e m w o u l d b e m o r e a p p l i c a b l e ,
a n d t h e s i m p l e s o n i c a t i o n / i n c u b a t i o n d e s c r i b e d h e r e i s a d -
e q u a t e f o r th i s p u r p o s e . A s i m i l a r a p p r o a c h h a s r e c e n t l y
b e e n d e s c r i b e d b y C h u n w o n g s e e t a l. ( 1 9 93 ) .
T h e l o c i t a r g e t e d i n t h e p r e s e n t s t u d y a r e a i m e d a t g e n -
e r a t i n g m a r k e r s t o a i d b r e a k i n g u p t h e a g r o n o m i c a l l y i m -
p o r t a n t w h e a t - r y e t r a n s l o c a ti o n 1 B L . 1 R S ; n e v e r t h e l e s s ,
a n y r ye c h r o m o s o m e , o r in d e e d n o n - w h e a t c h r o m o s o m e ,
c a n b e t a r g e t e d i n a s i m i l a r w a y b y ju d i c i o u s c h o i c e o f l o c i
o n t h e b a si s o f th e w e l l - d e v e l o p e d R F L P m a p s o f th e T r i -
t i c e a e g e n o m e s . F u r t h e r m o r e , t h e R I S p r im e r s c a n b e u s e d
a s a n i n i t ia l s c r e e n , t o e l i m i n a t e s e g r e g a n t s l a c k i n g a n y r y e
c h r o m a t i n , t h e r e b y l i m i t i n g t h e n u m b e r o f i n d i v id u a l s
w h i c h n e e d t o b e s c r e e n e d w i t h t he m o r e s p e c i f i c p r im e r s .
Acknowledgements The author thanks Axis Genetics for the syn-
thesis of some o f the oligonucleotides used in this study, and Dr. R.Appels for helpful comments on the manuscript.
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