Generation of PCR-Based Markers for the Detection of Rye Chromatin

8/2/2019 Generation of PCR-Based Markers for the Detection of Rye Chromatin

http://slidepdf.com/reader/full/generation-of-pcr-based-markers-for-the-detection-of-rye-chromatin 1/6

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



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



742

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



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

743

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



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.

References

Appels R, McIntyre CL (1985) Cereal genome organization as re-vealed by molecular probes. O xford Surveys Plant Mol Cell Biol2:235-252

Appels R, Gerlach WL, D ennis ES, Swift H, Peacock WJ (1980) Mo-lecular and chromosomal organization of sequences coding forthe ribosomal RNAs in cereals. Chromosoma 78:293-311

Appels R, Dennis ES, Sm yth DR, Peacock W J (1981) Two repeatedDNA sequences from the heterochromatic regions o f rye (Secalecereale) chromosomes. Chromosoma 84:265-277

Appels R, M oran LB, Gustafson JP (1986 a) Rye heterochromatin.I. Studie s on clusters of the major repeating sequence and theidentification of a new dispersed repetitive sequence element.Can J Genet Cytol 28:645-657

Appels R, M oran LB, G ustafson JP (1 986 b) The structure of DNAfrom the rye (Secale cereale) NOR-R1 locus and its behaviour ina wheat background. Can J Genet Cytol 28:673-685

Appels R, Reddy R McIntyre CL, Moran LB, Frankel OH, ClarkeBC (1989) The mo lecular-cytogenetic analysis of grasses and itsapplication to studying relationships among sp ecies of the Triti-ceae. Genome 31:122-133

Barker RF, Harberd NR Jarvis MG, Flavell RB (1988) Structure andevolution of the intergenic region in a ribosomal D NA repe at unitof wheat. J Mol Biol 201:1-17

Chunwongse J, Martin GB, T anksley SD (1993) Pre-germination

genotypic screening using PCR-am plification of half-seeds.Theor Appl Genet 86:694-698Cuadrado A, Jouve N, Heslop-Harrison JS (1995) Physical mapping

of the 5s rRNA m ultigene fam ily in 6x-triticale and rye: identifi-cation of a new rye locus. Genome (in press)

Dhaliwal AS, Mares DJ, Marshall D R, Skeritt JH (1988) Protein com -position and pentosan content in relation to dough stickiness of1B/1R translocation wheats. Cereal Chem 62:143-149

Driscoll CJ, Sears ER (19 71) Individual additions of the chromo-somes of 'Im peria l ' rye to wheat. Agron Abstr 1971:6

Hopp e BL, Conti-Tronconi BM , Horton RM (1992) Gel-loading dyescompa tible with PCR . BioTechniques 12:679-680

Kanazin V, Ananiev E, B lake T (1993) The genetics of 5S rRNA mul-tigene families in barley. Genome 36:1023-1028

King IP, Purdie KA, Rezanoor H N, Koebner RM D, Miller TE, Read-er SM, Nicholson P (1993) Characterization of Thinopyrum bes-sarabicum chromosome segments in wheat using random ampli-



fled polymorphic DNAs and in situ hybridization. Theor ApplGenet 86:895-900

Koebner RMD, Shepherd KW (1985) Induc tion of recombinationbetween rye chromosome 1RL and wheat chromosomes. TheorAppl Genet 71:208-215

Koebner RMD, Shepherd KW (1986) Controlled introgression towheat of genes from rye chromosome arm 1RS by induction ofallosyndesis. 1. Isolation of recombinants. Theor Appl Genet

73:197-208Koebner RMD, Shepherd KW, Appels R (1986) Controlled intro-gression to wheat of genes from rye chromosome arm 1RS by in-duction of allosyndesis. 2. Characterisation of recombinants .Tbeor Appl Genet 73:209-217

McNeil D, Lagudah ES, Hohmann U, Appels R (1994) Amplifica-tion of DNA sequences in wheat and its relatives: the Dgas44 andR350 families of repeated sequences. Genome 37:320-327

Moore G, Abbo S, Cheung W, Foote T, Gale M, Koebner R, LeitchA, Leitch I, Money T, Stancombe R Yano M, Flavell R (1993)Key features of cereal genome organization revealed by the useof cytosine methylation-sens itive estriction endonucleases. Ge-nomics 15:472-482

Olson M, Hood L, Cantor C, Botstein D (1989) A common languagefor physical mapping of the human genome. Science245:1434-1435

745

Reddy P, Appels R (1989) A second locus for the 5S multigene fam-ily in Seca le L.: sequence divergence in two lineages of the fam-ily. Genome 32:456-467

Rogowsky PM, Guidet FLY, Langridge P, Shepherd KW, KoebnerRMD (1991) Isolation and characterization of wheat-rye recom-binants involving chromosome arm 1DS of wheat. Theor ApplGenet 82:537-544

Sharp PJ, Kreis M, Shewry PR, Gale MD (1988) Location of B-am-

ylase sequences in wheat and its relatives. Theor Appl Genet75:286-290Shepherd KW (1973) Homoeology of wheat and alien chromosomes

controlling endosperm protein phenotypes. In: Sears ER, SearsLMS (eds) Proc 4th Int Wheat Genet Symp, University of Mis-souri, Columbia, Missouri, pp 745-760

Shepherd KW, Islam AKMR (1988) Fourth compendium of wheat-alien chromosome lines. In: Miller TE, Koebner RMD (eds), Proc7th Int Wheat Genet Symp, Institu te of Plant Science Research,Cambridge, UK, pp 1373-1398

Xin Z-Y, Appels R (1988) Occurrence of rye (Seca le ce rea le ) 350-family DNA sequences in Agropyron and other Triticeae. PlantSyst Evol 160:65-76

Zeller FJ, Hsam SLK (1983) Broadening the genetic variability ofcultivated wheat by uti lizing rye chromatin. In: Sakamoto S (ed),Proc 6th Int Wheat Genet Symp, Kyoto, Japan, pp 161-173

Generation of PCR-Based Markers for the Detection of Rye Chromatin

Documents