High-lysine gene segregation distorted in the barley cross Riso 1508 x Crypt CI 1090: Patterns of endosperm proteins by an electrophoretic method

He edi as 108: 129-131 (1988) B ie epo High-lysine gene seg ega ion dis o ed in he ba ley c oss Ris@ 1508 x C yp CI 1090: Pa e ns o endospe m p o eins by an elec opho e ic me hod HANNU AHOKAS Depa men o Gene ics, Uni e si y o Helsinki, A kadianka u 7, SF-00100 Helsinki, Finland (Recei ed Ap il 29, 1987) The ba ley c oss be ween he Indian line C yp (CI 1090) and 1508 (Ris~ high-lysine mu an no. 1508 in c . Bomi) shows, in F, spikes, an abe an seg ega- ion o abou 1:63 o sh unken o plump g ains in- s ead o he expec ed a io o 1:3 (AHOKAS 1978, 1979a). A di e en kind o dis o ion was obse ed in he c oss o c . Glacie and 1508 (AHOKAS 1979b). The Glacie supp ession, which seems o be p omis- ing o b eeding, will be desc ibed la e . P og ess in b eeding o 1508 g ain plumpness and yield has been epo ed by BANG OLSEN e al. (1986). The p esen a icle deals wi h he case o C yp . Below, he c osses a e ma ked acco ding o PURDY e al. (1968), and he gene a ion o seed, ac- co ding o AHOKAS (1976). When an F, spike o C yp /1508 o he ecip ocal c oss is pollina ed wi h 1508, he BC-F,,, g ains seg ega e sh unken and plump in a a io o 1:1 wi h expe imen al esul s 5850 o he c oss C yp /2*1508 (P>0.30) and 35:36 o 1508/C yp //1508 (P-I). Thus he mu an gene o sh unken pheno ype seg ega es no mally in his F,, hough i s pheno ypic e ec is masked in sel ed F, spikes, leading o 1:63 a io in he F(2) g ains. Ad- di ionally, when he equency o he mu an gene (o he p opo ion o 1508 genome) eaches 75 % in he zygo es on a C yp /1508 he e ozygous plan , he dis o ion is no longe appa en . I was o in e es o know hen, whe he he geno ypically homozygous sh unken g ains wi h a plump pheno ype on F, spikes ha e a mu an -like p o ein pa e n in endo- spe ms. This ques ion will be answe ed below. Sample p epa a ion o SDS-PAGE om single g ains All emo able husks we e pealed o wi h inge nails om he g ain. The emb yo was cu away wi h a scalpel (i s o al emo al may be checked unde s e eomic oscope). The g ain was i ed in o a ans- pa en plas ic pipe e- ip (o ange 50 o 200 kl) o se e as a g ip while d illing. Excess plas ic was cu away. The endospe m was g ound o a powde by d illing wi h an F1 cu ing edge a ached o a minia- u e d ill (Mini Plus o equi alen ). Meal samples o 15 o 25 ng we e weighed in a 1.5 ml Eppendo ube and suspended in a modi ied LAEMMLI (1970) sample bu e con aining 65.8 mM T is-HC1 (pH 7.0), 2.1 YO SDS, 9.2 '/o glyce ol and 0.001 % b omophenol blue. The amoun o sample bu e (~1) used o he suspension was 14.25 imes he mil- lig ams o he meal. The meal was suspended in he e ening wi h a mic opipe e- ip and by o exing, and allowed o s and o less han hal -hou . The ubes we e hen spun o 5 seconds in a mic ocen- i uge (12500 pm) o a oid ex ensi e swelling du - ing he subsequen hea ing o 2 min in boiling wa e . The caps we e pie ced be o e boiling. The boiled ubes we e le o e -nigh a 18-22°C. Any in e up ion in boiling may lead o anomalies in he p o ein pa e n ollowing elec opho esis wi h some high-molecula weigh p o eins o ally missing. The esul is simila , i boiling is omi ed. On he ollow- ing mo ning, 2-me cap oe hanol was added (PI) o 0.75 imes he weigh o he meal in millig ams, and he pelle was b oken and pa ially esuspended as abo e. Be o e loading, he samples we e cla i ied by spinning in mic ocen i uge a 12000 o 14000 pm o 10 min a oom empe a u e. The dissol ing p ocess can also be done du ing a couple o hou s be o e beginning he gel un wi hou any g ea e - ec on he banding pa e n. The gels used he e we e essen ially acco ding o LAEMMLI (1970) wi h 9.5 % ac ylamide in he sepa a ion gel. A e elec- opho esis, he gels we e s ained wi h B illian blue R (Sigma) ollowing a modi ica ion o FAIRBANKS e al. (1971). The gels we e agi a ed in solu ion 1 o 5 h, solu ion 2 o 10 h, and in se e al changes o solu- ion 4 o cla i y he backg ound. In my hands, he me hod abo e e eals mo e p o ein bands han he apid one desc ibed by SMITH and PAYNE (1984) and He edi as I08 (1988) Fig. 1. SDS-PAGE dis ibu ion o endospe m p o eins om meals wi h he p esen me hod (a-e, g-i) o wi h he me hod o SMITH and PAYNE (1984) on lanes ii and hh. Solu ion olumes used o ex ac ion we e he same on a meal-weigh basis. 60 pl was loaded pe lane. Lane hal es we e p in ed wi h wo di e en exposu e imes. Lane a = Bomi, b = C yp , c = C yp /1508 F,,,, d = 15081C yp F,,,, e = 1508, = molecula weigh ma ke s in kilodal ons (kd), g-h-i = F,,, endo- spe ms om C yp 11508 c oss wi h majo p o eins be ween 24-45 kd o F,,,-, Bomi- and C yp -like pa e ns, espec i ely. Lanes ii and hh = spli samples o meals o i and h, co espondingly, wi h less bands isible and wi h di e en mobili ies han wi h he p esen me hod. Angles and do s indica e unique bands o mobili y di e ences be ween Bomi and C yp Some o he mobili y di e ences a e exp essed codominan ly in F,,,’s o C yp and 1508. SMITH e al. (1986). Also mul iple bands a e e- ealed whe e he la e displays a single di use band (Fig. 1). G ound meal o ba ley can also be used, i whole-g ain p o eins a e o be s udied. The me hod desc ibed he e ca ied ou on indi idual g ains is a good es o a ie al pu i y o seeds o small g ain ce eals. Resul s and discussion Wi h SDS-PAGE, Bomi shows wo majo p o ein bands be ween 24 and 45 kd (Fig. 1 a), which a e also sppa en when alkyla ed B ho dein p epa a- ions o Bomi a e elec opho esed (DOLL and AN- DERSEN 1981; ULLRICH e al. 1986). C yp has a com- posi e o h ee majo bands a he B ho dein posi- ion (Fig. 1 b). F,,, endospe ms be ween 1508 and C yp a e in e media y be ween Bomi and C yp a hei 24-45 kd bands, whe e 1508 is de oid o ol- uminous bands (Fig. 1 e). The F,,, endospe ms, being iploid, show some dosage e ec in he eci- p ocal c osses (Fig. 1 c and d). The F,,, pa e n wi h 1508 as seed pa en (Fig. 1 d) dis inc ly displays, also a he B ho dein posi ion, he p esence o mos Bomi bands no appea ing in 1508 (Fig. 1 e). This is in acco dance wi h he lack o dele ions in he B ho - He edi as 108 (1988) BRIEFREPORT 131 dein locus o 1508 (FORDE in KREIS e al. 1983) and wi h he appea ance o B ho dein mRNA in 1508 (THOMPSON and BARTELS 1983) as well as wi h some immuno eac i e end p oduc s o B ho deins in 1508 (ULLRICH e al. 1986). When F zJ g ains o he c oss C yp A508 seg ega - ing abou 1:63 sh unken o plump we e s udied on gels, no 1508-like banding pa e n was obse ed among he 100 plump endospe ms s udied. These g ains a e expec ed o seg ega e 1:3, o p obably mo e exac ly (-):( - 1:3.2 o mu an o non-mu an pheno ypes (P<0.001). Ins ead, he h ee pa e ns o he majo p o eins (Fig. 1, g, h, i) we e ound. All he a e sh unken F,*, endospe ms s udied had he 1508-like pa e n. Thus, he pheno ypically plump, bu geno ypically sh unken endospe ms a e no ghos -g ains de oid o p ope s o age p o eins. The 1508 allelic Ris0 mu an s no. 18 and 19 (JEN- s N 1979) we e also dis o ed in he F zJ g ain seg e- ga ion in c osses wi h C yp . Some o he old Indian ba ley lines, CI 1085, CI 1086, CI 1087, CI 1088, CI 1089. C1 1091, CI 2229, all om Cawnpo e as is C yp , and CI 3405 om Que a (p esen ly in Pakis- an) ailed o show dis o ion wi h 1508. The sh unken g ains o BC-F, spikes (see in o- duc ion) we e he sou ce o a sea ch o he pu a- i e homozygous supp esso s. Supposing ecombi- na ion o occu be ween sh unken gene and pu a- i e complemen a y supp esso genes (AHOKAS 197X), he sh unken BC-F,,, seeds should gi e ise o plan s seg ega ing sh unken and plump BC-F ,, g ains. Such plan s showing abou 1:15 seg ega ion o sh unken o plump we e de ec ed. Thei descen- den s we e selec ed o s able supp esso s du ing se e al seasons. Such de i a i es om 1508/C yp // 1508 c oss a e s ill en i onmen ally somewha sensi- i e, small g ained, six- owed, wi h pedicella e la - c als (Fig. 2). Thei endospe m p o ein pa e ns on SDS gels a e mos ly simila o hose o 1508, wi h h ee di e ences conce ning he majo p o eins ( e- sul s no desc ibed in his pape ). P esen ly, some backc oss lines based on he supp essi e geno ype o c . Glacie a e unde s udy. These Glacie /l508 de i a i es ha e be e g ain and plan cha ac e is- ics han he C yp de i a i es, and a e expec ed o be use ul as high-lysine eeding ba ley. 16-1 3x16) 64 64 Acknowledgemen s. - I am obliged o Ms. Leena Naskali, M. Ag ic. Fo es ., o help a sample p epa a ions. and o D . A. H. Schulman o eading and e ising he manusc ip . The s udy was ca ied ou unde he auspices o he Academy o Finland ( he Re- sea ch Council o Ag icul u e and Fo es y). Fig. 2. G ain sample o a high-lysine de i a i e om he c oss lSOX/C yp //1508. Na u al size. Li e a u e ci ed AHOKAS, H. 1976. A way o ma k he gene a ion o he seed. ~ Ba ley Gcne . Newsl. 6; 96 AHOKAS. H. 1978. Gene ic supp ession o sh unken endospe m in Ris@ mu an 1.508. -Ba ley News/ 21: 62-64 AHOKAS, H. 1979a. Fu he esul s on he supp ession o sh unk- en endospe m in high lysine mu an s. ~ Ba ley Gene . Newsl. Y; 3-7 AHOKAS, H. 1979b. Dis o ion o FI?] ke nel seg ega ion iii he c osses Ris@ ISOXiGlacic and Rise 1508/High Amylo Glacie . -Ba ley News/. 22: 129-131 BANG OLSEN, K., STILLING. B. and MUNCK, L. 1986. B eeding o yield in high-lysine ba ley. h i h In . Ba ley Gene . Symp., Okayama, Abs m , p. 222 DOLL. H. and ANDERSEN, B. 19x1, P epa a ion o ba ley s o age p o ein. ho dein. o analy ical sodium dodecyl sul a e-poly- ac ylamide gel elec opho esis. -Anal. Biochem. 11.5: 6146 FAIRBANKS, G . STECK, T. L. and WALLACH, 0. . H. 1971. Elec iopho e ic analysis o he majo polypep ides o he huinan e y h ocy e memb ane. - Biochemis y 10. 2hOk26 17 JENSEN. J. 1979. Loca ion o a high-lysine gene and DDT- esis - ance gene oil ba ley ch omosome 7. - Euphy icu 28: 47-S6 KREIS. M., SHEWRY, P. R., FORDE. B. G., RAHMAN. S. and MIFLIN, B. J. 1983. Molecula analysis o a mu a ion con e ing he high-lyaine pheno ype on he g ain o ba ley (I o d um w/- gu e). - Cell34: 161-167 LAEMMLI, U. K. 1970. Clea age o s uc u al p o eins du ing he assembly o he head o bac e iophage T4. - Nu u e 227: 68(& 685 KING, W. 0.. KONZAK. C. F.. PETFR~ SON, C. J. and ALLAN. R. E. 1968. A p oposed s anda d me h- od o illus a ing pedig ees o small g ain a ie ies. ~C' op Sc . 8: 405406 SMITH. D. B. and PAYNE, P. I. 1984. A p ocedu e o he ou ine de e mina ion o elec opho e ic band pa e ns o ba ley and mal endospe m p o eins. - J. Null. /m . Ag c. Bo . 16: 487- 49 8 SMITH, D. R., LISTER, P. R. and HANSSON, P. R. 1986. Disc i- mina ion o ba ley a ie ies by elec opho esis o endospe m p o eins ex ac able in o a mix u e o sodium dodecyl sulpha e, 2-me cap oe hanol and dime hyl o mamide. .- J. Ce eal Sci. 4: 107-1 16 THOMPSON, R. D. and BARTELS, D. 1983. Ho dein messenge RNA le els in he wild ype and mu an ba lcy endospe m. - Plan Sci. Le . 29: 295-304 ULLRICH, S. E., RASMUSSEN. U.. HBYER-HANSEN, G. and BRANDT, A. 1986. Monoclonal an ibodies o ho dein polypep- ides. - Cu bh g Res. Commun. 51: 381-399