Genetic instability of a barley shrunken mutant

ie di u 114 281-284 ilY9I) B ie epo Gene ic ins abili y o a ba ley sh unken mu an HANNU AHOKAS Depa men o Gene ics, Uni e si y o Helsinki, A kadianku u 7, SF-00100 Helsinki, Finland, and Plan B eeding Ins i u e, ARC, SF-3 I600 Jokioinen, Finland Recei ed No embe 30, 1990. Accep ed Ma ch 7, 1991) A pu a i e ansposable gene ic ins abili y in ba ley (Ho deum ulga e L.) has p oduced a sh unken mu- a ion showing xenia, i.e., obse able seg ega ion in spikes o he he e ozygo es. The enzymology o his mu an in a Bomi-like gene ic backg ound has p e- iously been s udied (SCHULMAN and AHOKAS 1990). Ex ac s o i s imma u e sh unken endos- pe ms show 14 % o he ac i i y o soluble s a ch syn hase o a no mal Bomi cul i a , which may mean ha he gene o soluble s a ch syn hase has been mu a ed (SCHULMAN and AHOKAS 1990). Six sh unken endospe m, xenia o sex genes ha e been lis ed o ba ley in he e iew by SDGAARD and VON WETTSTEIN-KNOWLES (1 987). Typical sex/sex plan s a e iable. A special class o endospe mal mu an s called des causes de ec i e endospe ms, xenia, and le hali y (RAMAGE and CRANDALL 1981). The p e- sen sh unken mu an displays educed iabili y, and he supp esso mu an desc ibed below may all in o he dex ca ego y. In ba ley, WISE and ELLINGBOE (1985) unde - s ood he occu ence o he esis ance gene ecom- bina ion be ween M1u6 and Mla13 o be caused by a ansposi ion. SIMONS and SOMERVILLE (1988 and e a um 1990), using he same ba ley accessions as WISE and ELLINGBOE (1983, ob ained esul s which di e ed, howe e . While ansposable elemen s a e a e o undocumen ed o ba ley (ROHDE e al. 1987; MOORE e al. 1989; FUERSTENBERG and JOHNS 1990), in maize, a c oss-pollina ing species, hey a e dy- namic ac o s c ea ing new gene ic ma e ial in popula ions whe e a ia ion is chosen (GRIEL e al. 1989). The p esen mu a ion and di e en mu an s, which a e in e ec simila ly sh unken, ha e a isen independen ly in he ba ley s ocks ca ying he pu- a i e gene ic ins abili y, he sh unken mu an being one o he mos common mu an ypes along wi h a mu an called ligh -g een. Though p ecise assess- men s o he equencies o hese ins abili ies a e wan ed, i is ob ious ha he gene ic backg ound a ec s he ins abili y equencies in he p esen case. A hei mos equen , hey all in he ange o 1/500 o 1/1000 game es, he occu ences p ob- ably being meio ic in o igin. The p esen sh unken mu an shows some ins ab- ili y. I can e iden ly mu a e o a hinne sh unken o m. Fu he , he sh unken s ocks ha e p oduced a s ipe-shoo mu an , and a mu an wi h emb yos leading o in iabili y a ge mina ion ( esul s no dis- cussed he e). On he o he hand, plan s seg ega ing nea ly plump, sh unken and s e ile lo e s in hei spikes ha e independen ly appea ed and been ob- se ed h ee imes in sh unken s ocks (Fig. 1). When g own, he nea ly plump g ains gi e ise o plan s seg ega ing he wo ypes o g ains and s e il- i y as abo e. The sh unken g ains gi e ise o con- sis en ly sh unken plan s. Because he seg ega ing spikes ca ied mainly sh unken g ains, some nea ly plump g ains, and a he less s e ile lo e s, he hy- po hesis ha he ypically sh unken g ains ep esen wo mu a ions whose he e ozygo es show pa ial complemen a ion esul ing in he nea ly plump g ains was es ed, bu his explana ion was w ong. A supp esso gene o allele making he he e ozyg- ous g ains nea ly plump bu causing zygo ic le hal- i y when homozygous migh explain he nea ly plump pheno ype be e . Ma e ial and me hods Seg ega ion da a we e eco ded om c osses o ho- mozygous sh unken mu an wi h c . Ado a. The ba ley line used below has an isogeny o abou 75 % wi h c . Bomi, being F6 o la e gene a ion om he c oss, o mula unknown/2*/Bomi. Plan s we e g own in he g eenhouse ei he on Vapo B2 pea om win e o sp ing (en i onmen 1) o on Kekkila po - illing loam om au umn o win e (en i on- men 2). In bo h en i onmen s, addi ional e ilize s He edi as 113 (1991) Fig. 1. Spike o ba le! a la e dough age. - Le : 3 pike wi h all : ains 4i unkc.n. caused h! he ypical h unken iiiu ani. - Middle a id igh : >pike> seg ega ing i unhen and nca l> plump g ain and e ile lo e s as a csul o a ying he supp esso gene. we e supplied o a oid nu i ional s esses. and sup- plemen a y ligh was applied wi h sodium apou lamps. Emascula ions and pollina ions we e made by he au ho , who has almos 30 yea s o expe i- ence o c ossing ba ley. Fo SDS-PAGE (sodium dodecyl sulpha e. polyac ylamide gel elec opho e- sis). endospemis o indi idual de-emb yona ed. ma u e endospemis we e sampled om seg ega ing spikes and g ound as desc ibed elsewhe e (AHOK.AS 1988). De-emb yona ed endospemis a he incipien >cllou.-ma u i y s age we c homogenized in 1 .S-ml bppendo ubes wi h Eppendo inic opes les in I,IEVW !'s ( 1970) sample bu e wi hou 2-me cap- oe hunol. To de e mine he necessa y bu e Lolu ne in mic oli es, he esh heigh in milli- p am5 a mul iplied by 7.3. The samples we e hea ed a 97°C o 2 min. a e which 2-me cap o- e hanol was added o make 5 % o he o al olume. The p e iously epo ed SDS-PAGE p ocedu e (.A mK 5 1988) was ollowed in o he espec s. He e ozygo es as well as F, plan s display a 1:3 a io o seg ega ion o sh unken o no mal g ains in hei spikes. The F, spikes o he sh unken mu- an x c . Ado a displayed 769 sh unken g ains and 237 1 plump g ains: x' = 0.435. P>0.50. When hese plump g ains we e plan ed, he e was a seg e- ga ion in he F? o all-plump o sh unken-and- plump g ained plan s in he a io o 7 1 o 137 which i s wi h I:?, x2 = 0.060, P>0.70. The sh unken g ains esul ed in F2 plan s wi h slow g ow h, low e ili y and s un ed appea ance. Thcse homozygous sh unken plan s make o be e g ow h unde g eenhouse condi ions han in he ield. The Bonii- like gene ic backg ound is ela i ely ui ul in he g eenhouse en i onmen . The lowes g ain in a spike may a ely show a plump phenocopy. The ollowing sh unken mu an s exhibi ing xenia, Riss 13 in Bomi, Riso 56 in Ca lsbe p 11, Riso 1508 in Bomi. and ciniol in High-Amylose Glacie we e no ound o be allelic wi h he p esen mu an as judged om he F, spike seg ega ion. Fu he , he F, plan s g own in he ield did no display s un ed g ow h. The nea ly plump g ains o plan s seg ega ing sh unken. nea ly plump and s e ile (Fig. I) we e g own in he g eenhouse in wo en i onmen s. En- i onmen 1 esul ed in 820 sh unken, 674 nea ly plump g ains. and 230 s e ile lo e s. En i onmen 2 esul ed in 828 sh unken, 555 nea ly plump g ains. and 227 s e ile lo e s, he espec i e o als being 1648, 1229. and 357. Assuming ha he s e ile lo e s con ain a p opo ion o s e ili y in- duced by he lowe ed adap a ion o he geno ype, and ha he game ic ansmission o he gene nak- ing he e ozygo es nea ly plump is lowe ed, he seed popula ion may ollow Ha dy's and Weinbe g's equa ion, p2:2pq:q' = 1648: 1229: [457 - (numbe o s e ile lo e s due o o he easons)]. The p e- dic ed alue o q' would be 229, and he amoun o s e ili y due o o he easons would be 6.8 96. The a io 1648: 1229229 de ia es signi ican ly om 12: 1 in a way ha he game ic ansmission o he supp esso ac o leading o nea ly plump g ains and s e ile lo e s mus be educed. Sh unken seeds om he o iginal mu an plan seg ega ing sh unken, nea ly plump and s e ile lo e s we e aised o wo successi e gene a ions as lines. p o ing o be s able and yielding only sh unken g ains. C osses we e done o es whe he hese lines would ac ually be ca ie s o wo kinds o sh unken mu an s wi h di e en mu a ion si es o ep esen ing di e en s a es o a ansposable elemen (SCHWARZ-SOMMEK e al. 1985), esul ing He -edi us 114 (1991) BRIEF REPORT 283 in a nea ly plump g ain when he e ozygous. The se en sh unken lines we e c ossed in 18 o he 21 possible di e en pai s. Had he wo mu an s oc- cu ed in a 1:l a io among he se en sh unken lines, ei he o he mu an s should appea a leas in one o he lines a P=0.99. Fi e F1 plan s o each c oss we e g own and p o ed o be all- sh unken, he lines hus ca ying he ypical sh unken gene. This was also e idenced by c ossing wo o he lines wi h he ypical sh unken ca ie line. Two o he lines we e c ossed as a pollen pa en wi h plan s seg ega ing sh unken, nea ly plump and s e ile lo e s. Because emascula ed wi hou cu ing he husks, he F(l) hyb id g ains we e well- o med and pe mi ed he ollowing clas- si ica ion: wen y sh unken, eigh nea ly plump and i e s e ile. The seg ega ion was also con i med by he seg ega ion o he co esponding FI gene a ion spikes. The hypo he ical aa:Aa numbe s o 20:s i well wi h he a io o he ex ac ed alues o 1648:( 1229/2) om he sel ing da a abo e (x' = 0.002, P>0.95). The e a e se e al quan i a i e di e ences be- ween nea ly plump and sh unken endospe mal p o- ein bands e ealed by SDS-PAGE bo h in im- ma u e and ma u e g ains (Fig. 2). The SDS-PAGE also shows wo quali a i e di e ences. The blue band o 66.4 kg mol-' is p esen in sh unken and absen om nea ly plump endospe ms. Nea ly plump g ains ha e a pu ple s aining band o 61.3 kg mol-I, which zone is ep esen ed by a blue band in sh unken endospe mal p o eins. The s aining p o- cedu e used pe mi s isual dis inc ion o he wo shades o colou on gels. The accumula ion o he majo ho deins anging om 33 o 46 kg mol-' is g ea e in nea ly plump g ains han in sh unken g ains (Fig. 2). The p esen da a indica e ha he supp esso gene causing he nea ly plump pheno ype has a le hal e ec when homozygous, and has a dec eased ans- mission h ough game es. Two game ophy ic ac- o s causing abe an seg ega ion a ios ha e been desc ibed in ba ley (TABATA 1961: KoNIsHI and MATSUURA 1988; KONlSHI and ABE 1990). The e - ec o he supp esso gene in a non-sh unken gene ic backg ound is no known. I canno be con- cluded ye , whe he he supp esso gene is allelic wi h he ypical sh unken allele, o whe he i occu- pies ano he locus. This can p obably be esol ed when he co esponding genes ha e been cloned. AcP,io~,/ed~~emen s. - The s udy was mainly ca ied ou unde he auspices o he Academy o Finland ( he Resea ch Council o Ag icul u e and Fo es y). Fig. 2. Sh unken (b and d) and nea ly plump (c and e) endospe mal p o eins ac iona ed wi h SDS-PAGE. T ack a shows he molecula weigh s anda ds in kg mol-'. The a ow head indica es he 66.4 kg mol-' band, and he do , he 61.3 kg mol-' band, which s ains blue in sh unken and pu ple in plump samples. Re e ences AHOKAS, H. 1988. High-lysine gene seg ega ion dis o ed in he ba ley c oss Rise I508 x C yp CI 1090: pa e ns o endospe m p o eins by an elec opho e ic me hod. - He edi as 108: 129- 131 FUERSTENBERG, S. I. and JOHNS, M. A. 1990. Dis ibu ion o Bsl e o ansposons in Zea and ela ed gene a. - Th o . App/. Gene . 80: 68G686 GRIEL, A,, SAEDLER, H. and PETERSON, P. A. 1989. Maize anspoa- able elemen s. - Annu. Re . Gene . 23: 7 1-85 KONISHI, T. and ABE, K. 1990. Geog aphical dis ibu ion o he Gu2 gene o ce a ion in ba ley. - Bu ley Gene . Ne~,s/. 19: 4446 S(- i[ L I.. , '4. H. and A iw s, H. 1990. A no el sh unken endo- pemi mu an o ba ley. ~ Phwol. Plun . 7X: 583-589 S - iw*~L-So im~, 2s.. GKII-L. A.. BERNIYKXN, R. and SAEDLER, H. 19x5. Sequence co npa ision o 'Ela es' o a/-ml sugge. model o Sp n (En) ac ion. - EM80 J. 4: 2439-2443. Si io s. S. P. and SowxLi .i.E. S. C. 1988. In es iga ion o a gene ic ins abili y in ba ley. - Bu l ! Gene . N wd. 18: 37- 4s. and E a um 1990. - Bu -lcy Gene . Ne~,. /. 19: 86-87 Soc; ni~ B. and WI:II-S~I I~-KNOWLES, P. 'ON. 1987. Ba ley: gene and ch omosomes. - Cu -1,h -g R s. Conm . 52: 123- I96 T. B. I. . M. 1961. S udies o a game ophy e lac o in ba ley. ~ .lup .I Ge . 36: 1.57-167 Wis . R. P. and ELL.I GBOE. A. H. 1985. Fine s uc u e and ins abil- i ) o he MI-o Ioc i in ha lcy. - G i(.. Ill: 113-130