Mutants and duplication in chromosome 7 (syn. 5H) in the barley line HA21: duplications may enhance QTLs and serve to make constant linear cis-heterozygosity

He edi as 128: 167-171 (1998) Mu an s and duplica ion in ch omosome 7 (syn. 5H) in he ba ley line HA21: duplica ions may enhance QTLs and se e o make cons an linea cis-he e ozygosi y HANNU AHOKAS’.2 and MARIA J. ERKKILA2 Di ision o Gene ics, PL 56, FI-00014 Uni e si y o Helsinki, Finland C ops and Soil, ARC, FI-31600 Jokioinen, Finhnd Ahokas, H. and E kkila, M.J. 1998. Mu an s and duplica ion in ch omosome 7 (syn. 5H) in he ba ley line HA21: duplica ions may enhance QTLs and se e o make cons an cis-he e ozygosi y. - He edi as 128: 167- 171. Lund, Sweden. ISSN 0018-0661. Recei ed Ma ch 4, 1998. Accep ed Ma ch 6, 1998 Cy ological and linkage da a indica e a duplica ion in he sho a m o ch omosome 7 (syn. 5H) in he mu an line HA21 (ba ley, Ho deum uulga e, c . ‘Pi kka’). The associa ed mu an (ha21) shows a weigh ed a e age linkage o 22.1 cM wi h pld, hi he o an igno ed an hocyaninless gene, o c . Pi kka. Some c osses p oduce F, seg egan s wi h an exagge a ed ha21 pheno ype which may ep esen posi ion e ec o inc eased dosage o he mu an gene h ough ecombina ion. Compa ed wi h c . Pi kka, HA21 has changes in g ain chemis y (a- and P-amylase, P-glucanase), which may be caused by changed QTL dosage o QTL posi ion e ec due o duplica ion. The use o duplica ion in c ea ing cons an +m/+m o n + /m + linea cis-he e ozygo es is sugges ed. Linea cis-he e ozygo es may p oduce s able he e osis o a enua e he undesi ed e ec s o d as ic mu an s. Hannu Ahokas, C ops and Soil, ARC, Mylly ie 10, FI-31600 Jokioinen, Finland. E-mail: [email p o ec ed] A mo phological a ian was ound in a ield o c . ‘Pi kka’, a six- owed ba ley (Ho deum ulga e L.), by he i s au ho when he was a schoolboy in 1965. This ueb eeding a ian , HA21 (as a mu an ha21), has small, sessile o subsessile la e al lo e s ha may emain s e ile unde some condi ions. I some imes has an ab up ape ing lemma, ine awns, a endency o pis illody, and semidwa ism ha does no espond o gibbe ellic acid (GA) and GA, (AHOKAS 1973). HA21 has an inc eased p o ein con en (AHOKAS 1977). HA21 has also been class ied as a us ( usi o m o py amid-shaped spike) mu an (FRANCKOWIAK and PECIO 1992). Two di e en F2s, wi h HA21 as one pa en , showed a e seg egan s o dwa ed, s e ile plan s wi h exagge a ed ha21 cha ac e is ics. These seg egan s we e hypo hesized o ha e been due o an inc ease in gene dosage h ough ecombina ion, HA21 i sel possibly ca ying a duplica ion. Meiosis in he HA21 x Pi kka F, hyb id showed a he e omo phic bi alen o a sa elli e pai , which sup- po s he duplica ion explana ion (H.A., unpub- lished). When HA21 was s udied as pa o a gene ics labo a o y exce cise, i was ound ha HA21 has, o he wo possible sa elli e ch omosomes, somewha longe sho a m o ch omosome 7 [syn. 5H acco d- ing o he newes e ision (LINDE-LAURSEN e al. 1997)] han c . Pi kka a mi o ic me aphase (STENIUS e al. 1976). Linkage da a o ansloca ion es e s wi h he duplica ion o ch omosome 7 (5H) a e p e- sen ed below. The use o duplica ion in making ixed linea cis- he e ozygo es h ough ecombina ion is indica ed. Linea cis- he e ozygosi y, a anged by a duplica ion, may esul in a ixed he e osis, o i may a enua e he ad e se e ec s o d as ic mu an genes ep esen ed by bo h he mu an and wild allele. Dosage o quan i- a a i e ai loci (QTL) may also be inc eased by duplica ion. Cons an he e osis a anged wi h dupli- ca ions o o he ype has ecen ly been sugges ed by GRAMATIKOVA (1 995). MATERIAL AND METHODS Plan s. - T ansloca ion lines we e ob ained om D . P. Hagbe g (The Swedish Uni e si y o Ag icul u al Sciences, S alo , Sweden). The non-mu an geno ype o c . ‘KO A’ was a seg egan om he gene ic s ock hu 184a ob ained om D . T. Tsuchiya (Colo ado S a e Uni e si y, USA). The line ca ying he msg44cx gene (male s e ile) was a hyb id de i a i e om he c oss (Pi kka x AHOR 2680) x (AHOR 2680 x ‘Paa o’). Paa o SCSl is a seg ega ing cy o- plasmic s eak mu an (AHOKAS 1976). C ‘O a’ and Paa o we e om a comme cial seed lo . A single plan o each was used o c osses, excep om Pi kka, whe e se e al plan s ha e been used wi hou a iable esul s. Seg ega ions we e eco ded unde ield condi ions. G ain enzyme ac i i ies. - HA21 (also accessioned as PI 349682) and i s o iginal c . Pi kka (Tammis o line a4459) we e plan ed in single ows, side by side, boa ded by ows o o he ba leys in he ield on sil y clay e ilized wi h 400 kg ha-’ o 20-10-10 (NPK) in 168 H. Ahokas and M. J. E kkilu He edi as 128 (1998) Fig. 1. a and b. Me aphase I in he F, hyb id o HA21 x Pi kka. a A he e omo phic loose ing bi alen (a ow). b A he e omo phic od bi alen . The sho a m o he longe ch omosome has wo majo coils be ween he cen ome e (c) and he seconda y cons ic ion (s), while he sho e has one. Scale = 10 pm. Elimaki (in 1990 and 1993) and on sandy clay wi h 550 kg ha-' o 20-4-8 in Jokioinen (in 1996) in sou he n Finland. Ex ac s o enzyme ac i i ies we e made o husked, su ace s e ilized g ain asep ically ge mina ed o 5 days a 15"C, as desc ibed p e iously (AHOKAS and NASKALI 1990). The p o ein in ex ac s was measu ed wi h BSA as a s anda d by he UV me hod o AHOKAS (1978), a-amylase by Ce alpha, P-amy- lase by Be amyl, and endo-P-glucanase by azo-ba ley glucan (Biocon, Megazyme) as desc ibed p e iously (AHOKAS and NASKALI 1990; AHOKAS and ERKKILA 1992). Linkage and cy ology. - Linkage was analyzed wi h MAPMAKER and is epo ed in Haldane cM (LAN- DER e al. 1987). Cy ological meio ic ma e ial was ixed in ace o e hanol (1:3) and s ained using he Feulgen p ocedu e. RESULTS Cy ology. -The meio ic di ision o HA21 x Pi kka showed a he e omo phic bi alen , suppo ing he hy- po hesis ha HA21 is he esul o a duplica ion. Special cases showed a double-coil dimension on he sho a m o a sa elli e-ca ying ch omosome, i.e., no. 6 (6H) o 7 (5H) (Fig. la and b). The he e omo phic bi alen appea ed as a od wi h a highe p opo ion (8.1 YO) han all he o he bi alen s (2.4%) in 246 me aphase I PMCs s udied. Seg ega ion and linkage. -The c oss o HA21 x Pi kka, seg ega ed a F, ha21 and wild ype i ing a 1:3 a io (Table 1). C osses o HA21 wi h c . Pi kka, Paa o and wi h KO A did no p oduce exagge a ed seg egan s a F,. These exagge a ed seg egan s we e only obse ed in he c oss wi h c . O a and he line HA72-62. The pale au icle colou @la) o HA21 and c . Pi kka was ound o be linked wi h he ha21 pheno ype. Linkage es ima es anged om 10.8 o 38.2 cM (Table l), wi h a weigh ed a e age o 22.1 cM. No linkage was e iden be ween ha21 and (six- owed, syn. sl), s (sho achilla hai , syn. s h, ms h), o msg44cx (male s e ile) no be ween pla and The associa ion o ha21 wi h he ansloca ion b eak poin s was es ed in he F2 o each c oss. The da a suppo linkage o he sho a m o ch omosome 7 (5H) (Table 2). The ha21 locus shows linkages wi h he b eak-poin s o T3-7d, T6-7ae, and T6-7k on he sho a m, and ha o T1-7 appa en ly close o he cen ome e o he ch omosome 7 (5H). S. G ail1 enzyme ac i i ies. - HA2 1 had a highe seed mass. The ac i i y o P-amylase, a s o ed seed p o ein, was signi ican ly highe in HA21 han in c . Pi kka (Table 3). In con as , he ac i i ies o a-amy- lase and P-glucanase, which appea du ing ge mina- ion, we e lowe in HA21 han in c . Pi kka a e i e days o ge mina ion. The enzyme ac i i y di e ences a e consis en on bo h soluble p o ein and g ain mass basis. DISCUSSION Linkuge. -The locus ha21 is loosely linked o he pale au icle @la) gene o c . Pi kka (Table 1). The pla mu a ion makes he epigeal o gans ee om an hocyanins, o highly educes he con en s. Ab- sence o an hocyanins, caused by a ious loci, is a ea u e in many cul i a s, hough a locus assigned o ch omosome 7 (5H) is unknown (JENDE-STRID 1993, 1995). The exagge a ed seg egan s o wo o he HA21 c osses could be asc ibed o dosage o posi- ional e ec s. The mu an ha21 is p obably associa ed wi h he duplica ion. He edi as 128 (1998) Spon aneous mu an s in ba ley ch omosome 7 (557 169 Table 1. F2 seg ega ion o ha21 and o he genes Pedig ee O he Numbe Pheno ype numbe s o Fi o Numbe o huZl-plu genes o ha21 and o he mu an 3.1 o exagge - linkages (m2) plan s ha21 a ed ha21 (cM) ha21,m, hu21,+ +,m, +,+ seg egan s HA21/Pi kka - 852 Paa o/HA21 Plu 353 Paa o SCSl/HA21 Plu 39 HA21/Ko A Plu 95 Di o S Di o HA21/0 a Pla 154 HA72-62/HA21 msg44cx 104 Di o V - 72 6 13 9 8 26 2 5 204 17 5 7 11 12 14 23 20 - 648 P>O.30 0 - 16 248 P>0.90 0 10.8 4 24 P>0.50 0 34.9 13 62 P>O.50 0 32.9 22 53 22 53 25 89 P= 0.02”) 16 38.2 17 62 P>0.90 2 - 15 64 a Wi h he exagge a ed 16 excluded, P > 0.90 Th ee o he ou ansloca ion b eak-poin s show- ing linkage wi h ha21, iz. T3-7d (KASHA and BURN- T6-7ae (HAGBERG e al. 1978), and T6-7k (HAGBERG e al. 1978; LINDE-LAURSEN 1988) ha e been indi- ca ed o be on he sho a m o ch omosome 7 (5H), while ha o T1-7 has been indica ed o be on he long a m be ween S and he cen ome e (PERSSON 1969). The locus S was mapped wi h in e ening ma ke s on he long a m o ch omosome 7 (5H) o be 49.1 Kosambi cM om he cen ome e (KLEINHOFS e al. 1993: Fig. 7) and appa en ly somewha less in he in eg a ed map o QI e al. (1996). The obse ed ee ecombina ion o S wi h ha21, and wi h pla, is in acco dance wi h he maps. G ain enzyme ac i i ies. -Though c . Pi kka has a high P-amylase ac i i y (SIMBERG 1950; ALLISON and SWANSTON 1974), he P-amylase le el is signi ican ly HAM 1965; PERSON 1969; LINDE-LAURSEN 1988), Table 2. F2 seg ega ion o ha21 and pa ial e ili y o ansloca ion es e c osses T ansloca ion Fe ile Pa ially B eakpoin e ile linkage o ha21 (cM) (+) hu21/haZ (+) hu22/ha21 T1-3b TI-7 T2-3g T2-7b T3-7c T3-7d T2-5a T4-5e T6-7ae T6-7i T4-7b T6-7k 77 48 44 31 93 40 98 34 95 36 68 44 89 37 63 35 78 12 84 29 84 35 36 7 78 34 69 15 30.0 97 42 84 42 89 40 82 27 87 20 33.1 62 48 37 11 82 20 36.5 76 25 63 15 34.9 highe in HA21 (Table 3) in samples ge mina ed o i e days. Be a-amylase eaches i s maximum ac i i y le el a e abou i e days o ge mina ion (GRIME and BRIGGS 1995; EVANS e al. 1997). The seed-ex- p essed P-amylase locus is on he ch omosome 4 (4H) (KREIS e al. 1988). The 2/3 o 3/4 le el o a-amylase ac i i y in HA21, compa ed wi h Pi kka, a e i e days o ge mina ion, may be a ibu ed o se e al causes. HA21 may ha e an ele a ed le el o a-amylase inhibi o o an al e ed ime-scale o he induc ion o a-amylase du ing ge - mina ion. The coding loci o a-amylase appea on ch omosomes 1 (7H) and 6 (6H) (BROWN and JACOB- SEN 1982). The sho a m o ch omosome 7 (5H) has QTLs o a-amylase, soluble p o ein, g ain p o ein (TINKER and MATHER 1994; OZIEL e al. 1996), g ain mass/ olume, and plan heigh (TINKER and MATHER 1994). QTLs o dias a ic powe , which closely co e- la es wi h P-amylase ac i i y (ALLISON and SWANSTON 1974; SANTOS and RIIS 1996), appea in he sho a m o ch omosome 7 (5H) (OZIEL e al. 1996; THOMAS e al. 1996). O he epo s also show ha ch omosome 7 (5H) has QTLs La. o a-amylase and g ain p o ein (HAYES and IYAMABO 1994; HAYES e al. 1993; HAN and ULLRICH 1994; OZIEL e al. 1996; MATHER e al. 1997) o g ain ni ogen con en (BEZANT e al. 1997; MATHER e al. 19971, g ain mass, and soluble p o ein (MATHER e al. 1997). G ain size and p o ein, plan heigh , a- and P-amylase a e also a ec ed in HA21 as compa ed wi h c . Pi kka (Table 3 and e e ences in he in o- duc ion). Duplica ion o posi ion e ec s o QTLs in HA21 may be a eason o he changes. The e ap- pea s o be quan i a i e di e ences in g ain p o ein bands as e ealed by SDS-PAGE o ac iona ed p o eins in HA21 and c . Pi kka (unpublished). 170 H. Ahokas and M. J. E kkila He edi as 128 (1 998) Table 3. Ac i i ies o enzymes a e i e days o ge mina ion in he ex ac s o HA21 and he o iginal cul i a Pi kka. Means o ha es s o h ee seasons, 1990 and 1993 in Elimaki, and 1996 in Jokioinen Ba ley Mean g ain Ex ac ed a-Amylase (U) p-Amylase (U) P-Glucanase (U) mass (mg) p o ein (”/.) (g g ains)-’ (g ex ac ed (g g ains)-’ (mg ex ac ed (kg g ains)-’ (g ex ac ed p o ein)- ’ p o ein)- ’ p o ein)-’ HA21 49.3k1.7 2.9k0.7 96.6 2.3 2166+13 1005k9 22.1i1.2 1221 54.3 28.8 2.0 Pi kka 41.8 k 1.0 2.3 0.2 127.7 1.8 3443 _+ 11 698 9 18.6 1.4 1446 6.2 39.1 1.3 Tes F= 8.362 U = 3 Sign es P P = 0.01 P = 0.35 P < 0.01 Sign es P < 0.02 Sign es P < 0.01 Duplica ion induc ion. - Duplica ions ha e been p o- duced by c ossing pa ially o e lapping ansloca- ions (HAGBERG and HAGBERG 1992). A special cha ac e is ic, le el o oo -associa ed bac e ia, o a duplica ion ca ie has been ound in ba ley (HAG- BERG and HAGBERG 1987; LILJEROTH and BAATH 1988; LILJEROTH e al. 1994). While duplica ions may inc ease he dosage o desi ed loci, con ol o he ansc ip ional le el by DNA me hyla ion may occu in associa ion wi h duplica ions (SUBRAHMANYAM e al. 1994; PRADHAN and SUBRAHMANYAM 1995). A iplica ion wi h ixed he e ozygosi y o he es e ase 4 locus may ha e occu ed in an Is aeli H. ulga e ssp. spon aneum (C. Koch) A. & G . (KAHLER e al. 1981; SOLIMAN and ALLARD 1989). Radia ion can induce duplica ions in ce eals (MACKEY 1954). C . Pi kka was eleased in 1952, and HA21 was ound in 1965, du ing he busy nu- clea es ing pe iod. O he o med and moni o ed adionuclides, 90S and I3’Cs deposi ions in he 1961- 1965 pen ad we e, espec i ely, 81 and 67 imes ha de ec ed in 1981-1985; he maximum occu ed in 1962, when he a e age a e was 850 Bq mP2 o 90S + 13’Cs in Finland (PAAKKOLA 1988). Signi ican di e ences exis be ween ba ley cul i a s a accumu- o e , he o iginal Pi kka ield was in an a ea (Elimaki) whe e he ex e nal, mos ly ock-emi ed adia ion shows a ela i ely high mean dose a e, 17.5 pR h-’ a 1 m heigh (LEMMELA 1984). la ing O 137cS (BHLENSCHLEGER e al. 1993). Mo e- B eeding wi h duplica ions. - Linea cis-he e ozygo - es may be p oduced by a anging di e en allele composi ion in he duplica ed segmen s. This migh esul in a ou able cases o ixed he e osis o possi- bly a ennua e he e ec o d as ic mu an s. S able a angemen s o he cons i u ions +m/ + m o m + / m + could be use ul o b eeding. The ela ionship o HA21 duplica ion wi h lys3 (high lysine) locus is being s udied. The lys3 locus shows a igh linkage wi h he T3-7d b eakpoin (JENSEN 1979). I may also be possible o inc ease he QTL numbe a ec ing o he loci wi h duplica ion. In HA21, changed le els in a- and P-amylase, P-glucanase, g ain mass and plan heigh may ha e occu ed by QTLs. REFERENCES Ahokas H, (1973). 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