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

Hereditas 128: 167-171 (1998) 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 HANNU AHOKAS’.2 and MARIA J. ERKKILA2 Division of Genetics, PL 56, FI-00014 University of Helsinki, Finland Crops and Soil, ARC, FI-31600 Jokioinen, Finhnd Ahokas, H. and Erkkila, M.J. 1998. Mutants and duplication in chromosome 7 (syn. 5H) in the barley line HA21: duplications may enhance QTLs and serve to make constant cis-heterozygosity. - Hereditas 128: 167171. Lund, Sweden. ISSN 0018-0661. Received March 4, 1998. Accepted March 6, 1998 Cytological and linkage data indicate a duplication in the short arm of chromosome 7 (syn. 5H) in the mutant line HA21 (barley, Hordeum uulgare, cv. ‘Pirkka’). The associated mutant (ha21) shows a weighted average linkage of 22.1 cM with pld, hitherto an ignored anthocyaninless gene, of cv. Pirkka. Some crosses produce F, segregants with an exaggerated ha21 phenotype which may represent position effect or increased dosage of the mutant gene through recombination. Compared with cv. Pirkka, HA21 has changes in grain chemistry (aand P-amylase, P-glucanase), which may be caused by changed QTL dosage or QTL position effect due to duplication. The use of duplication in creating constant +m/+m or rn + /m + linear cis-heterozygotes is suggested. Linear cis-heterozygotes may produce stable heterosis or attenuate the undesired effects of drastic mutants. Hannu Ahokas, Crops and Soil, ARC, Myllytie 10, FI-31600 Jokioinen, Finland. E-mail: [email protected] A morphological variant was found in a field of cv. ‘Pirkka’, a six-rowed barley (Hordeum vulgare L.), by the first author when he was a schoolboy in 1965. This truebreeding variant, HA21 (as a mutant ha21), has small, sessile to subsessile lateral florets that may remain sterile under some conditions. It sometimes has an abrupt tapering lemma, fine awns, a tendency to pistillody, and semidwarfism that does not respond to gibberellic acid (GA) and GA, (AHOKAS 1973). HA21 has an increased protein content (AHOKAS 1977). HA21 has also been classfied as a fus (fusiform or pyramid-shaped spike) mutant (FRANCKOWIAK and PECIO 1992). Two different F2s, with HA21 as one parent, showed rare segregants of dwarfed, sterile plants with exaggerated ha21 characteristics. These segregants were hypothesized to have been due to an increase in gene dosage through recombination, HA21 itself possibly carrying a duplication. Meiosis in the HA21 x Pirkka F, hybrid showed a heteromorphic bivalent of a satellite pair, which supports the duplication explanation (H.A., unpublished). When HA21 was studied as part of a genetics laboratory excercise, it was found that HA21 has, of the two possible satellite chromosomes, somewhat longer short arm of chromosome 7 [syn. 5H according to the newest revision (LINDE-LAURSEN et al. 1997)] than cv. Pirkka at mitotic metaphase (STENIUS et al. 1976). Linkage data of translocation testers with the duplication of chromosome 7 (5H) are presented below. The use of duplication in making fixed linear cisheterozygotes through recombination is indicated. Linear cisheterozygosity, arranged by a duplication, may result in a fixed heterosis, or it may attenuate the adverse effects of drastic mutant genes represented by both the mutant and wild allele. Dosage of quantitatative trait loci (QTL) may also be increased by duplication. Constant heterosis arranged with duplications of other type has recently been suggested by GRAMATIKOVA (1 995). MATERIAL AND METHODS Plants. - Translocation lines were obtained from Dr. P. Hagberg (The Swedish University of Agricultural Sciences, Svalov, Sweden). The non-mutant genotype of cv. ‘KO A’ was a segregant from the genetic stock hut 184a obtained from Dr. T. Tsuchiya (Colorado State University, USA). The line carrying the msg44cx gene (male sterile) was a hybrid derivative from the cross (Pirkka x AHOR 2680) x (AHOR 2680 x ‘Paavo’). Paavo SCSl is a segregating cytoplasmic streak mutant (AHOKAS 1976). Cv ‘Otra’ and Paavo were from a commercial seed lot. A single plant of each was used for crosses, except from Pirkka, where several plants have been used without variable results. Segregations were recorded under field conditions. Grain enzyme activities. - HA21 (also accessioned as PI 349682) and its original cv. Pirkka (Tammisto line a4459) were planted in single rows, side by side, boarded by rows of other barleys in the field on silty clay fertilized with 400 kg ha-’ of 20-10-10 (NPK) in 168 H. Ahokas and M. J. Erkkilu Hereditas 128 (1998) Fig. 1. a and b. Metaphase I in the F, hybrid of HA21 x Pirkka. a A heteromorphic loose ring bivalent (arrow). b A heteromorphic rod bivalent. The short arm of the longer chromosome has two major coils between the centromere (c) and the secondary constriction (s), while the shorter has one. Scale = 10 pm. Elimaki (in 1990 and 1993) and on sandy clay with 550 kg ha-' of 20-4-8 in Jokioinen (in 1996) in southern Finland. Extracts for enzyme activities were made of husked, surface sterilized grain aseptically germinated for 5 days at 15"C, as described previously (AHOKAS and NASKALI 1990). The protein in extracts was measured with BSA as a standard by the UV method of AHOKAS (1978), a-amylase by Ceralpha, P-amylase by Betamyl, and endo-P-glucanase by azo-barley glucan (Biocon, Megazyme) as described previously (AHOKAS and NASKALI 1990; AHOKAS and ERKKILA 1992). Linkage and cytology. - Linkage was analyzed with MAPMAKER and is reported in Haldane cM (LANDER et al. 1987). Cytological meiotic material was fixed in aceto ethanol (1:3) and stained using the Feulgen procedure. RESULTS Cytology. -The meiotic division of HA21 x Pirkka showed a heteromorphic bivalent, supporting the hypothesis that HA21 is the result of a duplication. Special cases showed a double-coil dimension on the short arm of a satellite-carrying chromosome, i.e., no. 6 (6H) or 7 (5H) (Fig. la and b). The heteromorphic bivalent appeared as a rod with a higher proportion (8.1 YO) than all the other bivalents (2.4%) in 246 metaphase I PMCs studied. Segregation and linkage. -The cross of HA21 x Pirkka, segregated at F, ha21 and wild type fitting a 1:3 ratio (Table 1). Crosses of HA21 with cv. Pirkka, Paavo and with KO A did not produce exaggerated segregants at F,. These exaggerated segregants were only observed in the cross with cv. Otra and the line HA72-62. The pale auricle colour @la) of HA21 and cv. Pirkka was found to be linked with the ha21 phenotype. Linkage estimates ranged from 10.8 to 38.2 cM (Table l), with a weighted average of 22.1 cM. No linkage was evident between ha21 and v (six-rowed, syn. vrsl), s (short rachilla hair, syn. srh, msrh), or msg44cx (male sterile) nor between pla and The association of ha21 with the translocation break points was tested in the F2 of each cross. The data support linkage to the short arm of chromosome 7 (5H) (Table 2). The ha21 locus shows linkages with the break-points of T3-7d, T6-7ae, and T6-7k on the short arm, and that of T1-7f apparently close to the centromere of the chromosome 7 (5H). S. Grail1 enzyme activities. - HA2 1 had a higher seed mass. The activity of P-amylase, a stored seed protein, was significantly higher in HA21 than in cv. Pirkka (Table 3). In contrast, the activities of a-amylase and P-glucanase, which appear during germination, were lower in HA21 than in cv. Pirkka after five days of germination. The enzyme activity differences are consistent on both soluble protein and grain mass basis. DISCUSSION Linkuge. -The locus ha21 is loosely linked to the pale auricle @la) gene of cv. Pirkka (Table 1). The pla mutation makes the epigeal organs free from anthocyanins, or highly reduces the contents. Absence of anthocyanins, caused by various loci, is a feature in many cultivars, though a locus assigned to chromosome 7 (5H) is unknown (JENDE-STRID 1993, 1995). The exaggerated segregants of two of the HA21 crosses could be ascribed to dosage or positional effects. The mutant ha21 is probably associated with the duplication. Hereditas 128 (1998) Spontaneous mutants in barley chromosome 7 (557 169 Table 1. F2 segregation of ha21 and other genes Pedigree Other Number Phenotype numbers of Fit to Number of huZl-plu genes of ha21 and other mutant 3.1 for exaggerlinkages (m2) plants ha21 ated ha21 (cM) ha21,m, hu21,+ +,m, +,+ segregants HA21/Pirkka - 852 Paavo/HA21 Plu 353 Paavo SCSl/HA21 Plu 39 HA21/Ko A Plu 95 Ditto S Ditto v HA21/0tra Pla 154 HA72-62/HA21 msg44cx 104 Ditto 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 With the exaggerated 16 excluded, P > 0.90 Three of the four translocation break-points showing linkage with ha21, viz. T3-7d (KASHA and BURNT6-7ae (HAGBERG et al. 1978), and T6-7k (HAGBERG et al. 1978; LINDE-LAURSEN 1988) have been indicated to be on the short arm of chromosome 7 (5H), while that of T1-7f has been indicated to be on the long arm between S and the centromere (PERSSON 1969). The locus S was mapped with intervening markers on the long arm of chromosome 7 (5H) to be 49.1 Kosambi cM from the centromere (KLEINHOFS et al. 1993: Fig. 7) and apparently somewhat less in the integrated map of QI et al. (1996). The observed free recombination of S with ha21, and with pla, is in accordance with the maps. Grain enzyme activities. -Though cv. Pirkka has a high P-amylase activity (SIMBERG 1950; ALLISON and SWANSTON 1974), the P-amylase level is significantly HAM 1965; PERSON 1969; LINDE-LAURSEN 1988), Table 2. F2 segregation of ha21 and partial fertility of translocation tester crosses Translocation Fertile Partially Breakpoint fertile linkage to ha21 (cM) (+) hu21/haZf (+) hu22/ha21 T1-3b TI-7f 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 higher in HA21 (Table 3) in samples germinated for five days. Beta-amylase reaches its maximum activity level after about five days of germination (GRIME and BRIGGS 1995; EVANS et al. 1997). The seed-expressed P-amylase locus is on the chromosome 4 (4H) (KREIS et al. 1988). The 2/3 or 3/4 level of a-amylase activity in HA21, compared with Pirkka, after five days of germination, may be attributed to several causes. HA21 may have an elevated level of a-amylase inhibitor or an altered time-scale for the induction of a-amylase during germination. The coding loci of a-amylase appear on chromosomes 1 (7H) and 6 (6H) (BROWN and JACOBSEN 1982). The short arm of chromosome 7 (5H) has QTLs for a-amylase, soluble protein, grain protein (TINKER and MATHER 1994; OZIEL et al. 1996), grain mass/ volume, and plant height (TINKER and MATHER 1994). QTLs for diastatic power, which closely correlates with P-amylase activity (ALLISON and SWANSTON 1974; SANTOS and RIIS 1996), appear in the short arm of chromosome 7 (5H) (OZIEL et al. 1996; THOMAS et al. 1996). Other reports also show that chromosome 7 (5H) has QTLs La. for a-amylase and grain protein (HAYES and IYAMABO 1994; HAYES et al. 1993; HAN and ULLRICH 1994; OZIEL et al. 1996; MATHER et al. 1997) or grain nitrogen content (BEZANT et al. 1997; MATHER et al. 19971, grain mass, and soluble protein (MATHER et al. 1997). Grain size and protein, plant height, aand P-amylase are also affected in HA21 as compared with cv. Pirkka (Table 3 and references in the introduction). Duplication or position effects of QTLs in HA21 may be a reason for the changes. There appears to be quantitative differences in grain protein bands as revealed by SDS-PAGE of fractionated proteins in HA21 and cv. Pirkka (unpublished). 170 H. Ahokas and M. J. Erkkila Hereditas 128 (1 998) Table 3. Activities of enzymes afterfive days of germination in the extracts of HA21 and the original cultivar Pirkka. Means of harvests of three seasons, 1990 and 1993 in Elimaki, and 1996 in Jokioinen Barley Mean grain Extracted a-Amylase (U) p-Amylase (U) P-Glucanase (U) mass (mg) protein (”/.) (g grains)-’ (g extracted (g grains)-’ (mg extracted (kg grains)-’ (g extracted protein)- ’ protein)- ’ protein)-’ HA21 49.3k1.7 2.9k0.7 96.6f2.3 2166+13 1005k9 22.1i1.2 1221 54.3 28.8 f 2.0 Pirkka 41.8 k 1.0 2.3 f 0.2 127.7 f 1.8 3443 _+ 11 698 9 18.6 f 1.4 1446 f 6.2 39.1 1.3 Test F= 8.362 U = 3 Sign test P P = 0.01 P = 0.35 P < 0.01 Sign test P < 0.02 Sign test P < 0.01 Duplication induction. - Duplications have been produced by crossing partially overlapping translocations (HAGBERG and HAGBERG 1992). A special characteristic, level of root-associated bacteria, of a duplication carrier has been found in barley (HAGBERG and HAGBERG 1987; LILJEROTH and BAATH 1988; LILJEROTH et al. 1994). While duplications may increase the dosage of desired loci, control of the transcriptional level by DNA methylation may occur in association with duplications (SUBRAHMANYAM et al. 1994; PRADHAN and SUBRAHMANYAM 1995). A triplication with fixed heterozygosity for the esterase 4 locus may have occurred in an Israeli H. vulgare ssp. spontaneum (C. Koch) A. & Gr. (KAHLER et al. 1981; SOLIMAN and ALLARD 1989). Radiation can induce duplications in cereals (MACKEY 1954). Cv. Pirkka was released in 1952, and HA21 was found in 1965, during the busy nuclear testing period. Of the formed and monitored radionuclides, 90Sr and I3’Cs depositions in the 19611965 pentad were, respectively, 81 and 67 times that detected in 1981-1985; the maximum occurred in 1962, when the average rate was 850 Bq mP2 of 90Sr + 13’Cs in Finland (PAAKKOLA 1988). Significant differences exist between barley cultivars at accumuover, the original Pirkka field was in an area (Elimaki) where the external, mostly rock-emitted radiation shows a relatively high mean dose rate, 17.5 pR h-’ at 1 m height (LEMMELA 1984). lating Of 137cS (BHLENSCHLEGER et al. 1993). MoreBreeding with duplications. - Linear cis-heterozygotes may be produced by arranging different allele composition in the duplicated segments. This might result in favourable cases of fixed heterosis or possibly attennuate the effect of drastic mutants. Stable arrangements of the constitutions +m/ + m or m + / m + could be useful for breeding. The relationship of HA21 duplication with lys3 (high lysine) locus is being studied. The lys3 locus shows a tight linkage with the T3-7d breakpoint (JENSEN 1979). It may also be possible to increase the QTL number affecting other loci with duplication. In HA21, changed levels in aand P-amylase, P-glucanase, grain mass and plant height may have occurred by QTLs. REFERENCES Ahokas H, (1973). The possible use of mo-type mutant in hybrid barley production. Barley Genet. Newsl. 3: 4-5. Ahokas H, (1976). Two segregating cytoplasmic mutants of barley. Hereditas 82: 187192. Ahokas H, (1977). Increase in protein content by partial fertility. Barley Genet. Newsl. 7: 6-8. Ahokas H, (1978). A simple and rapid screening method for the determination of protein and tryptophan in kernel halves and small samples of barley meal. J. Sci. Food Agric. 29: 41-52. Ahokas H and Erkkila MJ, (1992). Barley P-amylase and Pglucanase activities at germination in vulgare-type lines from backcrosses of wild, spontaneum strains with cv. Adorra. Agric. Sci. Finl. 1: 339-350. Ahokas H and Naskali L, (1990). Variation of a-amylase, P-amylase, P-glucanase, pullulanase, proteinase and chitinase activity in germinated samples of the wild progenitor of barley. J. Inst. Brew. 96: 27-31. Allison MJ and Swanston JS, (1974). Relationships between P-amylase polymorphisms in developing, mature and germinating grains of barley. J. Inst. Brew. 80: 285-291. Bezant JH, Laurie DA, Pratchett N, Chojecki J and Kearsey MJ, (1997). Mapping of QTL controlling NIR predicted hot water extract and grain nitrogen content in a spring barley cross using marker-regression. Plant Breed. 116: 141-145. Brown AHD and Jacobsen JV, (1982). Genetic basis and natural variation of a-amylase isoenzymes in barley. Genet. Res. 40: 315-324. Evans DE, Wallace W, Lance RCM and MacLeod LC, (1997). Measurement of Beta-amylase in malting barley (Hordeum vulgare L.). 11. The effect of germination and kilning. J. Cereal Sci. 26: 241-250. Franckowiak JD and Pecio A, (1992). Coordinators’s report: semidwarf genes: a listing of genetic stocks. Barley Genet. Newsl. 21: 116-127. Gramatikova M, (1995). Duplikatsii pri echemika. (Summary in English: Barley duplications). Rast. Nauk. 23: 84-86. Grime KH and Briggs DE, (1995). Release and activation of barley P-amylase. J. Inst. Brew. 101: 337-343. Hereditas 128 (1998) Spontaneous mutants in barley chromosome 7 (559 171 Hagberg A and Hagberg G, (1987). Some vigorous and productive duplications in barley. Barley Genet. V: 423426. Hagberg A and Hagberg P, (1992). Coordinator’s report: Chromosome duplications. Stock list of lines to be included in the Nordic Gene Bank. Barley Genet. Newsl. 21: 130-136. Hagberg A, Lehmann L and Hagberg P, (1978). Segmental interchanges in barley. 11. Translocations involving chromosomes 6 and 7. Z. Pflanzenzucht. 81: 89-110. Han F, Ullrich SE and the North American Barley Genome Mapping Project, (1994). Mapping of quantitative trait loci associated with malting quality in barley. Barley Genet. Newsl. 23: 84-97. Hayes PM, Iyamabo 0 and the North American Barley Genome Mapping Project, (1994). Summary of QTL effects on the Steptoe x Morex population. Barley Genet. Newsl. 23: 98-143. Hayes PM, Liu BH, Knapp SJ, Chen F, Jones B, Blake T, Franckowiak J, Rasmusson D, Sorrells M, Ullrich SE, Wesenberg D and Kleinhofs A, (1993). Quantitative trait locus effects and environmental interaction in a sample of North American barley germ plasm. Theor. Appl. Genet. 87: 392-401. Jende-Strid B, (1993). Genetic control of flavonoid biosynthesis in barley. Hereditas 119: 187-204. Jende-Strid B, (1995). Coordinator’s report: anthocyanin genes. Barley Genet. Newsl. 24: 162-165. Jensen J, (1979). Location of a high-lysine gene and the DDTresistance gene on barley chromosome 7. Euphytica 28: 41-56. Kahler AL, Morris MI and Allard RW, (1981). Gene triplication and fixed heterozygosity in diploid wild barley. J. Hered. 72: 374-376. Kasha KJ and Burnham CR, (1965). The location of interchange breakpoints in barley. I. Linkage studies and map orientation. Can. J. Genet. Cytol. 7: 62-77. Kleinhofs A, Kilian A, Saghai Maroof MA, Biyashev RM, Hayes P, Chen FQ, Lapitan N, Fenwick A, Blake TK, Kanazin V, Ananiev E, Dahleen L, Kudrna D, Bollinger J, Knapp SJ, Liu B, Sorrells M, Heun M, Franckowiak JD, Hoffmann D, Skadsen R and Steffenson BJ, (1993). A molecular, isozyme and morphological map of the barley (Hordeum vulgare) genome. Theor. Appl. Genet. Kreis M, Williamson MS, Shewry PR, Sharp P and Gale M, (1988). Identification of a second locus encoding p-amylase on chromosome 2 of barley. Genet. Res. 51: Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE and Newburg L, (1987). MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181. Lemmela H, (1984). Ulkoisen taustasateilyn kartoitus Suomessa. Finnish Centre for Radiation and Nuclear Safety, STUK-B-VAL0 32: I - 15. Liljeroth E and BBBth E, (1988). Bacteria and fungi on roots of different barley varieties (Hordeum vulgare L.). Biol. Fert. Soils 7: 53-57. Liljeroth E, Hagberg P and Hagberg A, (1994). Bacterial abundance on roots of a high-yielding duplication line and some related varieties. Hereditas 121: 197198. 86: 705-712. 13-16. Linde-Ldursen I, (1988). Giemsa C-banding of barley chromosomes. V. Localization of breakpoints in 70 reciprocal translocations. Hereditas 108: 65-76. Linde-Laursen I, Heslop-Harrison JS, Shepherd KW and Taketa S, (1997). The barley genome and its relationship with the wheat genomes. A survey with an internationally agreed recommendation for barley chromosome nomenclature. Hereditas 126: 1-16. MacKey J, (1954). Neutron and X-ray experiments in wheat and a revision of the speltoid problem. Hereditas 40: 64-180. Mather DE, Tinker NA, LaBerge DE, Edney M, Jones BL, Rossnagel BG, Legge WG, Briggs KG, Irvine RB, Falk DE and Kasha KJ, (1997). Regions of the genome that affect grain and malt quality in a North American tworow barley cross. Crop Sci. 37: 544-554. Mhlenschlaeger M, Gissel-Nielsen G and Nielsen SP, (1993). Differences in the sensitivity of barley varieties to direct cesium contamination from the Chernobyl accident. Health Phys. 64: 535-537. Oziel A, Hayes PM, Chen FQ and Jones B, (1996). Application of quantitative trait locus mapping to the development of winter-habit malting barley. Plant Breed. 115: 43-51. Paakkola 0, (1988). Laskeuma ja sen ymparistovaikutukset. In: Sateily ja turvallisuus (eds H Toivonen, T Rytomaa and A Vuorinen) Valtion Painatuskeskus and Sateilyturvakeskus, Helsinki, p. 360-392. Persson G, (1969). An attempt to find suitable genetic markers for dense ear loci in barley 11. Hereditas 63: 1-28. Pradhan S and Subrahmanyam NC, (1995). Methyl cytosine and adenine in barley genome: changes in structurally altered karyotypes. Proc. Ind. Nat. Sci. Acad. B61: 347-354. Qi X, Stam P and Lindhout P, (1996). Comparison and integration of four barley genetic maps. Genome 39: 379-394. Santos MMM and Riis P, (1996). Optimized McCleary method for measurement of total P-amylase in barley and its applicability. J. Inst. Brew. 102: 271-275. Simberg NH, (1950). Undersokning av maltkorn. Department of Chemical Engineering, University of Technology. Helsinki. Soliman KM and Allard RW, (1989). Genetic control of the triplicate esterase 4 locus in the diploid Hordeum spontaneum x H. vulgare crosses. J. Hered. 80: 70-71. Stenius M, Piik K and Parkkinen E, (1976). Ohramutantin HA21 karyotyyppi. Division of Genetics, University. Helsinki. Subrahmanyam NC, Bryngelsson T, Hagberg P and Hagberg A, (1994). Differential amplification of rDNA repeats in barley translocation and duplication lines: role of a specific segment. Hereditas 121: 157-170. Thomas WTB, Powell W, Swanston JS, Ellis RP, Chalmers KJ, Barua UM, Jack P, Lea V, Forster BP, Waugh R and Smith DB, (1996). Quantitative trait loci for germination and malting quality characters in a spring barley cross. Crop Sci. 36: 265-273. Tinker NA, Mather DE and the North American Barley Genome Mapping Project, (1994). Main effects of quantitative trait loci in Harrington/TR306 two-row barley. Barley Genet. Newsl. 23: 72-78.