Segregation distortion in F 2 and doubled haploid populations of temperate japonica rice

c Indian Academy of Sciences RESEARCH NOTE Segregation distortion in F 2 and doubled haploid populations of temperate japonica rice MASUMI YAMAGISHI 1,2,6, YOSHINOBU TAKEUCHI 3,7, ISAO TANAKA 4, IZUMI KONO 3, KOJI MURAI 1,8 and MASAHIRO YANO 5 1 Research Institute of Agricultural Resources, Ishikawa Agricultural College, Nonoichi, Ishikawa 921-8836, Japan 2 Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan 3 Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-0854, Japan 4 Fukui Agricultural Experiment Station, Ryo-machi, Fukui, Fukui 918-8215, Japan 5 National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan 6 Present address: Research Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan 7 Present address: National Institute of Crop Science, Tsukuba, Ibaraki 305-8518, Japan 8 Present address: Department of Bioscience, Fukui Prefecture University, Matsuoka, Fukui 910-1195, Japan Introduction Segregation distortion of genes and markers is frequently observed in the populations derived from inter-specific and inter-subspecific (i.e. indica japonica) crosses of rice (Oryza sativa L.). In this study, linkage maps were constructed using F 2 and doubled haploid (DH) populations derived from the same cross between the two temperate japonica rice cultivars Akihikari and Koshihikari, and chromosomal regions showing the segregation distortion of markers were surveyed. In the F 2 population, 7% of markers showed skewed segregation. This frequency of skewed markers was lower than that in previously reported F 2 populations derived from indica japonica crosses. In the DH population, the proportion of skewed markers was 19%, which was lower than that in previously reported DH populations derived from indica japonica crosses, but was higher than that in the japonica japonica F 2 population in this study. Segregation distortion can be detected as a deviation from the expected Mendelian segregation ratio of marker genes and molecular markers. Such marker genes and molecular markers are often linked with segregation-distortion factors, such as hybrid sterility (S) genes (Sano 1990) and gametophyte competition (ga) genes (Nakagahra 1972). Seed fertility in inter-specific F 1 hybrids is usually low. This low-seed *For correspondence. E-mail: yamagisi@res.agr.hokudai.ac.jp. [Yamagishi M., Takeuchi Y., Tanaka I., Kono I., Murai K. and Yano M. 2010 Segregation distortion in F 2 and doubled haploid populations of temperate japonica rice. J. Genet. 89, 237 241] fertility is caused by S gene, resulting in the distortion of allele frequency in F 2 population in regions where S genes exist. On the other hand, ga genes that select male gametes during fertilization cause segregation distortion but do not reduce F 1 seed fertility (Nakagahra 1972). In rice, segregation distortion has been observed in progenies derived from indica japonica crosses (Nakagahra 1972; Matsushita et al. 2003) and inter-specific crosses (Sano 1990), and many distortion factors have been identified (Xu et al. 1997; Kinoshita 1998). Using high-density linkage maps, the chromosomal regions showing segregation distortion have been surveyed throughout the genome by analysing the segregation ratio of molecular markers used for the linkage map construction, and many new skewed regions have been identified (Xu et al. 1997; Harushima et al. 2001). Although segregation distortion has been well analysed in interspecific and indica japonica derived populations of rice, there are only few reports showing the segregation distortion among japonica populations. Segregation distortion occurs even in japonica crosses, as evidencedby the identification of ga genesamong japonica rice varieties (Nakagahra 1972; Maekawa and Kita 1985). The only report of segregation distortion is from F 2 population derived from the tropical japonica temperate japonica cross, showing distortion in 15 of 174 markers (9%) (Redona and Mackill 1996). Such skewed chromosomal regions in japonica japonica populations have not been studied well, compared to the indica japonica populations, because of the limited number of molecular markers showing polymorphisms among japonica germplasms (Mackill Keywords. anther culture; linkage map; skewed segregation; Oryza sativa L. Journal of Genetics, Vol. 89, No. 2, August 2010 237

Masumi Yamagishi et al. 1995). We need to first identify the regions showing skewed segregation in japonica populations for obtaining a clue to determine the causes of segregation distortion. DH populations are important materials for genetic analysis of interesting traits and for plant breeding. In rice, anther culture is the only procedure that enables the generation of DH plants. The optimum anther culture conditions including medium compositions and temperature regimes have been established in rice, but the ability of anther culture to generate fertile DH plants varies with varieties depending on their genetic backgrounds (He et al. 1998). Alleles that increase fertile plant regeneration can be preferentially transmitted by anther culture processes, so that, in addition to the segregation distortion factors, genes controlling the ability to generate fertile plants might cause segregation distortion in DH populations. Segregation distortion has been reported to occur more frequently in rice DH populations derived from indica japonica crosses than those in F 2 populations (Xu et al. 1997). Yamagishi et al. (1998) revealed that two of the five regions showing segregation distortion in an indica japonica DH population contributed to increase DH plant generation. However, segregation distortion in japonica japonica DH populations has not been analysed yet. In this study, we analysed the segregation distortion of molecular markers used for the linkage map construction in F 2 and DH populations derived from two temperate japonica rice cultivars Akihikari and Koshihikari. The frequencies of skewed markers were compared between the F 2 and DH populations, and between the japonica japonica populations in this study and previously reported indica japonica and indica indica populations. Materials and methods In this study, F 2 and DH populations used were derived from F 1 plants of the cross between two temperate japonica cultivars Akihikari and Koshihikari. By selfing the F 1 plants, F 2 population consisting of 190 plants was developed. A population of 212 DH lines was developed through anther culture of the F 1 plants by the method of Yamagishi et al. (1996). A linkage map of the F 2 population was constructed using 87 restriction fragment length polymorphism (RFLP) markers, and that of the DH population was constructed using 135 RFLP markers and 34 randomly amplified polymorphic DNA (RAPD) markers. RFLP markers were mainly selected from the high-density linkage map (Harushima et al. 2001), and 10-base random primers (Operon Technologies, Alameda, USA) were used for RAPD amplification. Linkage analysis was performed with MAPMAKER/EXP 3.0 (Lander et al. 1987). A threshold of LOD 3.0 and a maximum recombination value of 30% were used to determine the linkage between the two markers. The recombination frequency (%) was converted to genetic distance (cm) by the function of Kosambi (1944). Chi-square analysis was used to compare the observed segregation ratio of each marker with the theoretical ratio of 1:1, 3:1 or 1:2:1. The linkage map of the DH population has been used for the genetic analysis of agronomic traits (Takeuchi et al. 2001; Yamagishi et al. 2002; Tanaka et al. 2006) and seeds, and genotype data of all the DH lines are available for scientific researches (public data in the Rice Genome Project, http://rgp.dna.affrc.go.jp/e/publicdata.html). Results We constructed a linkage map of the F 2 population from the Koshihikari Akihikari cross, using 87 RFLP markers. The F 2 map consisted of 15 linkage groups and two unlinked markers, which were aligned with a high-density linkage map (Harushima et al. 2001), according to the positions of the common markers on both linkage maps (figure 1a). The F 2 map covered 30% of the total rice genome, and the total map length was 641 cm. Four chromosomal regions including six markers (7%) showed skewed segregation from the expected segregation ratios (figure 1a). The marker locus showing the most skewed segregation was R2549 on chromosome 6 where the frequency of the Akihikari homozygote increased markedly and that of the heterozygote decreased (table 1). The linkage map of the DH population from the two temperate japonica cultivars covered 60% of the total rice genome (Takeuchi et al. 2001). Eleven chromosomal regions including 32 of the 169 DNA markers (19%) showed skewed segregation at the 5% significance level (figure 1b). The region showing the most pronounced distortion in the DH population appeared on the long arm of chromosome 3. Marker C668 showed the segregation of 38 lines (19%) with the Koshihikari allele and 166 lines (81%) with the Akihikari allele (table 1). In the F 2 population, two markers, C393A and C393B, were mapped on the long arm of chromosome 3 (figure 1a). Although RFLP marker C1402B and a RAPD marker OPJ10B, both of which were dominant markers, were mapped on the long arm of chromosome 3 of the DH map, they did not link to the marker C393A in the F 2 population. Segregation ratio in the F 2 population of the OPJ10B was 128:38, which fitted the expected 3:1 ratio (chi-square was 0.39 ns ), indicating that skewed segregation of the long arm of chromosome 3 did not occur in the F 2 population. Discussion The linkage maps of the F 2 and DH populations evaluated in this study covered approximately 30% and 60% of the total rice genome, respectively. These percentages were not high mainly due to the low level of polymorphisms in DNA markers between the two japonica cultivars used in this study (Takeuchi et al. 2001). In addition, the number of linked markers was smaller in the F 2 populations than that in the DH population because most of RAPD markers analysed in the DH population were not tested in the F 2 population, and some dominant RFLP markers were not linked to the markers in the F 2 linkage map. Determining the precise position of dominant markers including RAPD markers is relatively 238 Journal of Genetics, Vol. 89, No. 2, August 2010

Segregation distortion in japonica rice populations Figure 1. (a) Linkage maps of F 2, and (b) DH populations derived from the cross between two temperate japonica cultivars Koshihikari and Akihikari, and chromosomal regions showing segregation distortion. Linkage groups (thick open lines) and unlinked RFLP markers were aligned using the high-density linkage map (thin closed lines, Harushima et al. 2001) according to the positions of the common markers on the linkage map in this study and that by Harushima et al. (2001). Symbols at the top and bottom of the thin closed lines indicate the chromosome number and its arm. Markers are shown on the right of the chromosomes. (a) Fifteen linkage groups and two unlinked RFLP markers of the F 2 population. The closed boxes on the chromosomes show the regions of segregation distortion. (b) Seventeen linkage groups and two unlinked markers of the DH population. The gray and black boxes on the chromosomes show the regions with significant distortion in segregation in favour of Koshihikari and Akihikari alleles, respectively. * And **; significance levels at 5% and 1%, respectively. Journal of Genetics, Vol. 89, No. 2, August 2010 239

Masumi Yamagishi et al. Table 1. The most skewed markers on each of the skewed chromosomal regions in the F 2 and the DH populations, and the number and percentage of plants with each genotype. Koshihikari Akihikari Chromosome Marker homozygote Heterozygote homozygote Chi-square F 2 population 1 R559 55 (29%) 77 (41%) 58 (31%) 6.92* 5 C1230 64 (34%) 90 (48%) 35 (19%) 9.33** 6 R2549 44 (23%) 67 (36%) 77 (41%) 27.1** 9 G293 60 (32%) 130 (68%) 4.39* DH population 1 R1545 124 (59%) 86 (41%) 6.88** 3 OPAD14B 112 (61%) 71 (39%) 9.19** 3 C668 38 (19%) 166 (81%) 80.31** 5 C1122 115 (60%) 76 (40%) 7.96** 7 OPW18 89 (42%) 122 (58%) 5.16* 7 R2394 90 (42%) 122 (58%) 4.83* 8 S10543 123 (60%) 83 (40%) 7.77** 10 R844 86 (41%) 126 (59%) 0.55** 11 OPAK15 124 (59%) 87 (41%) 6.49* 11 OPAL11A 119 (59%) 84 (41%) 6.03* 12 C1069 86 (42%) 117 (58%) 4.73* * And ** indicate significant difference at 5% and 1% level, respectively, from the expected segregation ratio of 1:2:1, 1:3 (for G293, a dominant marker) or 1:1. difficult for the linkage analysis of F 2 populations because dominant homozygotes cannot be distinguished from heterozygotes. Twenty-five of the 135 DNA markers (19%) showed segregation distortion in an indica japonica F 2 population (Mc- Couch et al. 1988). In this study, six of the 87 markers (7%) showed skewed segregation in the japonica japonica F 2 population (figure 1a; table 1). The frequency of segregation distortion in the japonica japonica F 2 population is lower than that in indica japonica F 2 populations. Analysis of the F 2 population from a cross between tropical japonica and temperate japonica rice showed that 15 of 174 markers (9%) showed segregation distortion (Redona and Mackill 1996), similar to the frequency in the temperate japonica F 2 population (figure 1a; table 1). Because F 1 and F 2 plants from japonica japonica crosses usually show high seed fertility, S genes may not be a major factor involved in segregation distortion in japonica japonica populations. This is one of the main reasons for the relatively low frequency of skewed markers in the japonica japonica populations. A F 2 population from Nipponbare (japonica) Kasalath (indica) cross, possesses a total of 33 distortion factors on all 12 chromosomes (Harushima et al. 2001), which showed high seed fertility at the F 1 generation. A smaller number of distortion factors other than S genes, including ga genes, might cause skewed segregation in the japonica japonica populations than in indica japonica populations. We examined the segregation distortion in the japonica japonica DH population. The proportion of skewed markers was found to be higher in the DH population (19%) than in the F 2 population (7%), and the regions of segregation distortion in the F 2 population were different from those in the DH population (figure 1). Segregation distortion in rice F 2 and DH populations derived from indica japonica crosses also revealed a higher percentage of distorted chromosomal regions in DH populations than in F 2 populations (Xu et al. 1997). In addition to the distortion factors such as ga genes, anther culture also affect the selective transmission of chromosomal regions, resulting in higher percentages of skewed markers and also different skewed regions in DH populations compared to F 2 populations. Genes that enhance fertile DH generation during the anther culture of japonica japonica hybrids might exist on some distorted chromosomal regions detected in this study. In indica japonica DH populations used for the construction of molecular linkage maps, the proportion of skewed markers has been reported to be 31% (Lu et al. 1996) and 41% (Huang et al. 1997). In the japonica japonica DH population, 19% of DNA markers showed segregation distortion (figure 1b), which was lower than the frequency of skewed markers in DH populations from indica japonica hybrids. The ability to generate fertile DH plants by anther culture is generally high in japonica cultivars compared with indica cultivars (Sathish et al. 1995), indicating that the ability to generate fertile DH plants is very different between japonica and indica cultivars. This large difference between japonica and indica cultivars might affect the percentage of skewed markers in the japonica japonica DH populations, which was lower than that in indica japonica DH population. 240 Journal of Genetics, Vol. 89, No. 2, August 2010

Segregation distortion in japonica rice populations References Harushima Y., Nakagahra M., Yano M., Sasaki T. and Kurata N. 2001 A genome-wide survey of reproductive barriers in an intraspecific hybrid. Genetics 159, 883 892. He P., Shen L., Lu C., Chen Y. and Zhu L. 1998 Analysis of quantitative trait loci which contribute to anther culturability in rice (Oryza sativa L.). Mol. Breed. 4, 165 172. Huang N., Parco A., Mew T., Magpantay G., McCouch S., Guiderdoni E. et al. 1997 RFLP mapping of isozymes, RAPD and QTLs for grain shape, brown planthopper resistance in a doubled haploid rice population. Mol. Breed. 3, 105 113. Kinoshita T. 1998 Report of the committee on gene symbolization, nomenclature and linkage group. II. Report from coordinator linkage mapping using mutant genes in rice. Rice Genet. Newsl. 15, 13 74. Kosambi D. P. 1944 The estimation of map distances from recombination values. Ann. Eugen. 12, 172 175. Lander E. S., Green P., Abrahamson J., Barlow A., Daly M. J., Lincoln S. E. and Newburg L. 1987 MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1, 174 181. Lu C., Shen L., Tan Z., Xu Y., He P., Chen Y. and Zhu L. 1996 Comparative mapping of QTLs for agronomic traits of rice across environments using a doubled haploid population. Theor. Appl. Genet. 93, 1211 1217. Mackill D. J. 1995 Classifying japonica rice cultivars with RAPD markers. Crop Sci. 35, 889 894. Maekawa M. and Kita F. 1985 New gametophyte genes located in the third linkage group (chromosome 3) of rice. Jpn. J. Breed. 35, 25 31. Matsushita S., Iseki T., Fukuta Y., Araki E., Kobayashi S, Osaki M. and Yamagishi M. 2003 Characterization of segregation distortion on chromosome 3 induced in wide hybridization between indica and japonica type rice varieties. Euphytica 134, 27 32. McCouch S., Kochert G., Yu Z., Wang Z., Khush G., Coffman W. and Tanksley S. 1988 Molecular mapping of rice chromosomes. Theor. Appl. Genet. 76, 815 829. Nakagahra M. 1972 Genetic mechanism on the distorted segregation of marker genes belonging to eleventh linkage group in cultivated rice. Jpn. J. Breed. 22, 232 238. Redona E. and Mackill D. 1996 Molecular mapping of quantitative trait loci in japonica rice. Genome 39, 395 403. Sano Y. 1990 The genetic nature of gamete eliminator in rice. Genetics 125, 183 191. Sathish P., Gamborg O. L. and Nabors M. W. 1995 Rice anther culture: callus initiation and androclonal variation in progenies of regenerated plants. Plant Cell Rep. 14, 432 436. Takeuchi Y., Hayasaka H., Chiba B., Tanaka I., Shimano T., Yamagishi M. et al. 2001 Mapping quantitative trait loci controlling cool-temperature tolerance at booting stage in temperate japonica rice. Breed. Sci. 51, 191 197. Tanaka I., Kobayashi A., Tomita K, Takeuchi Y., Yamagishi M., Yano M. et al. 2006 Detection of quantitative trait loci for stickiness and appearance based on eating quality test in japonica rice cultivar. Breed. Res. 8, 39 47 (in Japanese, with English summary). Xu Y., Zhu L., Xiao J., Huang N. and McCouch S. R. 1997 Chromosomal regions associated with segregation distortion of molecular markers in F 2, backcross, doubled haploid, and recombinant inbred populations in rice (Oryza sativa L.). Mol. Gen. Genet. 253, 535 545. Yamagishi M., Otani M., Higashi M., Fukuta Y., Fukui K., Yano M. and Shimada T. 1998 Chromosomal regions controlling anther culturability in rice (Oryza sativa L.). Euphytica 103, 227 234. Yamagishi M., Takeuchi Y., Kono I. and Yano M. 2002 QTL analysis for panicle characteristics in temperate japonica rice. Euphytica 128, 219 224. Yamagishi M., Yano M., Fukuta Y., Fukui K., Otani M. and Shimada T. 1996 Distorted segregation of RFLP markers in regenerated plants derived from anther culture of an F 1 hybrid of rice. Gene Genet. Syst. 71, 37 41. Received 11 January 2010, in revised form 19 January 2010; accepted 28 January 2010 Published on the Web: 14 June 2010 Journal of Genetics, Vol. 89, No. 2, August 2010 241