To map the GA3-insensitive semi-dwarf gene, tentatively designated as Rht-IC12196, and microsatellite markers on chromosome 4BS, produced F2 hybrid of T. aestivum ‘Novosibirskaya 67 (N67)’ - T. polonicum ’IC12196’ and F2 hybrids of T. polonicum ’IC12196’/Triticum durum Desf. ‘LD222’. Plant height of LD222 was tall. To investigate the allelic relationship between Rht-IC12196 and Rht-B1b alleles, F2 hybrids of T. polonicum ’IC12196’/T. durum ’Cando’ were also obtained. Cando has the Rht-B1b allele derived from Norin 10. Using the F2 generation of N67/IC12196, a small-scale bulk breeding method was applied to develop introgression lines. The procedure was described by Kosuge et al. (2010). Two hundred F2 plants were grown in an area of 20 m2. All the seeds were harvested from the F2 plants as a bulk. Subsequently, 200 F3 seeds were randomly chosen from the bulk, and were grown in the same area size in the subsequent cropping cycle. This method was applied until reaching the bulked F6 generation. Single seed descents of 132 F6 plants were used to advance the population from F7 through F10 generation. One-hundred and twelve hexaploid F10 lines were available for the present experiment. All populations were grown in the experimental field at the College of Agriculture, Ibaraki University, Japan. Culm lengths of individuals were measured after ripening
Microsatellite mapping of the Rht-IC12196 and allelic composition of the experimental lines
An intended to map of Rht-IC12196 using the F2 hybrid of N67/IC12196. However, it was found that segregation for plant height showed irregularity probably due to aneuploidy in the F2 generation. The chromosome numbers of the F2 of N67/IC12196 was expected to vary from 2n = 28 to 42, depending on the transmission of the D-genome univalents (Alston and Jones, 1968). Alternatively, there are used F2 hybrids of IC12196/LD222 to establish a template linkage map of chromosome 4BS including the semi-dwarf locus. It was expected that the ratio of IC12196 and non-IC12196 (N67 and non-parental) allele at the given locus would be 1:1 in the F10 generation provided there was lower natural selection during segregating generations. It was supposed non-parental allele was caused by a spontaneous mutation at the given locus. Genomic DNA was extracted from seedling leaves of N67, IC 12196, LD222, F2 hybrids of IC12196/LD222 and 112 hexaploid introgression lines, according to Dellaporta et al. (1983). To map the Rht-IC12196 gene and allelic composition of the introgression lines, used the wheat Xgwm, Xbarc and Xhbg microsatellite markers located on chromosome 4BS. The Xgwm microsatellite markers were developed by Röder et al. (1998). The Xbarc and Xhbg microsatellite markers were available from Song et al., (2005) and Torada et al. (2006), respectively. The PCR conditions, electrophoresis of PCR products and detection of amplified fragments followed Kosuge et al. (2008). Multipoint linkage values were calculated using Map Manager QTX (Manly et al., 2001). Minimum LOD scores of >3.0 were used to develop the linkage map. The software calculated genetic distances in centiMorgans (cM) by applying the Kosambi (1944) mapping function.