Detection Of Wheat Heading-time Related Genes And Cloning Of The Upstream Sequence Of Novel Allelic Variation Ppd-B1

Photoperiod genes determine the heading date of wheat,which not only affects the growth and yield of wheat,but also is important to determine its adaptability.The study of the photoperiod gene has important significance for the introduction and breeding of wheat,so it is very important to study the photoperiod genes.In this study,photoperiodic genotypes of 997 Chinese wheat cultivars(lines)were identified using the molecular markers.The differences on the composition of photoperiodic genotype in different wheat regions were analyzed and compared.For some of these varieties,vernalization genes were identified,and their heading time in field was recorded.The upstream sequence of new alleles Ppd-B1 was cloned,and its molecular marker was developed.The main results were as follows:1.In this study,997 wheat varieties form different Chinese wheat regions were identificated and analyzed on photoperiod genes.The variation type of no PCR amplified band was found in the wheat photoperiod Ppd-A1 locus(0.5 %),and the other materials were recessive genotype Ppd-A1 b.2,accounting for 99.5 %.The variation type of no PCR amplified band was found in the wheat photoperiod Ppd-B1 locus(0.3 %),dominant genotype Ppd-B1 a.1 accounted for 0.1 %,and other materials were recessive genotype Ppd-B1 b.1accounting for 99.6 %.The dominant allele Ppd-D1 a.1 was dominant(80.3 %),and the other materials were recessive genotype Ppd-D1 b.2,accounting for 19.7 %.Most of the breeding varieties planted in Northeast Spring Wheat Region,Xijiang Winter-Spring Region and Qinghai-Tibet Spring-Winter Region which is located on high latitude and has long sunshine hours were photoperiod insensitive Ppd-D1 b.2,but sensitive Ppd-D1 a.1 was dominant in Chinese other wheat regions.The frequency of Ppd-D1 a.1 in Chinese ten wheat regions from low to high,Xijiang Winter-Spring Region(40.0 %)<Northeast Spring Wheat Region(43.3 %)< Northern Spring Wheat Region(45.5 %)<Qinghai-Tibet Spring-Winter Region(53.4 %)< South China Winter Wheat Region(69.2 %)< Northwestern Spring Wheat Region(78.1 %)< Southwestern Winter Wheat Region(87.2 %)< Middle and low Yangtze Valley Winter Wheat Region(91.0 %)< Northern Winter Wheat Region(92.2 %)< Yellow and Huai River Winter Wheat Region(93.6 %).The frequency of Ppd-D1 a.1 in Chinese wheat cultivars(lines)increased gradually from north to south,and then decreased,showing a trend of both sides are low but the middle is high.The frequency of Ppd-D1 a.1 increased gradually from west to east.In general,the frequency of Ppd-D1 a.1 in Yellow and Huai River Winter Wheat Region was the highest and had a downward trend in other directions.There were three combinations of photoperiod genes in 309 tested materials which were analysed the relationship between the photoperiod gene and the heading date.The type Ppd-A1 b.2/bpd-B1 b.1/Ppd-D1 b.2 accounted for 33.7 %,and the average heading date was206.5 ± 5.4 d;the type Ppd-A1 b.2/Ppd-B1 b.1/Ppd-D1 a.1 accounted for 69.0 %,and the average heading date was 199.2 ± 4.2 d,respectively.2.Combinations of photoperiod and vernalization genes of 158 experimental materials were identificated by the molecular markers.New alleles that were no PCR amplified band had the role of delaying heading date of wheat in photoperiod response Ppd-A1 and Ppd-B1 loci,and the heading date were delayed 5.9 d and 8.1 d,respectively.We assumed that the new allelic variation of Ppd-B1 locus was sensitive and more effective in delaying the heading of wheat.In 158 experimental materials,there were 96(60.8 %)cultivars(lines)carrying insensitive alleles Ppd-D1 a.1,the other 62(39.2 %)showed sensitive type Ppd-D1 b.2,and the insensitive varieties Ppd-D1 a.1 significantly advanced 9.4 d of the heading date comparing with sensitive varieties Ppd-D1 b.2.There were three combinations of different genotypes in the three photoperiod loci,the Ppd-A1 b.2/bpd-B1 b.1/Ppd-D1 b.2 accounted for 35.8 %,and the average heading date was208.6 ± 5.7 d;the Ppd-A1 b.2/Ppd-B1 b.1/Ppd-D1 a.1 accounted for 63.6 %,and the average heading date was 199.1 ± 3.5 d.The average heading date of Ppd-A1 b.2/Ppd-B1 b.1/Ppd-D1 a.1 was earlier than Ppd-A1 b.2/bpd-B1 b.1/Ppd-D1 b.2 type by about 9.5 d.Through the identification of Vrn-A1,Vrn-B1,Vrn-D1 and Vrn-B3 vernalization genes,we found that frequencies of dominant Vrn-A1,Vrn-B1,Vrn-D1 and Vrn-B3 were 50.6 %,42.4 %,36.1 % and 5.7 %,respectively.There were 11 combinations of different genotypes in the four vernalization genes,the combination of Vrn-A1/Vrn-B1/vrn-D1/vrn-B3 had the highest frequency,accounting for 24.7 %;the recessive genotypes combination of Vrn-A1/vrn-B1/vrn-D1/vrn-B3 had the second highest frequency,accounting for 21.5 %.The average heading date of different combinations were different.The earliest combination ofaverage heading date was dominant allels combination Vrn-A1/Vrn-B1/Vrn-D1/Vrn-B3 for 195 d,and the latest combination of average heading date was vrn-A1/vrn-B1/Vrn-D1/Vrn-B3 for209±3.2 d.When the average heading date was the same,the combinations of vernalization genotypes may be different.There were 19 combinations of different genotypes in the four vernalization genes and photoperiod Ppd-D1 locus.The combination of Ppd-D1 a.1/vrn-A1/Vrn-B1/vrn-D1/vrn-B3 had the highest frequency,accounting for 20.3 %;the combination of Ppd-D1 a.1/Vrn-A1/Vrn-B1/vrn-D1/vrn-B3 had the second highest frequency,accounting for17.1 %.The average heading date of different combinations were different,the earliest combination of average heading date was dominant genotypes combination Ppd-D1 a.1/Vrn-A1/Vrn-B1/Vrn-D1/Vrn-B3 for 195 d,and the latest combination of average heading date was recessive Ppd-D1 b.2/vrn-A1/vrn-B1/vrn-D1/vrn-B3 for 218 d.The average heading date of dominant genotypes combination Ppd-D1 a.1/Vrn-A1/Vrn-B1/Vrn-D1/Vrn-B3 was earlier than recessive Ppd-D1 b.2/vrn-A1/vrn-B1/vrn-D1/vrn-B3 type by 23 d.The average heading date of the combination with dominant gene Ppd-D1 a.1 was earlier than the combination with recessive gene Ppd-D1 b.2.3.The promoter sequence of the novel allele at Ppd-B1 locus was cloned.The base insertion of 4256 bp in the promoter region was found comparing to Ppd-B1 b.1.Based on the variation in the area,a new molecular marker was developed to detected new variation of Ppd-B1 locus.