| Flowering time greatly reflects the adaptation of wheat cultivars to diverse environme ntal conditions in a certain extent, and is mainly regulated by vernalization and photoperio d genes. In wheat cultivars from the Yellow and Huai Valleys, which represent60%-70%of the total wheat production in China, the wheat genotypes wheat varieties of vernalizatio n and photoperiod is not very clear, limiting the use of Chinese wheat germplasms to ace rtain extent. Therefore, this study identified vernalization and photoperiod genes of local v arieties in Huang Huai wheat area, widely cultivated varieties at present and wheat landrac es, and analyzed their distribution, in order to find new alleles and new polymorphism co mbinations. Results of this study have the potential to provide useful information for scree ning relatively superior wheat cultivars for better adaptability and maturity.In this study,173winter wheat cultivars from the Yellow and Huai Valleys,51spring wheat cultivars from Gansu province and191wheat landraces were used to identify,whi ch were provided by Henan Agricultural University Wheat Breeding Laboratory. Using SL S method to extract DNA, Trizol method to extract RNA, specific primer PCR amplificati on, Restriction endonuclease technique, DNA cloning and Sequencing technology, the verna lization gene and photoperiod gene allelic types of different locus were identified and anal yzed. And at the same time, in order to further analyze these roles of new variation types in wheat growth process, we explored the new allelic function from the RNA level and protein level by RT-PCR and Western-blot techniques.The main results of this study are as follows:1. Using a series of molecular markers of Wheat Vernalization related genes Vrn-Al, Vrn-Bl, Vrn-D1and Vrn-B3, we identified173winter wheat cultivars from the Yellow and Huai Valley of China.When we used the specific molecular marker of Vrn-B3locus to PCR, compared these amplified segmentes, The cultivar name of Chadianhong had approxi mately900-bp longer than the others.After recycled and sequenced the amplified fragment, one novel allele at the Vrn-B3locus were discovered and were respectively designated Vrn-Bib. Vrn-B3b which was discovered in the cultivar Chadianhong, had an890-bp insertion in the promoter region of the recessive allele vrn-B3. Use Vrn-B3locus specific molecula r markers to ji874-109material during the amplification effect is not clearness, and accor ding to the Vrn-B3a sequence design for5primers, respectively to amplification, ji874-10 9material found the material sequence compared with dominant allele Vrn-B3a, its promot er region there are two pieces deletion,20bp (bp) and4, respectively named Vrn-B3c. B ased on these sequences of Vrn-B3b and Vrn-B3c, two new co-dominant markers were dev eloped in order to more precisely examine Vrn-Alb, Vrn-Bla, and Vrn-Blb. Cultivar Hema i26lacked the Vrn-A1gene. RT-PCR and western-blot indicated that the890-bp insertion in the Vrn-B3b allele significantly reduced the transcription of the Vrn-B3gene. Cultivar Chadianhong with the Vrn-B3b allele and Hemai26with a Vrn-A1-null allele possessed re latively later heading and flowering times compared to those of Yanzhan4110, which harb ored recessive vrn-B3and vrn-A1alleles. The results showed that, the890-bp deletion of Vrn-B3b in the promoter region and the loss of Vrn-Al loci all have an impact on delayin g the heading time and flowering time.2. After identification of photoperiod genotypes of173winter wheat cultivars from th e Yellow and Huai Valley,6kinds of Ppd-D1sequence polymorphism combinations were found,they were Hapl-Ⅰ, Hapl-Ⅱ, Hapl-Ⅲ, Hapl-Ⅳ, and another two novel combinations, which were designated Hapl-VII and Hapl-VIII, respectively. The distribution frequency of them were89.6%,1.7%,0.6%,4.6%,2.3%and1.2%of Chinese winter wheat cultivars s urveyed from the Yellow and Huai Valley of China. Of6polymorphism combinations, Ha pl-I was the most prevalent combination, and Hapl-VIII took the smallest part. Absence of2089-bp in exon8and presence of both TE in intron1and5bp in exon7for4cult ivars:Danmai1introduced from Danmark, Ji923235, Ji874-109, and Huapeiai, and we f ound Hapl-VII in these4cultivars. Hapl-VIII was found in2cultivars Damuzhiai and Xia n83(104)-ll Zhong "S", the Hapl-VIII combination was absence of2089-bp in exon8, T E in intron1, and5-bp in exon7. The two genotypes of heading time and flowering tim e are obviously different.3. Through identification of the allelic combination of vernalization genes, there were14combinations were found in173winter wheat cultivars, they are vrn-A1/vrn-B1/vrn-D1/vrn-B3, vrn-A1/vrn-B1/Vrn-D1a/vrn-B3, vrn-A1/vrn-B1/Vrn-D1b/vrn-B3, vrn-A1/Vrn-B1a/vrn-D1/vrn-B3, Vrn-A1a/vrn-B1/vrn-D1/vrn-B3, vrn-A1/Vrn-B1a/Vrn-D1a/vrn-B3, vrn-A1/Vrn-B1a/Vr n-D1b/vrn-B3, vrn-A1/Vrn-B1a/Vrn-D1b/vrn-B3, Vrn-A1a/vrn-B1/Vrn-D1b/vrn-B3, vrn-A1/Vrn-B1a/vrn-D1/Vrn-B3c, vrn-A1/vrn-B1/Vrn-Dlb/Vrn-B3b, vrn-B1/vrn-D1/Vrn-B3a, Vrn-A1b/vrn-B1/vrn-D1/vrn-B3and Vrn-A1b/Vrn-B1a/vrn-D1/vrn-B3.92out of the173Chinese winter wh eat cultivars surveyed had vrn-A1/vrn-B1/vrn-D1/vrn-B3(53.2%),30cultivars possessed vrn-A1/vrn-B1/Vrn-D1a/vrn-B3(17.3%),37cultivars possessed vrn-A1/vrn-B1/Vrn-D1b/vrn-B3(21.4%), therefore, the former3kinds of combinations took the biggest part, were prevale nt genotypes in winter wheat cultivars from the Yellow and Huai Valley. The rest of11k inds of combinations were not prevalent.4. Analysis of vernalization genotypes of191wheat landraces,indicated that there wer e5cultivars876,347,351, DA8and DB9, whose amplified segmentes had approximately200-bp longer than the others. After sequenced these amplified fragments, we found that they belong to the same type, which was designated Vrn-D1c, the genotype has a175-bp insertion in the promoter region of the recessive allele vrn-D1.5. Through identification of the allelic combination of vernalization genes, there were6allelic combinations were found in191wheat landraces, they are vrn-A1/vrn-B1/Vrn-D1a/vrn-B3, vrn-A1/vrn-B1/Vrn-Dlb/vrn-B3, vrn-A1/vrn-B1/vrn-D1/vrn-B3, vrn-A1/Vrn-B1a/vrn-D1/vrn-B3, vrn-A1/Vrn-Bla/Vrn-Dlb/vrn-B3, vrn-A1/Vrn-Blb/Vrn-D1b/vrn-B3, Distribution freq uency of the former3kinds of combinations are35%,46.6%and14.7%, respectively.Afte r surveyed and analyzed the heading time and flowering time of these cultivars having an y one genotype of the two combinations vrn-A1/vrn-B1/Vrn-D1a/vrn-B3or vrn-A1/vrn-B1/V rn-D1b/vrn-B3, we found that dominant allele Vrn-D1a and dominant allele Vrn-D1b had a n impact on delaying the heading time and flowering time. |
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