Analysis Of Myb-related Genes In Vitis Amurensis And Its Hybrids

This experiment first optimized the method for genome DNA extraction from Vitis amurensis toguarantee the accuracy of the subsequent tests by using high quality and high purity DNA. Weresearched genes which regulate anthocyanin biosynthesis of V. amurensis and its hybrids to identify thegenotype of Myb-related genes in V. amurensis, to enrich the study of Myb genes and to confirm thespecific gene regulating anthocyanin biosynthesis of V. amurensis. The comparison and the SNPanalysis of VvmybA1gene segments from V. amurensis and its hybrids confirmed the genetic diversityamong experimental material for the better classification of V. amurensis. In this study, from molecularlevel, we speculated that hermaphroditic V. amurensis ‘Shuangqing’ was maybe derived from theintraspecific variation of V. amurensis. The amin results of this research are as follows:1. The extraction of genome DNA from V. amurensis by improved CTAB methodTake5strains of V. amurensis and a V. vinifera as experimental material. The CTAB method byWang Jun (2000), DNA extraction kit and the improved CTAB method were carried out to extractgenome DNA of the leaves in uniform conditions, respectively. Some samples’ genome DNA extractedby traditional CTAB method was browning, and some were milky yellow. The result of DNA extractionkit showed a too low quality and purity DNA to be used. The samples’ genome DNA extracted byimproved CTAB method was white. The test result of purity and quality showed that the genome DNAextracted by improved CTAB method had a higher content, and OD260/OD280was1.8-1.95, which wassuperior to the value of another group of genome DNA. In addition, the result of agarose gel showedthat the integrity of genome DNA extracted by old CTAB method was destroyed and had more impurity.While the integrity and purity of the DNA gained by improved CTAB method was better. High-qualityDNA laid a good foundation for the subsequent experiments.2. The identification of the genotype of Myb-related genes in V. amurensis10unisexual samples and3hermaphroditic samples from V. amurensis,5hybrids of V. amurensis×V. vinifera were used as experimental materials and ‘Kyoho’ grape (V. labrusca×V. vinifera) and ‘PinotNoir’(V. vinifera) was taken as control materials. Identify the genotypes of Myb-related transcriptionfactors regulating the composition of anthocyanins. The result showed that V. amurensis had VvmybA1gene. There was VvmybA1a gene in ‘Pinot Noir’(V. vinifera),‘Kyoho’(V. labrusca×V. vinifera) andtwo hybrids of V. amurensis×V. vinifera,‘Beimei’ and ‘Noir de Nord’.‘Noir de Nord’ had VvmybA1and VvmybA3genes, while other four hybrids of V. amurensis×V. vinifera,‘Beichun’,‘Beimei’,‘Noirde Nord’ and ‘Gongniang1’, had VvmybA1, VvmybA2and VvmybA3genes. There were VvmybA1andVlmybA2genes in two hybrids of V. amurensis×V. vinifera,‘Zuohong1’ and ‘Zuoyouhong’. Thegenotype of Myb-related genes in hermaphroditic samples and unisexual samples of V. amurensis was coincident that they only had VvmybA1gene. The coincidence explained that the reason of theproduction of anthocyanin in V. amurensis was the only regulating gene―VvmybA1gene. They didn’thave the feature genes of V. vinifera, and the character of bisexual flower might be generated from theintraspecific variation by speculating. Because of the hybridization, there was significant differencebetween the genotypes of Myb-related genes in the hybrids of V. amurensis×V. vinifera.3. SNP detection and analysis of VvmybA1gene segments from V. amurensisAfter the clones of VvmybA1gene segments from part experimental material and sequencing, wegot some967bp gene segments. By means of sequence alignment, we found that the similarity ofnucleotide sequence was beyond98%between the19samples, and there were2different fragmentdeletions in noncoding region in3samples. A total of23SNPs were detected which made15aminoacids change and defined14haplotypes. The analysis of SNP showed higher level of Haplotype geneticdiversity(Hd=0.965) and Nucleotide genetic diversity(Pi=0.00897).The neutrality tests indicated that there was a balance between genetic drift and the selectiveneutral mutation in V. amurensis. And the gene remained relatively stable. Due to the hybridization, thegene of the hybrids of V. amurensis×V. vinifera had a genetic drift was changed, due to thehybridization, and the gene had a higher polymorphism. The Phylogenetic tree showed that there weregenes overlap between V. amurensis and the hybrids of V. amurensis×V. vinifera. Hermaphroditicsamples in V. amurensis ‘Shuangqing’ and ‘shuangyou’were in a same branch which was classified to abig branch with a few unisexual V. amurensis， which indicated that they had a closer relationship.‘Shuangfeng’ got an assortment with other unisexual V. amurensis. So hermaphroditic and unisexual V.amurensis were closer.