Studies On The Identification Of S-SAP Marker For Green Ougan And The Strategy For Screening Polymorphic CSSRs From Chinese Bayberry Transcriptome

Ougan (Citrus suavissima Hort. ex Tanaka) is the most special cultivar of citrus in the south of Zhejiang Province. Green Ougan is a newly variety found in2005of mandarin derived from the bud mutation of Ordinary Ougan, but there still was no available molecular markers for early distinguishing of the new variety. Chinese bayberry (Myrica rubra Sieb. et Zucc., Myricaceae) is one specialty of fruit tree which is native to China, and there are plenty of the resoureces in the country, but the available polymorphic cSSRs (cDNA-cSSRs) were scarce. Therefore, this research was armed at developing the S-SAP marker specific for Green Ougan and establishing a strategy for screening polymorphic cSSRs from those derived from Chinese bayberry transcriptome. The main results are as follows:1. Some modifications were made to S-SAP technique, and a system of S-SAP technique for Ougan was established, in which genomic DNA was digested solely with EcoR I. From the selective amplification products generated with the primers of C12and EAT, an about250bp band specific for Green Ougan was screened out, which size was confirmed to be247bp with cloning and sequencing. The results could be used to proof that Green Ougan is an authentic bud sport of Ordinary Ougan and also provide technical supports for the cultivar registration and the seedling identification of Green Ougan. The S-SAP technique based on digestion solely with EcoR I, could also provide some technical references to the similar studies of other fruit trees. Meanwhile, four RNaseH-LTR fragment sequences of mandarin were obtained by cloning, and they should be able to be applied to the S-SAP analysis of the varieties of citrus close-related to mandarin.2. In the UniGenes derived from RNA-seq of the transcriptome of Chinese bayberry cv. Biqi,6883cSSR loci were identified with1/3.2Kb in frequency.≤12bp,14-22bp and≥24bp cSSR loci respectively accounted for58%,35%and7%or so; di-and tri-nucleotide cSSR loci took up over75%; AG/CT and AAG/CTT were the most in corresponding cSSR loci and their number decreased with the repeat number increasing. In all the cSSRs derived from the transcriptome of Chinese bayberry, there were594compound cSSRs about accounting for10%, and5914 cSSRs were divided comprehensively into eleven types. The5914cSSRs contained6330cSSR loci occupying92.0%of all loci.3. A strategy was devised to screen for polymorphic cSSRs based on the analysis of polymorphisms in each type of cSSR. And it was that the2-ntL, Compound B, Compound A, and Compound D cSSRs should be preferentially selected for screening polymorphic cSSRs. In addition,3-ntL cSSRs could be suitable for obtaining abundant polymorphic cSSRs efficiently, and2-ntM cSSRs suitable for screening highly polymorphic cSSRs. Using the strategy, the efficiency of screening polymorphic cSSRs could be greatly raised and that could greatly facilitate consequently screening polymorphic cSSRs in large scale.4.109polymorphic cSSRs were also obtained and they can generate389alleles with polymorphism ratio being93.8%. Using these cSSR markers, the relationships among some Chinese bayberry samples were furtherly confirmed. And the109cSSR markers should be a promising genomic resource for molecular breeding of Chinese bayberry.