Development And Application Of SSR Primers Based On The Transcriptome In Dimocarpus Longan Lour.

This study focused on the research of EST-SSR molecular marker in Dimocarpus longan Lour.(Honghezi)with the basis of Embryonic Callus(EC) transcriptome data. 11 pairs of polymorphism primers were developed from the EC transcriptome data, and the EST-SSR-PCR experiment system was optimized, which was then successfully applied to the longan genetic relationship analysis. Finally, the EST-SSR-PCR was compared with ISSR molecular marker. The detailed results were as follows:1 EST-SSR loci characteristics analysis of longanThe EC transcriptome data of Dimocarpus longan Lour.(Honghezi)were used as materials to retrieve and analyze the SSR by using MISA software.5710 EST-SSR loci in total were found, and its occurrence frequency was 8.28%, the distance between EST-SSRs was 5.41 kb in average, and 796 types of EST-SSR motifs were found. In the aspect of numbers, Dinucleotide and Trinucleotide accounted for the largest proportion which was 43.35% and 32.73%, respectively, and the total proportion of Dinucleotide and Trinucleotidethe was 76.08%, indicating they werepreponderant nucleotide motifs. In the aspect of motif types of EST-SSR, Hexanucleotide and Pentanucleotide contained the most, and there were 389 types and 244 types in Hexanucleotide and Pentanucleotide, respectively, which accounted for 48.87% and 30.65% respectively, and the total proportion for the both was 79.52%. The retrieval results of ESR-SSR also showed that, among the Dinucleotide, (AG)n=(TC)n and (CT)n=(GA)n were accounted for 17.55% and 15.17%, respectively, which belonged to preponderant repeat types; frequency of (CG)n=(GC)n was only 0.000725% which was extremely low, showing obvious GC bias. Among the Trinucleotides, (AGA)n=(TCT)n was the most preponderant repeat types, which accounted for 2.92% of the total EST-SSR, the other two preponderant types were (CTT)n=(GAA)n and (AAG)n=(TTC)n, accounting for 2.71% and 2.59%, respectively. To sum up, the EST-SSR from EC transcriptome data of Dimocarpus longan Lour.(Honghezi) was rich.2 Optimization of EST-SSR-PCR system for longanAfter L16(44) orthogonal experiments, the most optimal reaction system of EST-SSR-PCR for longan was obtained:DNA template 80 ng,0.15 mmol/L dNTPs 2 μL,0.1 μmol/L primers (Forward and Reverse) 1 μL, Tap enzyme 1.5 U;and 10×PCR Buffer (1.5 mM Mg2+) which was not optimized experiment factor was 2.5 μL, add ddH2O to 25 μL. Then 3 randomly selected pairs of primers and 8 samples of longan germplasm(used as template) were used to test the optimized EST-SSR-PCR reaction system, the electrophoresis results showed that the amplification bands was clear and flat, no primer dimmers and nonspecific amplification were present, and the background interference was low, which suggested that the optimized reaction system of EST-SSR-PCR was stable and reliable.3 EST-SSR polymorphic analysis of longanAccording to the sequence characteristics of EST-SSR of longan, to 50 pairs of primes with a length varied from 18 to 24 bp were designed using Primer Premier 5, their amplification fragment length was 100-300 bp. Then the DNA ofDimocarpus longan Lour.(Lidongben) used as template to test the usability of primers.19 pairs of qualified primers were obtained out of 50 primers, whose amplification length was the expected sizes. Then 95 DNAs from different longan germplasms were used as materials to check the primer polymorphisms,10% Native-PAGE results showed that,11 pairs out of 19 had good polymorphism and their amplification bands were bright and clear, no tail and no dispersion and low background interference, and the development rate of polymorphic primers was 22%. Analysis of 11 pairs of EST-SSR primers on 95 longan germplasms showed that 114 amplification bands were obtained and their polymorphic percentage reaches 100%. Numbers of polymorphic amplification bands ranged from 5 to 25, and primers named LY-43 got 25 polymorphic bands which was the most among that of the whole primers; primers named LY-9 got only 5 polymorphic bands which was the least. The average polymorphic amplification bands of EST-SSR primers was 10.36. Threrefore, t the polymorphism of EST-SSR molecular marker which was developed by longan EC transcriptome was rich.4 Analysis between EST-SSR and ISSR of longanEST-SSR and ISSR molecular markers technology were used to study the genetic relationship of 95 longan germplasms. Numbers of amplification bands of the 11 pairs of EST-SSR primers were ranged from 5 to 25, and these amplification bands had a wide distribution range, the average numbers of polymorphic amplification bands of each primer was 10.36. In the results of ISSR, numbers of amplification bands which were amplificated by 10 pairs of UBC primers distributed in the range of 8-12, and these amplification bands showed a tiny distribution range and 9.50 polymorphic bands of each primer was found on average. The average numbers of polymorphic amplification bands of EST-SSR was more than that of ISSR. In addition, electrophoresis results showed that the specific primer developed by EST-SSR were more efficient combined to DNA sample than that of ISSR; 5 specific markers were obtained from 11 pairs of developed EST-SSR primers:LY-5, LY-38, LY-43, LY-45, LY-48, if much more efforts were paid to the development of specific longan EST-SSR markers, and that will be good for identification of longan germplasm resources.Clustering results based on EST-SSR showed 95 tested longan germplasms can be effectively separated from others, which were divided into 2 groups and 8 taxa. ’Lizhilongyan’ was clustered alone in a group, indicating that ’Lizhilongyan’ had a unique genetic background, and ’Lizhilongyan’ and ’Lilong’ were different species. Some local varieties such as’Nanan 1’ and ’Nongkesuo 3’ were clustered together, showing they had a similar genetic background. Some tested ’Dongbi’ varieties were clustered into different groups, which showed genetic polymorphisms also exist between different ’Dongbi’ varieties. In addition, EST-SSR also revealed that some varieties such as ’Matou 1’ and ’Matou 2’ had genetic difference. Conclusions above fit with clustering results from ISSR, which showed that both of the two molecular markers can reveal the commonness and difference of clustering results of longan. A comprehensive analysis of electrophoresis amplification and clustering results showed that that our development of EST-SSR markers of longan was effective, and it can be applied to the genetic relationship analysis of longan.