Development And Utilization Of EST-SSR Molecular Markers For Three Wild Fruit Trees In Xinjiang

Cerasus tianschanica Pojark,Prunus divaricata and Prunus domestica L.are important wild fruit tree resources in Xinjiang Yili area,which play an important role in maintaining and improving the local ecological system.At present,three kinds of fruit trees are faced with the double test of fragile ecological environment and severe man-made damage.In this study,EST-SSR molecular marker is used to protect these wild resources.By searching the EST sequences of three fruit trees,the specific primers were designed,the optimal SSR-PCR reaction system was screened,the primers were validated,and then the primers will used to study the genomics of three wild fruit trees.The result were as follows:(1)There were 111850 Cerasus tianschanica Pojark EST sequences in the NCBI public database and 8288 ESTs containing SSR,accounting for 7.41% of the total ESTs.Among them,dinucleotide is mianly,CT and TC are the main types of nucleotides,accounting for 40.17% of the total.30 pairs of primers were synthesized and detected,the effective amplification rate was 70.00%.(2)There were 347 Prunus divaricata EST sequences in the NCBI public database and 270 ESTs containing SSR,accounting for 77.81% of the total ESTs.There are only three types of nucleotides in the limited sequence of Prunus divaricata.The SSR type is not abundant,but the frequency is the highest in the three fruit trees.The number of repeats was dominated by dinucleotide and trinucleotide,and in dinucleotide repeats was the highest,accounting for 87.40% of the total SSR.30 pairs of available primers were synthesized and the effective amplification rate was 66.67%.(3)In the NCBI database,there are only 54 EST sequences in Prunus domestica L,which are not sufficient to design primers.Therefore,the primers are designed with the EST sequences of its close relatives,such as Prunus spinosa L,Prunus salicina Lindl,Prunus insititi L.and Prunussimonii Carrière.Then,692 SSRs were found in 4379 EST sequences,accounting for 15.80% of the total EST.Dinucleotides and trinucleotides are the main ones.Among them,the dinucleotide account for 82.5% of the total SSRs and trinucleotide are the most abundant repeat elements.Finally,54 pairs of primers were synthesized and the effective amplification rate was 64.82%.(4)In this study,114 pairs of specific primers were designed for the Cerasus tianschanica Pojark,Prunus divaricata and Prunus domestica L.Most primers were able to amplify a clear band at 56°C,only the Cerasus tianschanica Pojark primers had fewer bands at this temperature.So,the primers without amplification products were then subjected to annealing temperature gradient screening.A total of 12 temperature gradients were designed with a range of 48~66°C.The result showed that most of the primer annealing temperature concentrated between 55.9~60.3°C.(5)In this study,an optimized SSR-PCR reaction system was established from the stability and economy.The optimal combination of dNTPs,primers,Taq enzymes and template DNA were screened out.20 ?L system reaction system: dNTP 0.2 mmol/L,primer 0.4 ?mol/L,Taq enzyme 0.75 U,template DNA 60 ng.SSR amplification program: 95? pre-denaturation 5 min;30 cycles(95? denaturation 30 s,56? annealing 30 s,72? extension 45 s),72? extension 10 min.The experiment confirmed that the system was stable and reliable.(6)The results showed that the Cerasus tianschanica Pojark,Prunus divaricata and Prunus domestica L.EST-SSR primers were have some versatility,and the versatility among the same genera plants was significantly higher than the same family plants.The amplification rate of Prunus divaricata primers in Prunus domestica L.was 90.00%,shows that the difference genome between Prunus divaricata and Prunus domestica L.was small.