Development Of Peach EST-SSR Primers And Their Transferability In Partial Rosaceae Species

Peach is an important economic fruit tree and garden tree specie in the world, and it is also a woody model plant of the Rosacea. It is of great significance to study the germplasm resources and genetic background of peach. In the process of scientific research, SSR marker is still one of the windows that explore the germplasm resources and genetic background of species. The development scales of SSR primers are important for any species. Unfortunately, The SSR primers of peach are too less, and only a handful in other Rosaseae species. The whole transcriptome of peach was sequenced though different organs by Verde, producing a total of 80797 ESTs together with results of previous studies and providing abundant sequence resources. In this study, we used the MISA to identify SSRs from the 80797 ESTs, then designed SSR primers by primer 3 modules and took validation of PCR amplification with 100 primers on 12 cultivars of different types. Last, we also explored the transferability of 48 primers on 30 commonly Rosacea species(1) Identification of EST-SSRsIt found that 12.3% of redundancy rate was hid in 80797 non-redundant ESTs after rechecking with CD_hit process.12519 SSRs were identified from 10272 ESTs by MISA, that with an occurrence frequency of 14.60%. All the SSR types included 159 different motifs which floated between dinucleotide and hexanecleotide. The most three length types were single nucleotide, dinucleotide and hexanucleotide sharing the distribution frequency of 35.80%,36.43% and 25.56%, respectively. Others, the more the repetition number of motif was, the lower the distribution frequency was. The different motif types, which possessed the same length, distributed unevenly. Furtherly it had the preference for a few motif types.(2) Design of SSR primersIt obtained 4526 SSR primers from 12519 EST-SSRs by primer 3 modules included 2598 dinucleotide and trinucleotide in total. The primer development efficiency only depended on the cardinal number size of EST-SSRs. The total development efficiency was 36.15%.(3) SSR-PCRverificationIn total,237 bands were amplified on 12 peaches of different types with 100 selected primers. It was 2.37 in average that single pair of primer amplified the bands, and 79% primers could get two or more. The expected heterozygosity and observed heterozygosity ranged from 0-0.7659 and 0-0.09167, with average of 0.3817 and 0.1230 respectively. It had a range of 0-0.8056 and an average of 0.4054 for these primers discrimination.(4) Transferability analysisAfter the transferability analysis, we found that 66.67% SSR primers had amplified on 30 Rosaceae species entirely.16 species had lost the band with primer 018, which touched highest deletion rates. The species with the highest deletions degree was R30 that lost 7 loci. Furtherly,95% and 90% primers had the effectively amplification of more than 66.67% and 93.33%, respectively.