Using the fluorescent labeled SSR markers to establish the molecular ID of pear germplasm resources in Shandong

Abstract：【Objective】As the origin and diversity center of oriental pears, China is very rich in Pyrus germplasm, and more than 3 000 cultivars have been recorded. According to the origin and geographical distribution, pears cultivated in China are generally divided into four systems:Chinese White Pear (P. bretschneideri Rehd.) , Sand Pear, Ussurian Pear and Sinkiang Pear.Shandong is one of the main cultivation areas of pear in China, with long cultivation history and rich germplasm resources, such as the famous local varieties of'Chili'in Laiyang, 'Xiangshui'in Qixia and'Changba'in Huangxian.However, it is difficult to distinguish different cultivars or germplasm accurately only by the traditional morphological identification method.Establishing the multi-level molecular identification techniques can improve the accuracy of cultivar identification.In this paper, 45 pear germplasm resources collected from different regions of Shandong province were used as materials to establish molecular ID code by simple sequence repeat (SSR) markers.【Methods】The experiment was conducted from June 2016 to October 2017 at Shandong Institute of Pomology.The 45 germplasm resources tested were collected from the germplasm resources nursery of Tianping Lake core demonstration orchard, Tai'an Comprehensive Experimental Station.40 pairs of primers were developed and designed from SSR-enriched library, and 10 pairs of primers with high polymorphisms and good repeatability were selected, and then were labeled with FAM fluorescent for amplification and capillary electrophoresis.Genomic DNA was extracted from fresh leaves according to the CTAB protocol.The PCR was carried out in a final volume of 20 μL containing 2 μL DNA template (10 ng· μL^-1) , 2 μL 10×PCR buffer (including MgCl₂) , 0.4 μL dNTPs (10 mmol· L^-1) , 0.3 μ L of each of the two primers (20 µmol· L^-1) , 0.2 μL Taq DNA polymerase, and 14.8 μL sterile distilled water.PCR reaction was programmed as:one cycle of 5 min at 94 ℃ as initial denaturation, followed by 35 cycles, in which each cycle consisted of a denaturation step at 94 ℃ for 30 s, an annealing step at suitable temperature for 35 s, and an extension step at 72 ℃ for 40 s, followed by final extension at 72 ℃ for 5 min.The products of 45 samples with each primer pairs were analyzed.Each band pattern was coded by different single digit or lowercase letters.Meanwhile, according to the number of band patterns, in an ascending order, the 10 primer pairs were decided, and molecular ID codes of 45 pear germplasm were established.The software POPGENE 32 was used to analyze the data, and the number of alleles amplified by the primers, the amplification band type, and the gene diversity and polymorphism information content of the samples at different SSR sites were obtained.【Results】The results showed that a total of 111 polymorphic alleles and 189 polymorphic patterns were revealed by the 10 primer pairs, with an average of 11.1 alleles and 18.9 patterns for each primer pairs.The length of the amplified fragment was 85-295 bp.The molecular ID codes of 45 tested germplasm were different, which could distinguish all germplasm resources.This study indicated that the detection technology by using fluorescent labeled SSR markers had the merits of reliable, efficient and highthroughput, and that it is convenient and efficient to establish the molecular ID of pear gemplasm using the above encoded mode of patterns.【Conclusion】The molecular ID codes of 45 pear germplasm resources in Shandong were established using the fluorescent labeled SSR markers, and the constructed molecular ID was different from one another and could completely distinguish 45 pear germplasm resources.It not only provides a rapid, accurate and efficient molecular identification method for pear germplasm resources in Shandong, but also contributes to provide a reference for varieties identification, evaluation and utilization, and genetic relationship analysis.