Genetic Diversity Analysis And Cultivar Indentification Of Loquat (Eriobotrya Japonica Lindl.) Based On SSR

Loquat [Eriobotrya japonica (Thunb) Lindl.], a rare and specialty fruits of subtropical areas, originated in China and has been cultivated for a Long history, with high economic value. Zhejiang and Jiangsu provinces are the main known producing areas of loquat, Also belong to highly evolved type of cultivation area, a lot of varieties and cultivars were developed. Therefore, study on the genetic diversity and phylogenetic relationship of Chinese main loquat germplasm resources based on the local cultivars and types of loquat cultivars in these areas, will has important guiding significance for innovative use and effective protection of loquat germplasm.In this study, a total of 54 cultivars or types were used, including main cultivars or types of loquat collected from Zhejiang and Jiangsu provinces, and cultivars of Fujian and Sichuan, two wild species E. prinoides var. daduheensis and E. Prinodides also included, and 110 located apple SSR markers were screened for genetic analysis of loquat SSR primers. The loquat microsatellite system was established to test for the genetic diversity and cultivar identification of 54 materials, genetic diversity among natural triploid strains of 'Dawuxing' loquat also studied, and the primers that can be used for identification of natural triploid loquat were screened, the main results as follows:1. The SSR system was estabilished with eight cultivars as 'Jidanhong','Jiajiao','Changhong', 'Tongpi','Dawuxing','Zaohuang','Dahongpao' and 'Dameiguihongpao'. Seventy eight pairs of polymorphism primers were screened from 110 ones, the polymorphism ratio was 71 percent, and the appropriate annealing tempretures of these 78 were optimized with gradient PCR.2. Four markers (AT000400, CH01d03, CH01h02, and CH03c02) were judged to be multi-locus and not used for genetic diversity analysis. The 39 polymorphic SSR markers gave a total of 155 alleles, two to seven alleles per marker with an average of 3.38. The effective number of alleles varied from 1.04 for CH01f02 to 4.01 for CH03a09, with an average of 2.21. The Shannon index, as a measure of gene diversity, ranged from 0.10 for CH01f02 to 1.49 for CH03a09, with an average of 0.84. The observed heterozygosity ranged from approximately zero for CH01f02 to 0.83 for Hi08a04, with a mean of 0.47. Similar values were calculated for experted heterozygosity, the highest was 0.76 with CH03a09 and CH02d12, the lowest was 0.04 of CH01f02, ith a mean of 0.50. The fixation index ranged from-0.70 for Hi08a04 to 1.00 for CH01f02, with an average of 0.07. All these indicated that the genetic diversity of seleted materials was abundant. In the dendogram constructed from UPGMA cluster analysis of the similarity matrix, with 155 SSR alleles, the accessions are clustered in two groups, the commonly cultivated loquats and the two wild species at a 0.48 threshold of SM coefficient. The cultivated loquat accessions were further subdivided into three subgroups, at a threshold of 0.723, and generally reflected their geographic origin. Subgroup A included all. local accessions from the geographically close Zhejiang and Jiangsu provinces, being temperate zone accessions, plus the'Longquan No.l'from Sichuan and 'Changhong' from Fujian, and the two Spanish cultivars. Subgroup B included the subtropical Fujian cultivars ('Zaozhong No.6', 'Jiefangzhong', 'Xiangzhong', 'Taicheng No.4' and 'Baili'), but also the Sichuan cultivar 'Jinfeng' and the Japanese cultivar 'Moriowase'. The third subgroup included only one cultivar: 'Dawuxing' of Sichuan. The clusters thus generally reflected the geographic origin of their members. PCA analyses, carried out using the similarity matrices for the 39 SSR markers, confirmed the UPGMA cluster analysis. The two wild species were separated from the cultivated accessions, and the cultivated accessions were classified in four groups, mainly accordingto their geographical distribution. All the Zhejiang accessions, ten Jiangsu accessions, the Japanese cultivar'Moriowase', and two Spanish cultivars,'Marc'and'Peluches', were in groupⅠ. The 15 Jiangsu accessions clustered in groupⅡ, all the Fujian cultivars in groupⅢ, and'Dawuxing' and 'Longquan No.l', the two most commonly cultivated varieties in Sichuan province, in groupⅣ. Thirty nine SSR markers distinguished all accessions except four cultivars:the two Spanish cultivars 'Marc' and 'Peluches' could not be distinguished from each other, and 'Meiyu' could not be distinguished from 'Changlv No.2'. The scores of these markers confirmed pedigrees, such as that of'Zaozhong No.6'(a 'Jiefangzhong'×'Moriowase'),'Changlv No.5'(a 'Baiyu'×'Tianzhong'), and 'Xiangzhong'. Based on the number of effective alleles, we selected a set of five SSR markers (CH03a09, CH02c06, CH04g12, CH02d12, and CH05h05) able to distinguish all the accessions except bud sports.3. Fifty five pairs of polymorphism primers were screened from 78 ones, gave a total 135 alleles with ten strains of'Dawuxing'. The allele with 222 base pairs of CH01h02 was the specific one of the triploid. Three alleles of 222 bp,199 bp and 195 bp were gave with primer CH01h02 at strain A332, three alleles of 214bp,195 bp and 185 bp with primer CH04c06, and another three of 270 bp,266 bp and 246 bp with primer NZ02b01. Similar alleles of 217 bp,195 bp and 185 bp with primer CH04c06 at strain A368 and A379, also 238 bp,236 bp and 230 bp with primer Hil5hl2 at strain A322. New alleles emerged as compared diploid and each one of triploid strains, indicating foreign genes maybe introgressed along with the formation progress of triploid strains. All ten strains completely Distinguished from each other, the highest SM similarity coefficient was between A2x and A313, with 0.926, and the contrast one was between A313 and A332, with the similarity coefficient of 0.496. Principal component analysis divided 10 strains into three groups, GroupⅠ, including the A484, A376, A379 and A368 of four lines, A35, A322 and A332 are three lines constitute the groupⅡ, groupⅢconsists of A2x, A313 and A484. All these indicated that SSR can be used to reveal the genetic diversity of natural triploid loquat, and to identify some of triploid strains.