Analysis of genetic diversity of Malus species resources by TRAP markers

Abstract: 【Objective】Malus Mill. is a germplasm resource with important economic value in fruit cultivation and production. Breeders often use the interspecific hybridization for rootstock breeding. The genetic diversity analysis of parental materials would significantly effect apple breeding. Although there are abundant data available for the study of genetic evolutionary classification of apple plants, there are controversies on the standards of classification of different based on for different methods. The TRAP marker has the characteristics of good polymorphism, high stability and easy operation. In this study,we developed more genomes of 30 species of Malus and 3 species of Prunus by developing TRAP markers for phylogenetic evolution studies, from EST sequences. The genetic diversity information of apple genus was revealed horizontally, which would provide reference for the identification of genus relationship, parental selection, evolution analysis and utilization of germplasm resources.【Methods】DNA was extracted from young leaves of 30 species of Malus and 3 species of Pyrus by improved CTAB method. PCR amplification was performed using 4 fixed primers and 8 random primers, agarose gel electrophoresis detection. The genetic similarity coefficient(GS) was calculated by NTSYS-PC software, and cluster analysis was performed by unweight pair group method using arithmetic averages(UPGMA). The main coordinate analysis was performed using the EIGEN program. The population genetic structure of the test materials was analyzed using Structure 2.3.1 software. Three species of pear were used as tests for reliability in data statistics.【Results】In this study, 16 pairs of primer combinations amplified 407 effective fragments in 33 samples, with the fragment size ranged from 100 bp to 1 200 bp, of which 404 were polymorphic, and the average percentage of polymorphic was 99.26%. The number of alleles amplified by each primer combination was between 17 and 50, and the average number of alleles amplified by each primer pair was 25.44. UPGMA cluster analysis showed that the average genetic similarity coefficient GS of 33 species of Malus and Pyrus was 0.711, of which 30 species of Malus were GS=0.725. When GS=0.666, three species of Pyrus could be regarded as the first major group,while the other 30 species of Malus could be regarded as the second largest category. When GS=0.680,30 species of Malus could be divided into two subgroups, three species of M. doumeri, M. ioensis and M. angustifolia could be divided into one subgroup, and the other 27 species could be divided into another subgroup. Using the principal coordinate analysis method, which is the same as the UPGMA method, 33 test materials were divided into 3 categories. The three species of pear were in the first majorgroup; the three species of M. doumeri, M. ioensis and M. angustifolia of the apple genus were in the second largest category, and the other 27 species were in the third largest category. The third category coulf be divided into four sub-categories. The first sub-categories included M. komarovii, M. ombrophilia and M. yunnanensis, the M. ombrophilia and M. yunnanensis were closely related. The second subcategory included three materials of the M. sikkihensis, M. toringoides and M. transitoria. The third subclass includes: M. baccata, M. mandshurica, M. rockii, M. hupehensis, M. spectabilis, M. micromalus,M. sieboldii, M. kansuensis, M. prattii, M honanensis, M. soulardii and M. xiaojininensis; the fourth subcategory included M. halliana, M. sieversii, M. asiatica var. rinki, M. prunifolia, M. robusta, M. domestica subsp. chinensis, M. orientalis, M. sylvestris and M. fusca. Structure 2.3.1 software divided 33 test materials into three subgroups, the first subgroup included 13 species of M. baccata, M. mandshurica, M. rockii, M. sikkihensis, M. sieboldii, M. kansuensis, M. komarovii, M. toringoides, M. transitoria, M. ombrophilia, M. yunnanensis, M. xiaojininensis and M. ioensis. The second subgroup included 15 species of M. hupehensis, M. halliana, M. sieversii, M. asiatica var. rinki, M. prunifolia, M. spectabilis,M. micromalus, M. prattii, M. honanensis, M. soulardii, M. robusta, M. domestica subsp. chinensis, M.orientalis, M. sylvestris and M. fusca. The third subgroup included Malus doumeri, Malus angustifolia,and three pear species of P. bretschneideri, P. pyrifolia and P. communis. It seemed that the UPGMA clustering graph and the main coordinate clustering results were similar to the traditional pedigree, but there were obvious differences in the classification order of the group and the system. The results of population structure analysis were different from the cluster analysis of genetic similarity coefficient and the analysis of principal coordinate clustering.【Conclusion】The 16 pairs of TRAP markers were developed using TRAP marker technology for the genetic diversity analysis of apple germplasm materials. From the EST level, there were 33 species of apple species and 3 species of pear genus. The analysis results showed that the clustering results of UPGMA method and the main coordinate clustering results were similar, which is similar to the traditional pedigree, but there are some differences between the clustering results and the analysis of the population results. Relationship analysis provided molecular evidence for the relationship between the species of Malus genus.