Development Of Microsatellite Markers And Investigation Of Genetic Diversity In Tung Tree (Vernicia Fordii)

Tung tree (Vernicia fordii) is one of the important woody edible oil plants, especially in terms of biological energy has huge development value, so we will promote tung as woody oil species for long-term breeding research. With the depth of research such as the number, characters, genetic diversity and genetic relationship of tung germplasm resources largely restricts the breeding effect, and tung trees is peculiar in our country, has the extremely rich germplasm resources, so if we want to go into details of tung germplasm resources, first we should develop a large number of efficient molecular markers. However, lack of useful molecular markers hinders current genetic research. These new polymorphic genic-SSR markers will facilitate future studies on genetic diversity, molecular fingerprinting, molecular assisted breeding, comparative genomics and genetic mapping in tung tree. The genetic relatedness identified in V. fordii would provide potential clue to choose gerniplasms in interest as progenitors for cross breeding and variety improvement of V. fordii in practice. The main results were as follows:(1)This study derived 1,047 non-redundant ESTs from a total of 3202 original ESTs of V. fordii seed EST Library, and discovered 212 SSRs in 173 non-redundant ESTs. The di-and tri-nucleotide microsatellites, accounted for 94.34% of the SSRs. (AG/CT,43.87%)was the most abundant SSR motifs, followed by (AT/TA,19.34%), (AGC/GCT,5.66%) and(AAG/CTT,4.72%). Fourteen polymorphic EST-SSR markers were developed from 68 non-redundant ESTs loci tested in 10 cultivated Tung accessions by agarose gel and capillary electrophoresis.(2) We performed transcriptome sequencing of developing seeds and characterized microsatellites from transcriptome sequences to identify valuable markers for V. fordii molecular genetics research. A total of 81805 unigenes were identified, in which 6,366 SSRs in 5,404 unigenes were discovered. The di-and tri-nucleotide microsatellites accounted for 92% of the SSRs with (AG/CT,31.34%),(AAG/CTT,13.31%) and (AT/AT, 11.99%) being the most abundant SSR motifs. Fifteen polymorphic Genic-SSR markers were developed from 98 unigenes loci tested in 41 cultivated Tung accessions by agarose gel and capillary electrophoresis.(3) Polymorphism markers were applied to identify the monomorphism of 61 V. fordii inbred lines and eventually randomly selected the sample VF1-12 from 15 tung inbred lines with high homozygous, after 17-mer of analysis method and the low coverage of the initial sequencing, estimated tung genome size is 1.3 G, high homozygous,They have the same results.(4) 29 SSR (14 EST-SSR and 15 Genic-SSR) primer pairs were used to assess genetic diversity of 196 V. for dii individuals in this study. A total of 107 putative alleles were generated with a mean of 3.67 alleles per locus. The number of alleles per locus (Na) ranged from 2 to 11, the expected heterozygosity (He) ranged from 0.09 to 0.7, and the polymorphism information content (PIC) values ranged from 0.08 to 0.63, respectively.The 29 SSR markers exhibited a moderate level of polymorphism in V. fordii (He= 0.38, PIC= 0.35).(5) The UPGMA cluster tree illuminated the genetic similarity coefficients ranged from 0.9604-0.9986, the genetic distances were small ranging from 0.0022-0.0404, among V. fordii populations, indicating closeness of their genetic relationship。Cluster’s analysis showed that the phylogenetic relationships with HUNAN and GUIZHOU population were quite near. The genetic similarity-based dendrogram revealed that most of the 169 accessions from the same geographic region were mainly in the same cluster.