On EST-SSR Primer Design And Genetic Diversity In Eucommia Ulmoides Oliver

Eucommia ulmoides Oliv. is the only species of genus (Eucommia) in the Eucommiaceaefamily. It is a tree species of important economic and medicinal values with strong adaptabilitywidely distributed in27provinces (areas) of China, it also has strong resistance to diseases andinsect pests. Wild resources of E.ulmoides are on the verge of extinction. Related studies haveshown that E.ulmoides has abundant variation types, i.e variation in shapes, structures and eventhe embryonic types. Therefore the researches are urgently needed on its genetics from theperspective of molecular biology. In this study, we used the transcriptome sequencing data ofleaves and fruits of E.ulmoides from different growing period to develop primers suitable forthe SSR markers. After that, systematic analysis was conducted on its genetic diversity, geneticrelationship, the degree of differentiation between and within, genetic structure of grouppopulation of experimental materials included136samples from20groups of E.ulmoides. Theresults of the present study could provide theoretical basis for germplasm resources collection,conservation and for genetic breeding of E.ulmoides. The main results were as follows:1. The SSR primers of E.ulmoides were independently developed. SSR loci of2to6repeated units including1334strips distributed in150kinds were seeked out throughbioinformatics methods from49610Unigene sequences of transcriptome data extracted fromeucommia leaves and fruits, showing that frequency of SSR loci in the transcriptome data waslow by only2.9%. most repeated units were from dinucleotide and trinucleotide, accounts for92.72%of all SSR loci. A total of114pairs of Primer were designed to be synthesized usingPrimer5software according to Unigene sequences of ranscriptome, in which13primers ofhigh polymorphism and stability were selected through PAGE electrophoresis test. The lengthof the target fragment was between120bp to250bp.2. The genetic divisity of E.ulmoides SSR sites were detected.73alleles have beendetected from10SSR sites. Number of alleles (na) was in the range of2to13, with7.3allelesdetected on an average number of per site. Effective number of alleles (ne) was20.18, an average number was2.018. Expected heterozygosity (He) and observed heterozygosity (Ho)ranged from0.839to0.056and0.586to0.057,with the mean value0.362and0.290,respectively.The average Nei’s was0.361. According to the value of PIC, the sort of validity ofthe primers was: DZ64﹥EU12﹥E.17﹥EU108﹥EU69﹥EU96﹥E.29﹥EU29﹥E.37﹥E.19.Hardy-Weinberg detection showed that, the5sites, E.17, EU29, E.37, DZ64, EU1085, hadreached a significant level. Sites EU12, E.19, E.29, EU69, EU96did not reach significantlevels, indicating that Eucommia were intered by the evolutionary factors of genetic drift,mutation, migration and selection.3. The results of genetic diversity of E.ulmoides showed that the number of alleles (na)was in the range of2.0to3.6on a population level, the alleles on average for each group was2.6; effective number of alleles (ne) rangeed from1.426to2.299, the average effective numberof alleles (ne) was1.780; gene abundance (AR) was in the range of1.509to1.893, with anaverage of1.731; observed heterozygosity (Ho) ranged from0.167to0.450, with an average of0.292; expected heterozygosity (He) ranged from0.252to0.433, with an average of0.342.Hardy-Weinberg detection showed that in addition to three groups: Jiangya in Hunan, Shangqiu,Xinjiang, the remaining17groups have shown an unbalanced state, indicating that E.ulmoideshave been affected by natural selection, inbreeding, artificial selection, and many other factors;genetic differentiation studies have shown that97.40%of the genetic differentiation camewithin the group while only2.60%of the genetic variation from diffrernt groups.4. Population genetic structure analysis showed that E.ulmoides resources in China waswith low gene exchange and less pure gene pool, only groups of Guangxi, Jiangsu, Zhejiang,Luoyang, Shanxi and Shennongjia have less gene flow. There were65samples on theindividual level which had less gene flow, with a higher purity of the gene pool. Dominantgene pool of E.ulmoides resources had different colors in the South and North. The colors ofdominiant gene pool in the northern groups were red (the South: North=1:5), green (the South:North=1:2), Rose Red (South: North=1:3). In Southern groups, the colors were yellow (theSouth: North=2:1), blue and cyan. The distribution ratio of gene pool in the population of the South and the North was about1:1. The red rose gene pool mainly distributed in the groups ofHenan and Shennongjia in Hubei,with a rather narrow distribution.5. The clustering results suggested that20groups could be divided into four groups. Thefirst group was Shennongjia group, the second one was Zhejiang group, the third was Luoyang,Xinxiang, Shangqiu and Nanyang, the fourth was the rest of the14groups, indicated thatE.ulmoides had closegenetic relationship, high genetic exchange.