Genetic Diversity Of Elymus Species, Cloning Of P-genome Specific Repetitive DNA Sequences And Development Of EST-SSR Markers In Wheat

This report is composed of three parts.Part I is Genetic Diversity of Elymus species. Using twelve Elymus species collected from China, the usefulness of ISSR and SSR markers in genetic diversity analysis was compared. The genetic diversity of Elymus dahuricus complex species of China was analysed. The differentiation model and genetic diversity distribution of Elymus sibiricus in China were investigated using SSR markers. The main results were as follows. (1) The comparing of the usefulness of ISSR and SSR markers showed that both type markers appeared high polymorphism, and the results of cluster analysis based on ISSR and SSR markers were coincided highly with ploidy level and morphologic taxonomy. For 12 species, ISSR markers had higher marker utility than that of SSR markers. But SSR markers were fit to related species analysis. The relation of genetic similarity matrix between ISSR and SSR was significant for 12 species, but not significant for related species. (2) The genetic diversity of 39 populations of four E. dahuricus complex species collected in China was studied using ISSR and SSR markers. The results showed that the differentiation among E. dahuricus complex species was lower. E. excelsus was a relative independence specie. There was definite differentiation between E. tangutorum and E. dahuricus. E. cylindricus showed complex relationship with others species. The UPGMA cluster appeared obvious zone character. The east of Qinghai province might be the geographical differentiation boundary of E. dahuricus complex species. (3) The analysis of 6 E. sibiricus populations based on SSRs showed that there were plenty SSR locus variances within population and among populations. The main genetic variance existed among population. In another, there was only low percentage of heterozygotes within population. These showed that E. sibiricus was a kind of self-pollination plant. The study of 40 populations from the main distribution area showed that there was obvious zone character among population differentiation. And Qingzang altiplano was the area of complicated genetic variance.Part II is Cloning of P-genome Specific Repetitive DNA Sequences. Agropyron Gaertn (P genome) and other genome of Triticum were used to isolate P-genome specific repetitive DNA sequences. The main results were as follows: (1) Using the method of reclaiming the products of RAPD, A specific DNA repetitive sequence of P genome in Agropyron, OPC08₅₇₈, was isolated and cloned. Homologous comparison with the DNA sequences registered in GenBank indicated that it was a new P-genome specific DNA repetitive sequence. Southern hybridization showed that OPC08₅₇₈ had the same distribution in the genome W, H and F. But it has no hybridization signals detected in ABD, R, V, E, St, Ns, I, D and A genomes. A pair of specific primers SC-OPC08 were designed according to the sequence of OPC08₅₇₈. This primer could not only distinguish P genome from genomes ABD, R, V, E, St, W, Ns, D, A, C, S, U, I, M and AG, but also could be used in the detection of chromatin of P genome from Wheat-Agropyron addition lines. The results of PCR amplification were consistent with the results of Southern hybridization. (2) Using the method of reclaiming the products of ISSR. A P-genome specific DNA repetitive sequence in Agropyron, UBC811₅₃₅, was isolated and cloned. Homologous comparition indicated that it is a new P-genome specific DNA repetitive sequence. Southern hybridization showed that UBC811₅₃₅ had strong distribution in the P genome. But it has no hybridization signals detected in F, V, H, Ns, R and St genomes. A pair of specific primers SC-UBC811 was designed according to the sequence of UBC811₅₃₅. This primer could be used in the detection of P chromatin from Wheat-Agropyron addition lines.Part III is Development and mapping of EST-SSR markers in wheat. A number of 151695 wheat expression sequence tags (ESTs) that originated from GenBank/dbEST from July 14, 2003 to August 24, 2004 were used to search for simple sequence repeats (SSRs) with motif 2-5 bp, and 2038 simple sequence repeats (EST-SSRs), which accounted for 1.34% of EST database, were identified. Based on these SSR sequences, 249 EST-SSR primer pairs and 166 amplified clear bands in various wheat cultivars were designed. These EST-SSR markers can be used as new molecular markers in wheat and related species. Using Chinese Spring nulli-tetrasomic lines, 93 EST-SSR primer pairs and 193 EST-SSR loci were located on 19 wheat chromosomes except for 4A and 4B. Forty-three loci were mapped on 11 chromosomes of the genetic framework previously constructed using recombinant inbred lines. EST sequences (166) corresponding to EST-SSR markers were analyzed by the programs BLASTN and BLASTX to obtain their information of bio-function and express types. After sequence similarity assignment, 48 ESTs have 35 kinds of homologous sequences or proteins. The most homologous sequences or proteins are from wheat and rice. 23 ESTs with coding 20 proteins putatively were located on 14 chromosomes and 5 genes of coding proteins putatively with 12 loci were mapping on 6 chromosomes.