Development And Application Of Indeland Ssr Markers In Bottle Gourd(Lagenaria Siceraria(Molina)Standl.)

The Cucurbitaceae family, consisting of～800species falling to～120genera, includes many economically important vegetable crops such as cucumber (Cucumis sativus L.), melon (Cucumis melo L.), watermelon (Citrullus lanatus (Thunb.) L.) and pumpkin (Cucurbita maxima L.). Bottle gourd had long cultivated history in China, archaeological researches indicated that bottle gourd was one of the earliest plants domesticated by human. There were variety of molecular markers, SSR markers developed much deeper, and InDel markers is the representative of the new tag. By using molecular markers, more detailed and reliable genetic informations of some useful agronomic traits could been known, the scientificity of parent selection in breeding could been enhanced, while explore and discover the vegetable germplasm resources, understand the origin of the crop and evolutionary relationships. The research about bottle gourd molecular markers were greatly backward in cucumber and cabbage. The application of molecular markers to study genetic diversity and phylogenetic relationship of bottle gourd germplasm resources, had an important significance to promote the development and utilization of bottle gourd germplasm resources.Our experiments first used SSR markers on the genetic relationship analysis of the cucurbitaceae, and then selected the96representative domestic bottle gourd germplasm resources using InDel molecular markers to analyze their genetic diversity and phylogenetic relationship. And we explored the genetic differences of Chinese bottle gourds, the kinship of inter-subspecific and intra-subspecific. The main results are as follows: 1. The assembled con tigs/singletons sequences were screened for perfect microsatellites using the software mreps2.5(http://bioinfo.lifl.fr/mreps/). The software websat (http://wsmartins.net/websat/) was used to design primers flanking SSRs,400SSR markers were developed. Only sequences containing SSRs equal to or longer than20bp were used for primer design. In order to study the phylogenetic relationship of the cucurbitaceae, we use200bottle gourd SSR primers, DNA of pumpkin, watermelon, gourd and bitter gourd for PCR amplifications. Transferability of these markers to four other cucurbitaceae species showed obvious decline, only transfer rate of these markers to watermelon is higher, which showed that the watermelon and bottle gourd had closest genetic relationship.We BLAST CRY1and UDP-glucosyltransferase gene with bottle and cucumber, rice and wheat, soybean and common bean, bottle gourd and melon. BLAST N result showed that13370bottle gourd sequences matched the cucumber genome in at least100bp overlap. Notably, we found that the gene Cryprochrome1(CRY1)showed an identity value of as high as93.5%in the conserved C-terminus DAS domains between the two species, demonstrating that the two species are phylogenetically very close. Another conserved plant gene, the UDP-glucosyltransferase gene, showed85%sequence identity between bottle gourd and cucumber, showed that the cucumber and bottle gourd had close genetic relationship.2. PCR was carried out by the400SSRs in our experiments. We finally pick out14SSR markers that amplification of them were stable and separating clearly. Based on14polymorphic SSR markers, the dendrogram was constructed to analyze the kinship of44Chinese bottle gourd accessions. However, Chinese bottle gourd cultivars/landraces didn’t detect obvious clustering by geographical location, that clustering of bottle gourd accessions was based around fruit morphology much more than on geographical origin. Consequently, base on the different shapes,44bottle gourd were classified into three groups.3.947InDel markers were totally obtained from the RAD-seq project. PCR was carried out by the selected85InDels in our experiments that their sequences containing InDels equal to or longer than10bp. We finally pick out22InDel markers that amplification of them were stable and separating clearly. Based on22polymorphic InDel markers, the dendrogram was constructed by NTSYS-pc software to analyze the kinship of96Chinese bottle gourd accessions. The result showed the genetic similarity coefficients among these bottle gourd accessions were in the amplitude range of0.36-1.00. We classified the96bottle gourd accessions into eight groups, in terms of the genetic similarity coefficient of0.68. These materials distributed relatively evenly in the map of principal component analysis. However, Chinese bottle gourd cultivars/landraces didn’t detect obvious clustering by geographical location, that clustering of bottle gourd accessions was based around fruit morphology much more than on geographical origin. Consequently, base on the different shapes,96bottle gourd were classified into three groups.In summary, we have developed400SSR markers and947InDel markers, and they are applied to the cucurbitaceae kinship and the bottle gourd genetic diversity research, the results showed that the bottle gourd had closest genetic relationship with watermelon in our experiment, the bottle gourd can be classified according to the shape.