Development And Application Of Mitochondria SSR Markers In Cucumber(Cucumis Sativus L.)

Cucumber（Cucumis sativus L.）is an important economic crop in cucurbits and one of worldwide vegetables.The extensive application of DNA molecular marker technology has promoted the process of cucumber breeding,such as genetic map construction,genetic diversity and phylogenetic relationships,marker-assisted breeding and seed purity identification,etc.Cucumber genomics research was basically based on the nuclear genome,while the research of cytoplasmic genome was less,particularly in the mitochondrial genome.Cucumber mitochondrial genome had high frequency of intermolecular recombination,which led to the complex structure.The development of mitochondrial genome markers and their application have not been reported,which reason is probably uncertain phenotypic traits of mitochondrial genetics.Studies have shown that the mitochondrial genome of cucumber is paternally transmitted.Based on these characteristics,molecular markers can be developed and are used for tracking the source of mitochondria during hybridization.In addition,cultivated cucumbers with different ecological types have differences in the nuclear genome,but whether the differences were present in the mitochondrial genome sequence have not been reported.Based on PCR,simple sequence repeats（SSR）has been widely applied in various fields of life sciences including the breeding research,which has significant advantages including evenly distributed in whole genome,highly variable,highly polymorphic,easy to operate and abundant in eukaryotic organisms.Therefore,the development of SSR markers from cucumber mitochondrial genome has an important significance for the research of mitochondria.Sequencing of cucumber mitochondrial genome was completed in 2011.A large number of short repeat sequences laid the foundation for the development of cucumber mitochondrial genome markers.In this study,we developed the cucumber mitochondria SSR（mtSSR）markers based on mitochondrial genome and their versatility and polymorphisms were furtherly studied.The main results were as follows:1.Based on the cucumber mitochondrial genome sequence,mtSSR primers were developed with bioinformatics methods.The effectiveness and specificity of mtSSR primers were verified with an inbred line ’Beijingjietou’.Besides,the transferability was tested in cucurbit crops.We designed 71 pairs specific mtSSR markers that were all distributed in the intergenic region.Primers were proved to be the specific primers of cucumber mitochondrion genome by BLAST analysis and experimental verification.The results of the versatility showed that 9 pairs of primers had amplification products in watermelon and 18 pairs of primers had amplification products in summer squash.In addition,21 pairs of primers had amplification products in muskmelon.The sequences of primers that had amplification products were absolutely matched with the sequences of mitochondrial genome respectively by BLAST analysis.The flanking sequence of mtSSR is conservative,which could be used in the study of mitochondrion inheritance of cucumber and its related species.2.Markers were screened with different ecotypes of cucumber,and the purity of F₁ hybrid seeds was detected with polymorphic primers.The results showed that four pairs of polymorphic primers were screened from 71 pairs of mtSSR markers.The purity identification of F₁ hybrids showed that the bands of F₁ hybrids were consistent with the male parent,and it was also consistent with the results investigated in field（consistency reached 99.57%）.Contamination of F₁ hybrid seeds caused by self-inbred and other unknown pollens can be detected by using this method.The research demonstrates that the feasibility of the specific mtSSR markers in the C. sativus F₁ hybrids purity identification.