Construction Of A SNP Fingerprint Databases For Lentinula Edodes Cultivars

Lentinula edodes (Berk.) Pegler, as a edible and medicinal fungi, is one of the most popular edible fungi and widely cultivated in Asia due to its unique flavor and health care efficacy. The discrimination among cultivars is necessary and urgent to protect breeder’s right. However, there is no scientific, reliable, as the accepted methods of cultivars identification presently. In this study, we used22national recognized cultivars of L. edodes as materials, according to the known genes to be involved in the growth and development process of the shiitake mushroom, analyzed the single nucleotide polymorphisms existed in the population for the functional genes by sequencing directly. Through the SNP analysis, the aim is to reveal the genetic diversity of the tested germplasms, and to evaluate the feasibility of SNP for distinguishing among cultivars, in order to construct a SNP fingerprinting of cultivated varieties of L. edodes finally.First of all, through alignment of the sequences of protoplasted monokaryons, we verified that the double peaks appeared in the sequencing chromatograms of lcc2and ras were due to the base difference between the two monokaryons, while the overlapping peaks were the result of the insertion or deletion of base, which means the dikaryotic cells were heterozygous state consisted of two different nuclei.In the preliminary experiment,54polymorphism genes were identified from58candidate genes, further cbhll-l, cyp1, exg1, pyrG, rcb2and rnrBl were used to the distinguished work of22cultivars. It shown that there were5,8,27,14,4and4SNP detected in cbhII-1, cypl, exgl, pyrG, rcb2and rnrBl respectively among22cultivars, which divided the popular into9,8,11,7,8and9gene type. A total of72SNPs were detected from the six genes. There were59transition mutations and13transversion mutations in the72SNPs. SNP occurred at a frequency of0.96%in exons, far lower than the non-coding region with SNP frequency of2.78%. There were totally33SNPs in the exons, of which25were synonymous mutations and8were non-synonymous mutations caused corresponding change of coded amino acids. The different genetic properties among the different cultivars of a same species are attributed to the mutations of DNA sequences. The polymorphism loci found in this study would lay a foundation for the further correlation analysis between the SNP labeling and specific traits.According to the result of SNP cluster, there were1-55polymorphic SNPs loci in the22cultivars, which suggested that the difference of the relationship distance among the main cultivars in our country was relatively large and genetic backgrounds was diverse. xiangjiu, guangxiang-51, Huaxiang-5and L952as wild-domestication or imported strains, there were obvious differences from all other varieties; however Shenxiang-8, Shenxiang-10and Shenxiang-12as a series hybrid varieties, L135and Senyuan-10there were slight differences between each other because of having the same parental background. Integrated the SNPs of the six target genes, all the22tested samples were distinguished completely. The hot figure of SNPs exhibited that the specific SNP type was present in every cultivar of L. edodes, suggesting that the SNP analysis of functional gene could be used to distinguish the cultivars of the Shiitake mushroom.Finally, other three recognized cultivars Wuxiang-1, Huaxiang-8and Junxing-8were to used in the validation experiments, the results showed the three strains existed large difference from most strains, however displayed the same genotype with Shenxiang-8, Cr-04and Cr-62separately, which indicated that there were some limitations in the cultivars discrimination only by the six filtered genes. Looking for more functional gene fragments with high polymorphism and could cover the whole genome will enable SNP to become a powerful tool for variety identification.The present study is the first to analyze the SNPs of functional genes of the shiitake mushroom, and try to apply SNP to strain identification and discrimination. Compared with the former strain-typing methods, SNP information are highly stable, easy to storage and exchange for labs, in genetic specificity identification of a new breed without any reference, greatly improves the work efficiency. And the SNP in functional genes might be one of the general factors for the different traits between cultivars.