Genome survey analysis and SSR loci mining of Bupleurum falcatum / 中国中药杂志

Buplewrum falcatum is a traditional Chinese medicine,which is mainly used for the treatment of cold and liver protection. B. falcatum is dominantly cultivated in Japan as well as planted in China,Korea and other countries and regions. In order to determine the appropriate sequencing strategy,the genome survey before large-scale genome sequencing is needed. This survey can provide information about the size and complexity of the whole genome of the target species. In the present study,the next generation sequencing technology( Illumina Hiseq 2000) was used to analyze the genome size and complexity of B. falcatum. In addition,SSR loci were analyzed from the sequenced data. Primer 3 was used to design specific primers and 33 pairs of primers were randomly selected for PCR with template DNA of B. falcatum,and the PCR system and optimal annealing temperature were screened. A total of 288. 64 G genome sequence data was obtained,and the estimated genome size of B. falcatum was 2 119. 58 Mb. The measured genome data depth was138×; the rate of heterozygosity was 1. 84%; and the ratio of repeat sequence was 83. 89%. It is speculated that the genome of B. falcatum is complex. The preliminary assembly was performed with K-mer = 41,and the contig N50 was 224 bp,the total length 896. 97 Mb,the scaffold N50 313 bp,and the total length was 922. 67 Mb. A total of 91 377 SSR sequences were detected in the sequenced genome data which were distributed in 70 809 unigenes.The main type is dinucleotide repeats,with 49 680 sequences,accounting for70. 16%. Among the 33 pairs of primers randomly synthesized according to the obtained SSR sequences,21 pairs were successfully amplifying the target sequences. The results will be helpful for later large scale genome sequencing and SSR molecular markers development for germplasm identification and trait mapping.

Genetic diversity analysis of Tetrastigma hemsleyanum germplasm resources based on fluorescently labeled SSR markers / 中草药

Objective To understand the genetic diversity and genetic relationship of 64 samples of Tetrastigma hemsleyanum germplasm resources. Methods Fluorescently labeled SSR were used for PCR amplification, POPGENE32 software was used to analyze genetic diversity and genetic relationship of 64 samples of T. hemsleyanum germplasm resources, UPGMA method was used to construct their genetic tree map, NTSYS software was used to construct their two-dimensional principal component analysis map and three-dimensional scatter map. Result Eight pairs of primers with clear and reproducible bands were screened from 14 pairs of primers and used for genomic DNA amplification of 64 sample materials. The observed number of alleles (Na) ranged from 3 to 13, and the mean value was 7.875 0; The effective number of alleles (Ne) ranged from 1.424 9 to 6.087 4, and the mean value was 3.605 2; The Shannon information index (I) ranged from 0.689 5 to 2.082 4, and the mean value was 1.424 0; The observed heterozyghosity (Ho) ranged from 0.206 3 to 0.734 4, and the mean value was 0.524 7; The expected heterozygosity (He) ranged from 0.300 6 to 0.842 4, and the mean value was 0.658 4; The Nei’s gene diversity (H) ranged from 0.298 2 to 0.835 7, and the mean value was 0.653 2; The polymorphism information content (PIC) ranged from 0.288 0 to 0.817 5, and the mean value was 0.614 5; The genetic similarity coefficient ranged from 0.115 4 to 0.954 5; The genetic distance ranged from 0.000 0 to 3.218 1. It was indicated that the genetic relationship of 64 T. hemsleyanum germplasm resources was far, and the degree of genetic differentiation was high. At the genetic distance of 1.018 9, 64 T. hemsleyanum germplasm resources could be divided into five groups. Conclusion There was no necessary connection between geographical difference and genetic difference of germplasm. Themsleyanum germplasm resources are rich in genetic diversity. The results of fluorescently labeled SSR analysis can provide some references for the utilization and variety breeding of T. hemsleyanum germplasm resources.