Genetic Diversity Evaluation Of Germplasm Resources Of Crocus Sativus L. In China

As a valuable Chinese herbal medicine,saffron,the dried stigma of Crocus sativus L.,can be used as a medicinal material,which has the functions of promoting blood circulation,soothing the nerves,detoxifying,etc.,and the market demand is large.However,the planting conditions and technical requirements of saffron are high,the genetic background is still unclear,the saffron variety is relatively simple,and the quality of saffron in different producing areas is also different,which restricts the benign development of saffron industry.With the continuous development of sequencing technology,the use of bioinformatics methods to analyze high-throughput sequencing data provides an effective tool for the analysis of the genetic background of saffron.In this study,transcriptome sequencing of saffron flower buds was performed by high-throughput sequencing technology.The SSR loci in the saffron genome were searched by analyzing transcriptome sequencing data,and SSR primers were designed and synthesized.In this study,non-denaturing polyacrylamide gel electrophoresis and DNA sequencing were used to detect the nucleotide diversity of SSR loci in the main germplasm resources of saffron,and to study the genetic diversity of the main germplasm resources of saffron in China.It provides a theoretical basis for the breeding,genetic improvement and industrial development of saffron varieties in China.The main research results are as follows:1.This study collected 18 planting resources in the main saffron producing areas of Tibet,Shanghai and Zhejiang,and established a genomic DNA library for different saffron germplasm resources.2.Pre-denaturation at 94℃ for 5 minutes,32 cycles(denaturation at 94℃ for 40 seconds,renaturation for 40 seconds at each primer annealing temperature,extension of 90 seconds at 72℃),and finally at 72℃ for 10 minutes and then stored at 4℃.The PCR amplification products were electrophoresed and detected by electrophoresis in a non-denaturing polyacrylamide gel(concentration is 6%,thickness is 1mm)in 1×TBE buffer.The voltage was 200 V and the electrophoresis time was 1.5 h.After electrophoresis,the gel was stained in AgNO3 solution,and the obtained electrophoresis bands were clear and the effect was good,which facilitated subsequent strip statistics.3.Based on the Illumina HiSeq sequencing platform,148,686 Unigenes of the saffron flower bud transcripts were detected(Total length,average length,N50,and GC content were 142,177,942 bp,956 bp,1,645 bp,and 44.27%,respectively),and 33,211 SSRs were detected in 26,268 Unigenes.According to bioinformatics analysis,there are 33,211 SSR loci with repeat sequences greater than 10 bp.Among the identified SSRs,the number of di-nucleotide motifs was 11,153,of which the maximum number of repeats was(AG)n,and the number was 8,545;the number of tri-nucleotide motifs was 11,762,of which the maximum number of repeats was(AAG)n,and the number was 3,793;the number of tetra-nucleotide motifs was 793;the number of pentra-nucleotide motifs was 913;the number of hexa-nucleotide motifs was 1,502.4.The saffron SSR primers were designed by using the software Primer Premier 3.0,and a total of 4,227 pairs of primers were obtained,from which 105 pairs of primers were randomly synthesized for verification.Among them,there are 58 pairs of real SSR primers,and real SSR primers account for 55% of the total.Therefore,it is speculated that in the 4,227 pairs of primers,about 2,324 pairs of real SSR primers can be used for the genetic diversity analysis of saffron.5.The genetic diversity of saffron planting resources from different habitats in China was studied by using 58 pairs of saffron real SSR primers.The results showed that the total difference of saffron germplasm resources in 18 different producing areas in China was small,but there were also some differences.Their genetic distances ranged from 0.0862 to 0.2586.Among them,Tibet 1 of No.5 and the local 2 of No.6 can be clustered into one saffron germplasm;The saffron 1 of No.7,Tibet 2 of No.12,Haozhou 1 of No.14 and the Huzhou of No.17 can be grouped together;The 15-year selection of No.10,Tibet 2 generations of No.11,Tibet 1 of No.13 and Haozhou 2 of No.15 can be grouped together.Shanghai 2 of No.4 and Fanhong 1 test species of No.8 can be grouped together.These data provided a theoretical basis for the study of genetic diversity of saffron germplasm resources in China.6.In the comparative genomics study of safflower planting resources,the saffron sequence was subjected to gelatinization purification,the target fragment was recovered,TA clone sequencing and multiple sequence alignment were performed,and BLAST alignment homologous sequences were performed in the NCBI database.As a result,it was found that in the sequence obtained by amplification of 18 saffron SSR primers,the sequence bases located in the coding region differed little,and the sequence bases located in the non-coding region differed greatly.This study provides a molecular basis for understanding the genetic diversity of germplasm resources in the main saffron producing areas of China,and the related results contribute to the genetic improvement of saffron and the breeding of new varieties.