| Cannabis sativa L.is a kind of dioecious cross pollination plant,which usually includes fiber hemp and seed hemp.To explore the genetic relationship of seed hemp germplasm resources,develop sex-linked markers and gene mapping can provide theoretical basis for sex identification of hemp seedling,heterosis utilization and breeding of gynoecious line.In this study,local seed hemp Yushe was used as materials,First,genome survey technology was used and 93germplasm of hemp were analyzed by SSR primers.Then two representative varieties of seed hemp were selected to explore the sex genetic characteristics.Then,stable sex-linked SCAR（Random Amplified Polymorphic DNA）markers were developed from RAPD（Random Amplified Polymorphic DNA）markers,and Bulked segregant analysis（BSA）was carried out for male and female characters of seed hemp to screen out sex-related candidate genes.The details are as follows:1.In order to analyze the population structure of seed hemp germplasm resources in China,the male and female single plants of seed hemp called Yushe were selected for survey sequencing.The results showed that the size of male genome was 776.71Mb and the size of female genome was 810.64Mb,which was slightly smaller than the published draft genome of hemp and marijuana.15 pairs of SSR markers were developed and screened.Combined with 15 pairs of markers in previous studies,93 local hemp germplasm in China were analyzed for genetic diversity.A total of 993 alleles were detected by30 pairs of primers.The major allele frequency ranged from 0.04 to0.53,the gene diversity index was 0.65-0.98,and the range of polymorphism information content index was 0.60-0.98.The cluster analysis showed that 93 germplasm resources were divided into fiber hemp and seed hemp at the similarity coefficient of 0.64;at the similarity coefficient of 0.66,they were further divided into three subgroups:fiber hemp in Shanxi and the south,fiber hemp in the north of Shanxi and seed hemp.2.In order to study the genetic characteristics of seed hemp,the sex segregation ratio and hormone sensitivity of representative germplasm of seed hemp were analyzed.A study on the sex segregation ratio of two representative seed hemps（Fenma No.3 and Yushe）shows that,in this experiment,the number of female was more than that of the male,and the sex ratio was not consistent with the theoretical ratio of1:1.In order to explore the sex hormone sensitivity,Yushe was selected as the experimental material,to study the effects of two hormones（gibberellin and ethrel）on the sex differentiation of three leaf hemp seedlings.The results showed that different concentrations of ethrel（30,60,100mg/L）were all the effects of feminization,and the most effective concentration was possibly around 60mg/L.Gibberellin has a male promoting effect on seed hemp.Under the gradient concentrations（50,100,200mg/L）,only highest concentrations（200mg/L）of gibberellin can significantly reduce the male to female ratio of seed hemp.In addition,the stem length of hemp seedlings treated with gibberellin is significantly longer than that of the control group,and the male flower organs are obviously stunted.3.In order to develop stable sex-linked SCAR markers,the published RAPD markers OPB-07 and OPD-05 were used to identify the female and male specific bands of Fenma No.3 and Yushe.After the recycling,cloning and sequencing of the specific bands,we got the specific fragments of 292bp with female and 956bp with male.According to the sequence,sex specific SCAR markers were developed.The female and male SCAR markers were named SCAR292 and SCAR500,respectively.These two markers only showed a single band in the test material,which can accurately identify the sex of cannabis.4.In order to further develop the sex-linked markers with seed hemp and screen the candidate genes for sex determination,50 male and female plants of Yushe were used to establish extreme mixing pools for high-throughput sequencing.The results showed that 455 contigs with significant enrichment related sites were screened out with 0.83as the threshold value after data filtering,and the sex-linked markers were mainly distributed between 7527066-9950366 on chromosome 10,indicating that the male related region of cannabis was within 2.42Mb between MADC5 and MADC6.Further gene annotation was carried out in KEGG,GO,NR,Swiss Prot and COG databases,and it was found that three reproductive development genes were identified in GO database.This study not only evaluated the genetic diversity of seed hemp germplasm resources,but also analyzed the genetic characteristics of sex.The developed sex-specific markers and the selected sex candidate genes have important reference value for the breeding of gynoecious line,the early sex identification and the separation of sex development related genes of seed hemp.In the follow-up study,we should collect monoecious materials,try and optimize the multi PCR system of sex identification,verify the candidate genes related to gender development,and further explore the mechanism of sex determination in the molecular level. |
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