| Cannabis sativa L., a member of the Cannabinaceae, is an annual herbaceous plant. It is an important economic plant for the production of food, fiber and intoxicants. China has a long history and rich germplasm resources. In this study, we developed the SSR markers based on cannabis transcriptome sequence information for the first time. Genetic polymorphism analysis and the division of the group were carried out on the 115 cannabis varieties and germplasm resources domestic and overseas. The main results are as follows:(1) Development of SSR markers based on cannabis transcriptome. 32,324 cannabis EST sequences were obtained based on the cannabis transcriptome information published. The total length was 38.12 MB and average length was 1207 bp. The SSR loci were searched by Auto SSR software. 4577 SSR loci were obtained in 3624 sequence, 668 sequence contains at least two SSR loci, 121 sequences containing compound SSR loci, in which the largest number of repeats was trinucleotide, accounting for 50.99% of the total SSR loci. Followed by hexanucleotides and dinucleotides, accounting for 25.13% and 16.34% respectively. The highest frequency was AAG / CTT(18.00%), followed by the AG / CT(12.91%, 591), AAT / ATT(8.65%, 396). According to the characteristics of cannabis SSR loci distribution, 3442 cannabis SSR markers were developed.(2) The function prediction of EST sequence contained SSR loci. 1591 sequences were annotated, accounting for 43.90%. According to the COG function classification, EST sequences contained SSR loci can be divided into 23 categories. Among them, the largest number was general function prediction type, for 350. Followed by the type of transcriptional function and signal transduction mechanisms, for 207 and 177, respectively.(3) A total of 117 primers were designed from 3442 cannabis SSR markers. Among them, 108 primers(92.31%) can effectively amplify in cannabis. 7 primers(CAN0011, CAN0035, CAN0107, CAN0504, CAN0576, CAN0690 and CAN2915) had two amplification sites, 2 primers(CAN0001 and CAN0079) had three amplification sites. There were 62, 24, 8, 1, 2, 1 loci with two, three, four, five, six, seven alleles, respectively. 21 loci without polymorphism. Finally, 252 polymorphic loci were obtained in 87 primers, average 2.9 amplification polymorphism loci per primers. Therefore, the EST-SSR markers developed in this study had a high quality.(4) Screening 45 primers as core primers for the analysis of cannabis germplasm polymorphism. Taking genetic similarity(GS) was 0.74 as threshold, 115 cannabis germplasm were divided into four groups roughly in accordance with the geographical distribution: Northern China, Europe, Central China and Southern China. According to the geographic latitude distribution, 3 groups of Chinese cannabis germplasm were corresponding to cool temperate, warm temperate, and subtropical zones, respectively. In PCo A, the three main axes explained approximately 62% of the total variation, at 36.4%, 16.8%, and 8.7%, respectively. The 115 cannabis varieties could also be distinctly classified into 4 groups.(5) Analysis on different groups of genetic distance and genetic relationships. Among the 4 groups, Northern China and Europe had a closer relationship may be due to that the northern part of China and the European had a similar latitude. The genetic distance in Central China and Southern China was nearest, while the genetic distance between Northern China and Southern China was furthest. Central China group and has the highest genetic polymorphism. The results laid a good foundation for the collection and utilization of cannabis germplasm resources. |
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