| Wild B. juncea (L.) Czern. et Coss. (Brassicaceae) is an annual or biennial plant. It is distributed in 16 provinces in China particularly in disturbed wheat and rape fields, roadsides, gardens and abandoned land. In recent years it has become one of the most harmful weeds. Studies on B. juncea have mainly focused on the cultivated species and the knowledge of the wild populations is very limited. Its classification has remained problematic and there is confusion about the proper Latin name for wild B. juncea. It has been named B. juncea (L.) Czern. et Coss and B. juncea var. gracilis Tsen et Lee. The genetic diversity and structure of wild B. juncea in China is not well understood either. Thus it is necessary to analyse the taxonomy, genetic diversity and relationship among wild B. juncea populations collected from China to reveal its genetic structure as well as its origin and evolution. This may have important theoretical and practical significance in studying the origin and evolution of wild B. juncea.Research on wild B. juncea was conducted from autumn. A total of 250 individuals from 25 populations collected in 13 provinces of China were used. Additionally, wild B. juncea from Germany, American B. juncea, B. juncea var. juncea cultivated for its oil, B. juncea var. multiceps used as vegetable, the diploid species of U's triangle B. rapa, B. nigra, B. oleracea and the recombined species of U's triangle B. napus and B. carinata were collected. Seeds were planted at the Experimental Farm of Nanjing Agricultural University in Pailou district, Nanjing. Young leaves from seedlings of each individual were collected for DNA extraction. The sequences of ITS (internal transcribed spacer) and RPS2 in these plants were amplifed. The ITS sequences of B. juncea var. multisecta, B. juncea var. tumida, B. juncea var. megarrhiza, B. juncea var gracilis, and the ITS and RPS2 sequences of Arabidopsis thaliana were loaded from NCBI. The taxonomy of wild B. juncea was analyzed with ITS and RPS2 sequences. Twelve loci were obtained by the compound microsatellite marker technique. The genetic diversity and genetic structure populations and the origin of wild B. juncea were analyzed with selected 11 pairs of nuclear genome markers.Based on the ITS and RPS2 sequences, a cluster analysis was carried out by the MP method. The ITS sequence lengths of individuals was 614 bp, with 56 bp variable sites and 34 bp parsimony informative sites, accounting for 9.12% and 5.53% of the total length of sequence, respectively. The length of RPS2 of individuals was 1019 bp with 116 bp variable sites and 87 bp parsimony informative sites, accounting for 11.38% and 8.54% of the total length of sequence, respectively. In the MP tree, wild B. juncea and its relatives clustered into two groups. The first group included B. oleracea and B. carinata with the support of 81%. The second group included other species with the support of 58%. The second group is conformed by two subgroups. The first subgroup included wild B. juncea from Germany, American B. juncea, B. nigra, B. juncea var gracilis, and B. juncea var: tumida with the support of 84%. The second subgroup included other species also with the support of 84%. In the second subgroup, B. campestris, B. napus, wild B. juncea from Xizang were clustered with 52%. B. juncea var. junceas, B. juncea var. multiceps, B. juncea var. multisecta, B. juncea var. megarrhiza and the populations of wild B. juncea, except the population of Xizang were parallel. The MP tree based on ITS and RPS2 sequences suggested a close relation between B. juncea var. juncea, B. juncea var. multiceps and B. juncea var. multisecta. Considering our results and those of other researchers, we suggest to name wild B. juncea as B. juncea var. juncea.Use the sequence of RPS2 to identify the population where collected, the result showed two groups can be formed by 10 variable sites. The first group with the 10 variable sites included populations from Sichuan, Qinghai, Guizhou, and Henan provinces. The second group in which 10 variable sites had been changed included populations from Jiangsu, Anhui, Jiangxi, Zhejiang, Shannxi, Hubei, and Hunan provinces.Because the ITS sequences among the populations of wild B. juncea were similar, the phylogeny tree constructed by ITS and RPS2 had a low support rate, and could not clearly reveal the relationship among populations of wild B. juncea. We used the sequence of RPS2 with variable sites to construct the phylogeny tree. The UPGMA tree constructed by RPS2 with A. thaliana as outgroup reveals B. nigra and two populations of wild B. juncea from Germany clustered as the first group; populations of wild B. juncea from China, B. juncea var. juncea, B. juncea var. multiceps and B. campestris clustered as second group. The second group was formed by three subgroups. The first subgroup included B. campestris and the populations of wild B. juncea from Xinjiang and Xizang. The second subgroup included eight populations of Henan, Sichuan, Guizhou, and Qinghai. The third subgroup included thirteen populations of Hunan, Hubei, Jiangxi, Shannxi, Anhui, Jiangsu, Zhejiang, B. juncea var. juncea and B. juncea var. multiceps. The resullt of haplotypes is same. The phylogeny tree revealed the RPS2 sequences of wild B. juncea origining from Xizang and Xinjiang.The genetic distance among 25 populations was 0-0.033 measured by RPS2. The nucleotide diversity index (Pi), coefficient of gene differentiation (Nst) and gene flow (Nm) among 25 populations were 0.01302,0.959, and 0.01, respectively. It is suggested that 96% of overall genetic variation of 25 wild populations occurs among populations and 4% within populations. Gene flow among populations was limited.This study aimed to develop microsatellite markers for assessing the genetic diversity and population genetic structure of B. juncea. The compound microsatellite marker technique was used to develop markers for investigating population genetics of wild B. juncea. Twelve loci were obtained, each of which showed high polymorphisms when tested in two populations from Sichuan and Jiangsu provinces. The number of alleles per locus ranged from 4 to 27, with an average of 15.2 alleles per locus. The newly developed microsatellite loci would be informative for further investigations of the population genetics and evolutionary patterns of wild B. juncea.In the research, we applied eleven nuclear microsatellites (nSSRs) of twelve microsatellites which we developed to 25 populations from throughout the species'range to infer current patterns of genetic diversity and structure. A total of 90 alleles were generated with a mean of 8 alleles per locus. The range of Shannon diversity index (7) varied from 0.355 to 0.610, with an average of 0.45. The range of diversity index(Hs) was from 0.241 to 0.503, with an average of 0.319. Molecular diversity parameters showed higher values in Chinese accessions. NJ cluster and the principal coordinate analysis of SSR data indicated that the 25 populations of wild B. juncea were divided into 2 lineages (Southern and Northern) with specific geographic distribution, except Jiangsu and Anhui provinces.The Northern lineage included Xizang, Xinjiang, the northern of Anhui, the Northern of Jiangsu, Qinghai, Shannxi, Henan located along the Yellow River. The Southern lineage included Sichuan, Guizhou, Jiangxi, Hubei, Hunan, Zhejiang located along the Yangtze River. The Northern lineage is located along the Yellow River and the other is located along the Yangtze River. We inferred that the ancestors of wild B. juncea in China originating from Xizang and Xinjiang spread from west to east, and that during this process the two genetic lineages divided. The reason of the close relationship between Anhui and Jiangsu located north of the Yangtze river and the provinces located along the Yellow River needs to be further addressed. |
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