| Chinese traditional medicine Danshen is the perennial herbs of Salvia miltiorrhiza Bunge.Its radix has a variety of pharmacological effects,such as promoting blood circulation and removing blood stasis,reducing swelling and relieving pain,nourishing the heart and soothing the nerves,and is traditionally and extensively applied clinically to treat cardio-and cerebralvascular disorders,among many others.As one of China’s traditional bulk medicinal materials,Danshen has been playing increasingly important roles in clinical and health-care utilization.However,the random introduction of unidentified source varieties in production and nonstandard management have resulted in a chaotic variety sources and unstable quality;and studies concerning the genetic diversity and molecular identification of the germplasm resources have been also inadequate.So far as we known,no comprehensive reports of the genetic diversity based on the molecular markers of the chloroplast genomes of the cultivated Danshen populations have been seen.In this research,four intergenic spacer sequences,the external transcribed spacer(ETS)of the nuclear r RNA gene,the 3 chloroplast intergenic spacers between D1 protein of PSII and t RNAHis genes(psb A-trn H),between t RNALeu and t RNAPhe genes(trn L-trn F),and between the hypothetical chloroplast open reading frame1(ycf1)and the ribosomal protein S15 encoding gene(ycf1-rps15),as well as 2 functional genes,namely rubisco large subunit(rbc L)gene,and the maturase K(mat K)gene within the introns of the chloroplast t RNALys(trn K)genes of the 40 cultivated Danshen populations collected from more than 30 major Danshen producing regions of China,were PCR amplified for the first time;and the sequence feature,nucleotide variation site,genetic diversity,genetic distance,and phylogeny were systematically analyzed and compared both individually and in partial combinations.The main results are in below:1.Basic structural characteristics of the six molecular markers and their combined sequences of the cultivated S.miltiorrhiza populationsThe aligned ETS sequences of 40 S.miltiorrhiza populations ranged in length from 421 to 433 bp,with GC contents of 56.8 ~ 62.8%;there were 241 SNP variation sites with a variation rate of 54.8%,and 205 parsimony information sites,accounting for 46.6%.The psb A-trn H sequences were 335~348bp in length,with GC contents of 22.4~26.2%;there were 90 SNP variation sites with a mutation rate of 24.7%,and 71 parsimony information sites,accounting for 19.5%.The trn L-trn F sequences were 298~306bp in length,with GC contents of 35.0~36.9%;there were 25 SNP variation sites with a variation rate of 7.7%,and 24 parsimony information sites,accounting for 7.4%.The ycf1-rps15 sequences were 430~434bp in length,with GC contents of 25.9~30.5%;there were 105 SNP variation sites,of a rate of 23.1%,and 88 parsimony information sites,accounting for 19.3%.The aligned rbc L gene sequences were 1275 bp in length with GC contents of 43.9~44.6%;there were 43 SNP variation sites with a variation rate of 3.4%,and 42 parsimony information sites,accounting for 3.3%;the rbc L gene encodes 425 amino acids with 10 amino acid variation sites,of a variation rate of 2.4%,of which 5 sites are similar amino acid variations and the other 5 sites may have substantial differences.The mat K gene sequences were 1123/1129 bp in length,with GC contents of 33.6~34.3%;there were 96 SNP variation sites with a variation rate of 8.5%,and 86 parsimony information sites,accounting for 7.6%;the mat K gene encodes 376 amino acids with 59 amino acid variation sites,of a variation rate of 15.4%,including 2 deletions,17 similar amino acid variations,and the other 40 sites with large variation differences.The combined sequences of the four intergenic spacers(in the order of ETS+psbAtrn H+trn L-trn F+ycf1-rps15)were 1488~1517bp in length and the total matrix length was 1584 bp,with GC contents of 35.9~38.8%;there were 179 gaps and 466 SNP variation sites,with a variation rate of 29.4%,392 parsimony informative sites,accounting for 24.7%,and 74 single informative sites,accounting for 4.7%.The combined chloroplast function genes(rbc L+mat K)were 2398~2404bp in length;there were 6 gaps and 133 SNP variation sites,of a rate of 5.5%,128 parsimony informative sites,accounting for 5.3%,and 5 single informative sites.2.Genetic diversity of six molecular markers and their combined sequences of the cultivated S.miltiorrhiza populationsThe overall mean genetic distances of the 6 molecular markers(ETS,psb A-trn H,trn Ltrn H,ycf1-rps15,rbc L,and mat K),the combined sequences of the 4 intergenic spacers(ETS+psb A-trn H+trn L-trn F+ycf1-rps15),and the chloroplast functional genes(rbc L+mat K)were 0.151,0.102,0.038,0.104,0.012,0.033,0.102 and 0.21,respectively;the haplotype diversity(Hd)were 0.64,0.601,0.619,0.795,0.479,0.485,0.950 and 0.510,respectively,showed that the cultivated S.miltiorrhiza had abundant genetic diversity at the species level.Neutrality test showed that except for trn L-trn H,the other markers were in line with the neutral evolutionary model,indicating that the cultivated S.miltiorrhiza population did not have an expansion event recently.The number of populations that could be identified based on the SNP fingerprints of ETS, psbA-trnH,trnL-trnF,ycf1-rps15,rbcL,and mat K were 14,8,4,11,4,and 2,respectively,with ETS being the highest,accounting for 35%.And the combined sequences of the two chloroplast functional genes could distinguish 6 populations.However,the combined sequences of the four intergenic spacers could distinguish up to 21 populations with a discriminating rate of 52.5%.This is a big step forward,though still inadequate.Further exploration of more DNA markers is needed.3.Phylogenetic relationships of the six molecular markers of cultivated S.miltiorrhiza populations alone and their combined sequencesThis study,for the first time,constructed the phylogenetic trees of the 6 molecular markers and revealed their phylogenetic relationships from different perspectives.The phylogenetic tree based on the aligned ETS sequences of S.miltiorrhiza showed that the 40 S.miltiorrhiza populations clustered in 2 clades,24 populations clustered in one clade,and the remaining 16 populations clustered in the other.The phylogenetic tree based on psb A-trn H showed that 29 populations clustered in one clade,and the remaining 11 clustered in the other.The phylogenetic tree based on the trn L-trn H showed that 30 populations clustered in one,and the remaining 10 populations clustered in the other clade.The phylogenetic tree based on ycf1-rps15 showed that 30 populations clustered in one clade,and the 10 clustered in the other.The phylogenetic tree based on the rbc L showed that 30 populations clustered in one clade,and the other 10 clustered in the other.The phylogenetic tree based on the mat K gene showed that 30 populations clustered in one clade,and 10 others clustered in the other.The phylogenetic tree based on the combined sequences of the intergenic spacer showed that 29 populations clustered in one clade,and the remaining 11 clustered in the other.The phylogenetic tree based on the combined sequence of chloroplast functional genes showed that 30 populations clustered in one clade,and the remaining 10 populations clustered in the other.The phylogenic relationships of the 6 molecular markers and the two combined sequences of the 40 cultivated S.miltiorrhiza populations all showed similar topological structures of two clades with subtle differences.6 molecular markers of 40 cultivated S.miltiorrhiza populations were PCR cloned.By means of bioinformatics,comprehensive and comparative analyses of the sequence characteristics,nucleotide variation sites,genetic diversity,genetic distance,phylogeny,etc.of the individual and partially combined sequences,were conducted.The genetic diversity and pedigree relationship of the current cultivated S.miltiorrhiza populations in China were revealed from different perspectives.It has accumulated abundant data and laid a solid foundation for the genetic identification,resource evaluation,genetic creation,and evolutionary research of cultivated S.miltiorrhiza population resources,and also provided novel alternative molecular markers and methods for similar research on other resource plants. |
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