| Salvia miltiorrhiza Bunge.is a perennial herb of the genus Salvia and the family Labiatae,its roots and rhizomes have been used as medicines.S.miltiorrhiza has abundant germplasm resources and many populations.In this paper,a total of 42 S.miltiorrhiza populations were collected from 30 regions of China and used as materials.The seed shape,size and some agronomic traits were examined.The internal transcribed spacer(ITS)regions of the r DNA were PCR amplified and analysised.Based on the cDNA sequence or gene sequence of the 6 key enzymes in the salvianone and salvianolic acid biosynthetic pathways of S.miltiorrhiza,namely 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR),1-deoxy-D-xylulose 5-phosphate synthase(DXS),1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR),phenylalanine ammonialyase(PAL),rosmarinic acid synthetase(RAS)and tyrosine aminotransferase(TAT),degenerate homologous primers were designed and their corresponding genomic genes of the 42 populations were cloned in segments for further genetic diversity analyses.At the same time,by using W-SCHY-W-1 as the material,a rapid determination method of Salvia seed vigor was preliminarily explored.The main results are as follows:1.Observation and Determination of Seed Shape,Size and Some Agronomic Traits of S.miltiorrhiza PopulationsThe seed test of 42 S.miltiorrhiza populations showed that the long diameter range was2.40 to 4.05 mm,the short diameter range was 1.01 to 3.00 mm,and the long-to-short diameter ratio range was 1.08 to 2.99 mm and its 1000-grain weight range was 1.24 to 12.75g;there are four types of seeds:flat-oval,oval,triangular ovoid-oblong and ovate-oblong,while the S.miltiorrhiza seeds harvested from field cultivation are all flat-oval and much smaller.Morphologies of most of the S.miltiorrhiza populations were very similar,while the population W-SCHY-W-1 was special,its leaves and corolla are larger than those of other populations,it has typical large white flowers.2.Genetic Diversity Analysis of the Internal Transcribed Spacers(ITS)of the S.miltiorrhiza PopulationsITS sequence analysis of 42 S.miltiorrhiza materials showed that its ITS1,5.8S and ITS2 regions were 200-229bp,164bp and 226-229bp in length respectively.There are 19variation sites in S.miltiorrhiza ITS1 sequences,while no variation was observed in 5.8 S region.There are 37 variation sites in S.miltiorrhiza ITS2 sequences.Of the 42 populations,3 individual populations namely W-SCHY-W-1,V-YNLJ-V-1 and W-SXYA-V-3 can be discriminated by ITS-based SNP fingerprints,among which V-YNLJ-V-1 is especially rich in SNP mutation sites.The population W-SXYA-V-3 has 4 SNP mutation sites.The population V-HBCD-V-3,W-HBJM-V-1 and W-GZ-V-2 have one more common deletion site,while the ITS sequences of the remaining 36 S.miltiorrhiza populations are highly conserved.3.The Cloning and Genetic Diversity Analysis of Key Enzyme Genes for Bioactive Ingredient Biosynthesis of S.miltiorrhiza PopulationsThe sequence analyses of the cloned 6 key enzyme genes for the biosynthesis of active components of 42 S.miltiorrhiza showed that:the SmHMGR gene is about 1656bp in length,without introns.There are 108 SNP variation sites in 42 populations,with a variation rate of6.52%.SmHMGR encodes 552 amino acid residues,with a total of 35 amino acid mutation sites.The SmDXS gene is approximately 3383bp in length and consists of 10 exons and 9introns.The spliced exon sequence of the SmDXS gene is 2192bp in length,with a total of 48SNP variation sites and a variation rate of 2.19%.The spliced exon sequence of SmDXS encodes 730 amino acid residues with 33 amino acid mutations sites.The introns of SmDXS have 224 SNP variation sites.The SmDXR gene is about 3806bp in length and consists of 12exons and 11 introns.The spliced exon sequence of the SmDXR is 1375bp,with a total of 47SNP variation sites,and a variation rate of 3.42%.The spliced exon sequence of SmDXR of encodes 458 amino acid residues with 27 amino acid mutations.The introns of SmDXR have578 SNP variation sites.The SmPAL gene is about 2767bp in length and consists of two exons and one intron.The spliced exon sequence of SmPAL is 2118bp,with a total of 51variation sites and a variation rate of 2.41%.The spliced exons of SmPAL encodes 706 amino acid residues with a total of 46 amino acid mutation sites.Only one variation site in the intron of SmPAL.The SmRAS gene is about 1431bp in length and consists of two exons and one intron.The spliced exon sequence of SmRAS is 1199bp,with a total of 162 variation sites and a variation rate of 13.6%.The spliced exon sequence of SmRAS encodes 397 amino acid residues with a total of 90 amino acid mutation sites.There are 139 variation sites in the intron region of SmRAS with a variation rate of 58.2%.The SmTAT gene is about 2296bp and consists of 6 exons and 5 introns.The spliced exon sequence of SmTAT gene exon is 1236bp in length,having 24 SNP variation sites and a variation rate of 1.94%.The SmTAT exon encodes 412 amino acid residues with only 4 amino acid mutation sites.The introns of SmTAT have 290 SNP variation sites.The total discriminatory SNP fingerprints for S.miltiorrhiza from the 7 studied gene sequences were 31,with the discrimination rate was 74%.Three populations can be discriminated by ITS and 29 populations can be discriminated by the 6 genes.Statistics of the putative amino acid sequence differences of the cloned 6 genes showed that there are 25populations with amino acid variations.Which was highly consistent with the DNA discrimination fingerprint statistics of 42 populations.Also phylogenetic trees based on various nucleotide sequences and their putative amino acid sequences encoded by the 6 genes were for the first time constructed to reveal the phylogenetic relationships from different aspects.4.Rapid Determination of S.miltiorrhiza Seed VigorThe bromothymol blue method(BTB)was used to rapid determine the seed vigor of W-SCHY-W-1 as a model material and simultaneous in situ-like and conventional germination tests were performed.From the results the linear regression equation between the seed vigor estimated by BTB and the germination rate from in situ-like germination,y=0.9835x+1.8595(R~2=0.996),was established;the linear regression equation between the seed vigor estimated by BTB and the germination rate from conventional germination was y=0.3471x+51.95(R~2=0.9719).Its was concluded that BTB can be used for rapid determination of S.miltiorrhiza seed vigor. |
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