Specific DNA barcodes, germplasm resources, and genetic diversity of Eleutherococcus senticosus / 中国中药杂志

Eleutherococcus senticosus is one of the Dao-di herbs in northeast China. In this study, the chloroplast genomes of three E. senticosus samples from different genuine producing areas were sequenced and then used for the screening of specific DNA barcodes. The germplasm resources and genetic diversity of E. senticosus were analyzed basing on the specific DNA barcodes. The chloroplast genomes of E. senticosus from different genuine producing areas showed the total length of 156 779-156 781 bp and a typical tetrad structure. Each of the chloroplast genomes carried 132 genes, including 87 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes were relatively conserved. Sequence analysis of the three chloroplast genomes indicated that atpI, ndhA, ycf1, atpB-rbcL, ndhF-rpl32, petA-psbJ, psbM-psbD, and rps16-psbK can be used as specific DNA barcodes of E. senticosus. In this study, we selected atpI and atpB-rbcL which were 700-800 bp and easy to be amplified for the identification of 184 E. senticosus samples from 13 genuine producing areas. The results demonstrated that 9 and 10 genotypes were identified based on atpI and atpB-rbcL sequences, respectively. Furthermore, the two barcodes identified 23 genotypes which were named H1-H23. The haplotype with the highest proportion and widest distribution was H10, followed by H2. The haplotype diversity and nucleotide diversity were 0.94 and 1.82×10~(-3), respectively, suggesting the high genetic diversity of E. senticosus. The results of the median-joining network analysis showed that the 23 genotypes could be classified into 4 categories. H2 was the oldest haplotype, and it served as the center of the network characterized by starlike radiation, which suggested that population expansion of E. senticosus occurred in the genuine producing areas. This study lays a foundation for the research on the genetic quality and chloroplast genetic engineering of E. senticosus and further research on the genetic mechanism of its population, providing new ideas for studying the genetic evolution of E. senticosus.

Large-scale advances in SSR markers with high-throughput sequencing in Euphorbia fischeriana Steud

BACKGROUND: Euphorbia fischeriana Steud is a very important medicinal herb and has significant medical value for healing cancer, edema and tuberculosis in China. The lack of molecular markers for Euphorbia fischeriana Steud is a dominant barrier to genetic research. For the purpose of developing many simple sequence repeat (SSR) molecular markers, we completed transcriptome analysis with the Illumina HiSeq 2000 platform. RESULTS: Approximately 9.1 million clean reads were acquired and then assembled into approximately 186.3 thousand nonredundant unigenes, 53,146 of which were SSR-containing unigenes. A total of 76,193 SSR loci were identified. Of these SSR loci, 28,491 were detected at the terminal position of ESTs, which made it difficult to design SSR primers for these SSR-containing sequences, and the residual SSRs were thus used to design primer pairs. Analyzing the results of these markers revealed that the mononucleotide motif A/T (44,067, 57.83% of all SSRs) was the most abundant, followed by the dinucleotide type AG/CT (9430, 12.38%). Using 100 randomly selected primer pairs, 77 primers were successfully amplified in Euphorbia fischeriana Steud, and 79 were successfully amplified in three other related species. The markers developed displayed relatively high quality and cross-species transferability. CONCLUSIONS: The large number of EST-SSRs exploited successfully in Euphorbia fischeriana Steud for the first time could provide genetic information for research on linkage maps, variety identification, genetic diversity analysis, and molecular marker-assisted breeding.

Genetic diversity and antibacterial activity of endophytic fungi from Zanthoxylum nitidum / 中国中药杂志

In order to reveal the distribution and population characteristics of endophytic fungi from Zanthoxylum nitidum and the antibacterial potential,this study performed molecular identification and analyzed the genetic diversity and antibacterial activity of endophytic fungi from Z. nitidum in Guangxi. Through culture and molecular identification,35 strains,belonging to 15 genera,12 families,10 orders,4 classes,and 2 phyla,were isolated from various tissues of Z. nitidum,of which Colletotrichum and Fusarium were the dominant genera,respectively accounting for 20% of total strains. The diversity of endophytic fungi was significantly different among roots,stems,and leaves,as manifested by the significantly higher Shannon index( H') in stems( 1. 678) than in roots( 0. 882 1) and leaves( 0. 515 4). The antimicrobial activity analysis showed that 14. 28% of endophytic fungi inhibited at least one indicator pathogen. Among them,Fusarium sp. ZN-34 and Fusarium sp. ZN-26 separately demonstrated the strongest inhibitory effect on Escherichia coli and Staphylococcus aureus. In general,Fusarium sp. ZN-26 and Phialemoniopsis plurioloculosa ZN-35 were advantageous in suppressing the two bacteria owing to the broad spectrum and strong efficacy. In summary,Z. nitidum in Guangxi boasts rich endophytic fungi with the majority showing strong antibacterial activity,which can be used as candidates for the extraction and separation of basic antibacterial substances and the development of natural antibacterial agents.

Analysis of genetic variation diversity of porcine circovirus-2 virus genome isolated from Shanxi area / 中国兽医学报

In order to study genetic variation diversity of porcine circovirus type 2 (PCV2) strains in Shanxi,the genomic sequences of nine PCV2 strains including SXQX,SXCZ,SXTY2,SXJC,SXJX,SXLL,SXPY,SXPG and SXXY recently isolated from some areas of Shanxi from 2013 to 2016,was cloned,sequenced and received by GenBank.The amplified PCV2 genomic sequences,ORF2 sequences and Cap protein amino acid of these nine strains were analysed and compared with those of published 28 PCV2 strains by DNAStar,drawing phylogenetic tree.The results showed that the genomic sequences of SXJX,SXJC and SXXY PCV2 strains were 1 768 bp,and the others were 1 767 bp,which accounted for 33％ and 67％,respectively.The homologies of nucleotide sequences of the nine strains were 94.7％-99.8％,the homologies of nucleotide sequences of the nine strains with the 28 isolates from different regions of the world PCV strain were 93.9％-99.9％,and the homologies of nucleotide sequences of the nine strains with the domestic vaccine strains were 95.1％-99.8％.The phylogenetic analysed that SXJX,SXJC and SXXY belonged to genotype PCV-2D,SXLL,SXPY and SXCZ belonged to genotype PCV-1C,and SXTY14,SXPG and SXQX belonged to genotype PCV-1A/1B.Thus it proved that the epidemic strain of PCV2 was mainly PCV-2b in Shanxi.The homologies of ORF2 nucleotide sequences and Cap amino acid of the nine strains were 90.0％-100.0％ and 87.1 ％-100.0％ respectively,the homologies of ORF2 nucleotide sequences and Cap amino acid of the nine strains with the 28 isolates from different regions of the world PCV strain were 87.6％-100.0％ and 84.1％-100.0％ respectively,and the homologies of ORF2 nucleotide sequences and Cap amino acid of the nine strains with the domestic vaccine strains were 91.0％-100.0％ and 89.3％-100.0％ respectively.The Cap amino acids of SXQX,SXJX,SXTY14,SXPG,SXJC and SXXY PCV2 were 233,ORF2 of SXQX,SXTY14 and SXPG located at 1 033-1 734 bp,ORF2 of SXXY,SXJX and SXJC located at 1 033-1 734 bp,and the Cap amino acids of SXCZ,SXLL and SXPY PCV2 were 234,ORF2 of them located at 1 030-1 734 bp,in addition,the positions of 1 030-1 734 bp were more three bases TCA than other ORF2 genome sequence of 1 767 bp,resulting in increasing a K (Lys) of amino acid sequencein at the 234 position.Also Cap protein of 9 PCV2 strains showed more amino acid variation in addition to the only high-ly conserved glycosylation sites (NYS) (pp.143-145 amino acid).It provided theoretical basis for the PCV2 immune prevention of research in Shanxi,and the data of basic theory of molecular pathogenesis of PCV2.

Screening and diversity of resistance germplasm of Dendrobium officinale by CDDP markers / 中草药

Objective Conserved DNA-Derived Polymorphism (CDDP) markers were used in the study of the genetic diversity of 43 Dendrobium officinale and preliminary resistance screening, in order to provide a theoretical basis for the screening of D. officinale and the selection of fine varieties. Methods A total of 21 CDDP primers were used to amplify the genomic DNA of the test material with clear and polymorphic primers. Results Sixteen primers generated 151 bands, of which 144 (95.7%) were polymorphic. The results of data analysis on three population showed that the percentage of polymorphic locus (PPL) were between 65.56% and 82.12%, coefficient of genetic differentiation among natural populations (Gst) was 0.110 2, and the total gene flow (Nm) was 4.035 4. Indicating that anthropogenic factors may also promote the genetic diversity of D. officinale. Conclusion The genetic diversity of D. officinale was rich, and the results showed that there were eight plants with potentially good resistance among 43 materials.

Influence of PCR cycle number on microbial diversity analysis through next generation sequencing / 生物工程学报

Using of high throughput sequencing technology to study the microbial diversity in complex samples has become one of the hottest issues in the field of microbial diversity research. In this study, the soil and sheep rumen chyme samples were used to extract DNA, respectively. Then the 25 ng total DNA was used to amplify the 16S rRNA V3 region with 20, 25, 30 PCR cycles, and the final sequencing library was constructed by mixing equal amounts of purified PCR products. Finally, the operational taxonomic unit (OUT) amount, rarefaction curve, microbial number and species were compared through data analysis. It was found that at the same amount of DNA template, the proportion of the community composition was not the best with more numbers of PCR cycle, although the species number was much more. In all, when the PCR cycle number is 25, the number of species and proportion of the community composition were the most optimal both in soil or chyme samples.

Compilation of an informative microsatellite set for genetic characterisation of East African finger millet (Eleusine coracana)

Background Genetic diversity of finger millet (Eleusine coracana), a nutritious neglected staple cereal in Africa and South Asia is largely uncharacterized. This study analysed 82 published SSR markers for finger millet across 10 diverse accessions to compile an informative set for genetic characterisation. Extensive optimization compared single samples with bulked leaf or bulked DNA samples for capturing within accession genetic diversity. The markers were evaluated to determine (1) how efficiently they amplified target loci during high-throughput genotyping with a generic PCR protocol, (2) ease of scoring PCR products and (3) polymorphism and ability to discern genetic diversity within the tested finger millet germplasm. Results Across 88 samples, the 52 markers that worked well amplified 274 alleles, ranging from 2 to 14 per locus with a mean of 4.89. Major allele frequency ranged from 0.18 to 0.93 with a mean of 0.57. Polymorphic Information Content (PIC) ranged from 0.13 to 0.88 with a mean of 0.5 and availability varied between 64 and 100% with a mean of 92.8%. Heterozygosity ranged from 0 to 1.0, with a mean of 0.26. Discussion Five individual samples from an accession captured the largest number of alleles per locus compared to the four different bulked sampling strategies but this difference was not significant. The identified set comprised 20 markers: UGEP24, UGEP53, UGEP84, UGEP27, UGEP98, UGEP95, UGEP64, UGEP33, UGEP67, UGEP106, UGEP110, UGEP57, UGEP96, UGEP66, UGEP46, UGEP79, UGEP20, UGEP12, UGEP73 and UGEP5 and was since used to assess East African finger millet genetic diversity in two separate studies.

Phylogenetic and genetic diversity analysis in Leptospira species based on the sequence homology pattern of 16S rRNA gene.

Leptospirosis is a bacterial zoonosis, caused by pathogenic spirochete which belongs to the genus Leptospira. It exists in diverse ecological habitats and affects almost all the mammals including humans. Several online databases like NCBI etc will provide the complete genomic sequence data of various Leptospira species. However, the Phylogenetic and genetic diversity Analysis in Leptospira species based on 16S rRNA gene has not studied in detail. Therefore the present study was conducted. Sequences of various species related to genus Leptospira obtained from the NCBI database etc and aligned (CLUSTAL_X). Two Phylogenetic trees were constructed (MEGA-5) in which the first one is related to various serovars of L. interrogans and the other is related to various species of Leptospira. The Phylogenetic trees revealed the relationship and genetic diversity of various serovars of L. interrogans and the other Leptospira species, with their nearest phylogenetic relatives. In the first tree, two major clades were observed which were named as A and B, whereas in the second tree, three major clades were observed and named as A, B and C respectively. Aquifex pyrophilus strain has been used for out grouping in both the trees. The genetic distance between the species in the phylogenetic tree is presented by a bar which represents 0.5 nucleotide substitutions per alignment position in the 16S rRNA gene sequence among the various serovars of L. interrogans while 0.05 nucleotide substitutions in case of various species related to the genus Leptospira. Thus, the findings from the above study confirm that the genus Leptospira exhibits genetic diversity in the 16S rRNA gene.

Diversity of Endophtic Bacteria Isolated from Glycyrrhiza / 微生物学通报

120 strains of endophytic bacteria identified by ERIC-PCR were isolated from wild and cultivated Glycyrrhiza uralensis plants which collected from Erdos Innermongolia province.The identified results indicated that Glycyrrhiza uralensis plants has plenty of endophytic bacterium in density and population,and the density is higher in root and leave than in stem.Partial sequence analysis of 16S rDNA gene of 82 strains indicated that these strains were in a high similarity with 19 known genus which belong to?、?、 ?-Proteobacteria、Firmicutes and Actinobacteria.The dominant genus were Bacillus sp.,Pseudomonas sp., Pantoea sp.and Serratia sp..

A New Method of Soil Bacterial DNA Extraction and T-RFLP Analysis for Diversity / 微生物学通报

Obtaining soil bacterial DNA of good quality is a key step in soil bacterial ecology study.A quick, efficient,sensitive and stably method of DNA extraction from soil were established by combining strongpoints of two kits(Soilmaster kit and DNA IQ~(TM)kit).In addition,the 16S rDNA gene and T-RFLP(Terminal restriction fragment length polymorphism)were used in the analysis of soil bacterial community diversity and the result show that T-RFLP is a powerful tool for bacterial community study.