Characteristics of the chloroplast genome of Dracaena marginata and phylogenetic analysis / 生物工程学报

Dracaena marginata is a widely cultivated horticultural plant in the world, which has high ornamental and medicinal value. In this study, the whole genome of leaves from D. marginata was sequenced by Illumina HiSeq 4000 platform. The chloroplast genome were assembled for functional annotation, sequence characteristics and phylogenetic analysis. The results showed that the chloroplast genome of D. marginata composed of four regions with a size of 154 926 bp, which was the smallest chloroplast genome reported for Dracaena species to date. A total of 132 genes were identified, including 86 coding genes, 38 tRNA genes and 8 rRNA genes. Codon bias analysis found that the codon usage bias was weak and there was a bias for using A/U base endings. 46 simple sequence repeat and 54 repeats loci were detected in the chloroplast genome, with the maximum detection rate in the large single copy region and inverted repeat region, respectively. The inverted repeats boundaries of D. marginata and Dracaena were highly conserved, whereas gene location differences occurred. Phylogenetic analysis revealed that D. serrulata and D. cinnabari form a monophyletic clade, which was the closest relationship and conformed to the morphological classification characteristics. The analysis of the chloroplast genome of D. marginata provides important data basis for species identification, genetic diversity and chloroplast genome engineering of Dracaena.

Chloroplast genomic characterization and phylogenetic analysis of Pellionia scabra / 生物工程学报

Pellionia scabra belongs to the genus Pellionia in the family of Urticaceae, and is a high-quality wild vegetables with high nutritional value. In this study, high-throughput techniques were used to sequence, assemble and annotate the chloroplast genome. We also analyzed its structure, and construct the phylogenetic trees from the P. scabra to further study the chloroplast genome characteristics. The results showed that the chloroplast genome size was 153 220 bp, and the GC content was 36.4%, which belonged to the typical tetrad structure in P. scabra. The chloroplast genome encodes 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes in P. scabra. Among them, 15 genes contained 1 intron, 2 genes contained 2 introns, and rps12 had trans-splicing, respectively. In P. scabra, chloroplast genomes could be divided into four categories, including 43 photosynthesis, 64 self-replication, other 7 coding proteins, and 4 unknown functions. A total of 51 073 codons were detected in the chloroplast genome, among which the codon encoding leucine (Leu) accounted for the largest proportion, and the codon preferred to use A and U bases. There were 72 simple sequence repeats (SSRs) in the chloroplast genome of P. scabra, containing 58 single nucleotides, 12 dinucleotides, 1 trinucleotide, and 1 tetranucleotide. The ycf1 gene expansion was present at the IRb/SSC boundary. The phylogenetic trees showed that P. scabra (OL800583) was most closely related to Elatostema stewardii (MZ292972), Elatostema dissectum (MK227819) and Elatostema laevissimum var. laevissimum (MN189961). Taken together, our results provide worthwhile information for understanding the identification, genetic evolution, and genomics research of P. scabra species.

Genetic diversity analysis and fingerprints of Chrysanthemum×morifolium based on SSR molecular markers / 生物工程学报

The present study aims to explore the genetic diversity of germplasm resources of Chrysanthemum×morifolium (hereinafter, C.×morifolium) at the molecular level and to establish a fingerprint database of C.×morifolium varieties. We employed 12 pairs of primers with high levels of polymorphism, clear bands, and high degrees of reproducibility to analyze the SSR molecular markers and genetic diversity of 91 C.×morifolium materials and 14 chrysanthemum- related materials. With regard to constructing the fingerprints of the tested materials, we chose 9 pairs of core primers. The findings revealed that 12 primer pairs detected 104 alleles in 105 samples, ranging from 2 to 26. The average number of observed alleles (Na) per site was 9.25. The average number of effective alleles (Ne) per site was 2.745 6, with its range being 1.276 0 to 4.742 5. Shannon genetic diversity index (I) values ranged between 0.513 3 and 2.239 9 (M=1.209 0). Nei's gene diversity index (H) ranged between 0.216 3 and 0.789 1 (M=0.578 0). The observed heterozygosity (Ho) ranged between 0.223 3 and 0.895 2 (M=0.557 5). The expected heterozygosity (He) ranged between 0.217 4 and 0.793 3 (M=0.580 8). The polymorphism information content (PIC) ranged between 0.211 5 and 0.774 0 (M=0.532 9). The genetic similarity (GS) ranged between 0.228 5 and 1.000 0 (M=0.608 3). Cluster analysis revealed that when the genetic distance (GD) equals to 0.30, the tested materials can be classified into 2 groups. When the GD equals to 0.27, the first group can be divided into 6 subgroups; accordingly, 105 tested materials can be divided into 7 subgroups. The cophenetic correlation test was carried out based on the cluster analysis, and the corresponding results showed that the cluster map correlated with the genetic similarity coefficient (r=0.952 73). According to the results of Structure population analysis, we obtained the optimal population number, with the true number of populations (K) being 3 and the population being divided concerning Q≥0.5. Three subgroups, i.e., Q1, Q2 and Q3, included 34, 33 and 28 germplasms, respectively, and the remaining 10 germplasms were identified as the mixed population. During the experiment, 9 pairs of core primers were screened among the total of 12 for a complete differentiation regarding 105 tested materials, and the fingerprints of 91 C.×morifolium materials and 14 chrysanthemum-related materials were further constructed. Overall, there were significant genetic differences and rich genetic diversity among C.×morifolium materials, which would shed light on the garden application and variety selection fields of C.×morifolium. The fingerprint database of 105 C.×morifolium varieties and chrysanthemum-related species may provide technical support for future research regarding the identification and screening system of C.×morifolium varieties.

Characteristics of complete chloroplast genome and phylogenetic analysis of Cirsium souliei (Asteraceae) / 药学学报

italic>Cirsium souliei (Asteraceae) is a perennial medicinal herb of Cirsium with important medicinal and ecological values. Here, we sequenced the complete chloroplast (cp) genome of C. souliei based on high-throughput sequencing technology, then assembled and annotated it, and analysed the structure and characteristics of the cp genome. The result indicated that the cp genome of C. souliei was a typical quadripartite circular structure of 152 470 bp in length, and GC content was 37.7%. The cp genome of C. souliei encoded 134 genes, including 89 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Meanwhile, we detected 188 simple sequence repeats (SSR) loci in the cp genome, which were mainly composed of mononucleotide repeats. Codon bias analysis showed that leucine (Leu) was the highest amino acids with frequency (10.51%), and there were 30 codons with the value of relative synonymous codon usage (RSCU) above one, of which mostly ended with A/U. Additionally, the result from phylogenetic analysis based on 46 cp genomes of Carduoideae showed that C. souliei and C. vulgare were sister species, and had the closest relationship with 100% bootstrap within Cirsium. This study provides theoretical basis for future studying genetic diversity, population genetic structure, systematics and evolution, and speciation mechanism.

Genetic Diversity Analysis of Isatis indigotica Germplasm Materials by Chloroplast DNA Variation and ISSR Molecular Marker / 中国实验方剂学杂志

ObjectiveTo analyze the sequence variation and genetic diversity of 47 Isatis indigotica germplasm materials， and carry out the study on the genetic differentiation and structure. MethodGenomic DNA of 47 I. indigotica germplasm materials were extracted by kit extraction method. Two chloroplast DNA （cp DNA） sequences and five inter-simple sequence repeat （ISSR） primers were used for amplification and sequencing. Chromas， Mega 7.0， DanSP5， and GenALEx were used to calibrate， splice， and analyze the sequence characteristics. PERMUT and PopGen 1.31 were used to analyze the genetic diversity parameters and genetic structure， and NTSYS was used to obtain the unweighted pair-group method with arithmetic means（UPGMA） clustering tree plot of 47 I. indigotica germplasm materials. ResultA total of 129 samples from 47 I. indigotica germplasm materials were successfully amplified and sequenced. The length of 2 cp DNA sequences after spliced was 1 412 bp， and there were 377 polymorphic variation loci， and 36 haplotypes. Fu and Li's D* test was significant （P<0.01）. The values of Pi， HS， and HT based on cp DNA were 0.119 89， 0.787， and 0.891， respectively. The genetic differentiation coefficients of gene differentiation coefficient（Gst）， nucleotide differentiation coefficient（Nst）， and fixation index（Fst） were 0.117， 0.468， and 0.488， respectively， and the gene flow （Nm） was 0.615. The mean values of PPB， Shannon information diversity index（I）， Nei's genetic diversity index（H）， and Gst based on ISSR were 78.85%， 0.334 8， 0.218 6， and 0.754 4， respectively， and the Nm value was 0.162 8. ConclusionI. indigotica has high genetic diversity and abundant haplotypes at the species level， with abundant haplotypes. Genetic differentiation among different germplasm materials is obvious， and gene exchange is not frequent. Genetic variation mainly exists among populations. The population has accumulated various low-frequency gene mutations recently， suggesting that it has experienced significant regional expansion in the history.

Genetic Diversity Analysis of Isatis indigotica Germplasm Materials by Chloroplast DNA Variation and ISSR Molecular Marker / 中国实验方剂学杂志

ObjectiveTo analyze the sequence variation and genetic diversity of 47 Isatis indigotica germplasm materials， and carry out the study on the genetic differentiation and structure. MethodGenomic DNA of 47 I. indigotica germplasm materials were extracted by kit extraction method. Two chloroplast DNA （cp DNA） sequences and five inter-simple sequence repeat （ISSR） primers were used for amplification and sequencing. Chromas， Mega 7.0， DanSP5， and GenALEx were used to calibrate， splice， and analyze the sequence characteristics. PERMUT and PopGen 1.31 were used to analyze the genetic diversity parameters and genetic structure， and NTSYS was used to obtain the unweighted pair-group method with arithmetic means（UPGMA） clustering tree plot of 47 I. indigotica germplasm materials. ResultA total of 129 samples from 47 I. indigotica germplasm materials were successfully amplified and sequenced. The length of 2 cp DNA sequences after spliced was 1 412 bp， and there were 377 polymorphic variation loci， and 36 haplotypes. Fu and Li's D* test was significant （P<0.01）. The values of Pi， HS， and HT based on cp DNA were 0.119 89， 0.787， and 0.891， respectively. The genetic differentiation coefficients of gene differentiation coefficient（Gst）， nucleotide differentiation coefficient（Nst）， and fixation index（Fst） were 0.117， 0.468， and 0.488， respectively， and the gene flow （Nm） was 0.615. The mean values of PPB， Shannon information diversity index（I）， Nei's genetic diversity index（H）， and Gst based on ISSR were 78.85%， 0.334 8， 0.218 6， and 0.754 4， respectively， and the Nm value was 0.162 8. ConclusionI. indigotica has high genetic diversity and abundant haplotypes at the species level， with abundant haplotypes. Genetic differentiation among different germplasm materials is obvious， and gene exchange is not frequent. Genetic variation mainly exists among populations. The population has accumulated various low-frequency gene mutations recently， suggesting that it has experienced significant regional expansion in the history.

Genetic variability of Sargassum liebmannii on the coast of Jalisco in the central Mexican Pacific revealed by molecular markers and morphological traits

BACKGROUND Sargassum liebmannii is widely distributed throughout rocky, coastal upwelling areas in the tropical Mexican Pacific. This brown algae is of great environmental and industrial importance. However, no information is available that documents the genetic or phenotypic variability of the species, which is needed to determine how it may react to environmental variation related to climate change. In this study, S. liebmannii specimens were collected from the coast of Jalisco, Mexico, and molecular and morphological characterization was conducted. Intraspecific variability was estimated according to the study areas. RESULTS The inter-simple sequence repeat (ISSR) markers indicated a polymorphism percentage of 95%. The Shannon index and Nei index showed relatively low values among the populations (0.3569 and 0.081, respectively). On the other hand, the genetic differentiation coefficient indicated inter- and intrapopulation values of 36.69% and 63.31%, respectively. The Jaccard similarity coefficient was used to determine the degree of similarity among individuals by geographical area. The morphological characteristics and environmental variables that were used to correlate phenotypes and genotypes indicated that S. liebmannii showed low genetic flow because of the presence of geographical barriers due to substrate that was not optimal for algal development. CONCLUSIONS The ISSR markers were useful for detecting genetic differences among S. liebmannii individuals. The results indicate that a coupled genotypic-phenotypic study is beneficial for documenting the variation present in the little-studied algal species. These studies may be used in future research to clarify taxonomic controversies while generating additional genomic information

Complete plastid genomes of Bupleurum chinense DC. and B. boissieuanum H. Wolff, with comparative and phylogenetic analyses of medicinal Bupleurum species / 药学学报

italic>Bupleurum L. (Apiaceae) is an economically important genus, in which many species are of medicinal value. In this study, the complete plastid genomes (plastomes) of B. chinense DC. and B. boissieuanum H. Wolff were sequenced and their characteristics were investigated. Comparative and phylogenetic analyses were conducted with other published Bupleurum plastomes. The complete plastomes of B. chinense and B. boissieuanum were 155 458 and 155 800 bp in length, and both exhibited the typical quadripartite circular structure consisting of a large single copy region (LSC, 85 343 and 85 804 bp), a small single copy region (SSC, 17 495 and 17 410 bp), and a pair of inverted repeat regions (IRa/b, 26 310 and 26 293 bp), respectively. A total of 129 genes, including 84 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes were identified from each of the two plastomes. Repeat sequences detected were similar in types and distribution patterns, but the numbers were slightly different. Comparative analyses revealed that the Bupleurum plastomes were highly conserved in length, structure, the guanine and cytosine (GC) content, and gene content and order, both intraspecifically and interspecifically, and no obvious expansion or contraction of the inverted repeat regions occurred. Sequence variation was lower within the same species than among different species, noncoding sequences (including intergenic regions and introns) showed a higher divergence than the protein-coding sequences, and sequences in the LSC and SSC regions were more divergent than those in the IR regions. In addition, 11 sequences with higher nucleotide diversity among species were detected in the LSC and SSC regions. All studied Bupleurum species were inferred forming a monophyletic group with a 100% bootstrap value. Bupleurum chinense and B. boissieuanum were phylogenetically closest to B. commelynoideum and B. falcatum, separately, with all three B. chinense accessions clustered into a distinct clade. These results provide genetic information for further species identification, phylogenetic resolution, and will assist in exploration and utilization of medicinal Bupleurum species.

Genetic Diversity Analysis of Erigeron breviscapus Based on SSR Markers / 中国实验方剂学杂志

Objective:To explore the genetic diversity and population structure of <italic>Erigeron breviscapus</italic>， so as to provide a scientific basis for its resource protection and rational utilization. Method:Twelve pairs of simple sequence repeat（SSR） primers were screened out from 243 individuals in 16 natural populations to calculate the genetic diversity parameters of <italic>E. breviscapus</italic>， which were then subjected to principal coordinate analysis and cluster analysis. Result:Twelve SSR markers generated 209 alleles， with an average of 17.417 alleles per locus. Based on 12 SSR markers and 16 populations of <italic>E. breviscapus</italic>， the observed heterozygosity （<italic>H</italic>₀） values were determined to be 0.603 and 0.613， the expected heterozygosity （<italic>H</italic>_e）to be 0.658 and 0.659， and the Shannon's information index （<italic>I</italic>） to be 1.443 and 1.446， respectively. The Wright's fixation index （<italic>F</italic>_st） was 0.123 and gene flow （<italic>N</italic>_m） was 2.077. Analysis of molecular variance （AMOVA） and genetic differentiation revealed that genetic variation within populations was the main source of total variation. The Nei's genetic distance and genetic identity coefficients were within the ranges of 0.107 （YA and XY）-0.713 （SZ and XZD） and 0.490 （SZ and XZD）-0.899 （YA and XY）， respectively. As demonstrated by the principal coordinate analysis and cluster analysis， the 16 populations of <italic>breviscapus </italic>were divided into two clusters. Conclusion:The genetic diversity of <italic>E. breviscapus</italic> was relatively high and there existed certain genetic differentiation and gene flow within and among populations. The genetic variation was mainly present within populations. All these have provided reference for subsequent study on good germplasm selection of <italic>E. breviscapus.</italic>

De novo transcriptome assembly and mining of EST-SSR markers in Gloriosa superba

Gloriosa superba is an economical source of pharmaceutical colchicine, which is a mitotic poison used to treat gout, cancer and inflammatory diseases. It is important to study the genetic variations in this plant, but the progress is impeded due to limited number of molecular markers. In this study, we developed the expressed sequence tag-derived simple sequence repeat (EST-SSR) markers from the transcriptome sequence of the leaf samples of three different ecotypes of G. superba. De novo assembly was performed on these sequencing data to generate a total of 65,579 unigenes and 38,200 coding sequences (CDSs). These CDSs were annotated using NCBI Nr protein database, gene ontology terms and KEGG pathways. Differential gene expression was studied to yield differences in these ecotypes at the molecular level. Finally, a total of 14,672 potential EST-SSRs were identified from these unigenes, among which the dinucleotide (5754, 39.22%) and trinucleotide (5421, 36.95%) repeats were most abundant types followed by mononucleotides (3213, 21.83%). The most frequent motifs were CT/GA (1392, 9.48%), AG/TC (1219, 8.31%), and GA/CT (1146, 7.82%) among the dinucleotide repeats and CCG/ CGG (1487, 10.13%), AGG/CCT (1421, 9.68%), AGC/CTG (697, 4.75%) and AAG/CTT (621, 4.23%) among the trinucleotide repeats. Polymorphism study using a random set of 20 newly developed EST-SSRs revealed polymorphic information content value ranging from 0 to 0.5926 with an average of 0.4021. The large-scale ESTs developed in the current study will be useful as a genomic resource for further investigation of the genetic variations in this species

QTL identification for downy mildew resistance in cucumber using genetic linkage map based on SSR markers

Fourteen cucumber lines were tested for genetic homozygosity and performed pairwise comparison to identify a pair with the highest DNA polymorphic level. Cucumber accessions CSL0067 and CSL0139 were selected to generate 315 F2 populations. The genetic linkage map based on 66 polymorphic SSR markers was constructed. It composed of eight linkage groups (LGs) spanning 474.4 cM. Downy mildew disease reaction was evaluated in cotyledons, first and second true leaf on 7, 10, and 14 day after inoculation. The results showed that downy mildew resistance was controlled by multiple recessive genes. The susceptible to resistant ratio of F2 progenies fit 9:7 susceptible/resistant segregation types corresponding to duplicate recessive epistasis. Fourteen QTLs were detected. The phenotypic variance ranged from 5.0 to 12.5%, while LOD values ranged from 3.538 to 9.165. Two major QTLs and two QTL hotspots were identified. Moreover, the additive effects data explained that these QTL reduced downy mildew susceptibility

Population structure and diversity assessment of barley (Hordeum vulgare L.) introduction from ICARDA

This study was undertaken to measure the genetic diversity and population structure of 48 barley accessions introduced from ICARDA using 51 polymorphic simple sequence repeat (SSR) markers to select unique parents for breeding. The mean polymorphic information content was 0.491, suggesting high polymorphism for the selected SSR markers among the barley accessions. The population structure indicated a fine genetic base only with two major clusters. All accessions had 100% membership probability in their respective clusters. Analysis of molecular variance revealed that most (78%) of the variation was attributed between populations, while 22% was due to variation among individuals within populations. Neighbour-joining (NJ) tree was constructed using this distance matrix and two major clusters were observed in it. Cluster 1 had all hulled barley accessions and cluster 2 had all hulless barley accessions. Cluster 2 could be further divided into three subclusters. Principal coordinates analysis results were similar to the NJ tree, where the hulled and hulless barley accessions were grouped into separate clusters. This study established the existence of considerable genetic diversity among the 48 tested accessions. The selected genetic resources will be useful for barley breeding in India and other countries.

Genomewide analysis of microsatellite markers based on sequenced database in two anuran species

Eukaryotic and prokaryotic cell genomes exhibit multiple microsatellites. In this study, we characterized microsatellites in genomes and genes of Nanorana parkeri and Xenopus laevis. This characterization was used for gene ontology (GO) analysis of coding sequences (CDS). Compared to the genome of N. parkeri, the genome of X. laevis is larger and contains more number of microsatellites, but the diversity of both species are similar. Trinucleotide repeats in the genome of N. parkeri and dinucleotide and tetranucleotide repeats in the genome of X. laevis were the most diverse. In both the species, diversity of microsatellites was highest in intergenic regions, followed by intron and exon regions, and lowest in coding regions. Microsatellites in CDS are thus subject to higher selective pressure. Many microsatellites are concentrated upstream and downstream of genes in both species, suggesting suppression of repeats in the middle of protein–CDS. Repeats are enriched in regions near gene termini purely due to the biophysical constraints of protein structure. In GO analysis, two and five unique GO terms, only found in N. parkeri and X. laevis, respectively, indicate advantageous mutations during species evolution. Biological process, cellular component and molecular function ontology reflected in the GO analysis predicted that the microsatellites located in CDS can alter protein function and may provide a molecular basis for species adaptation to new and changing environments

Identification of QTLs in oil palm (Elaeis guineensis Jacq.) using SSR markers through association mapping

Oil palm (Elaeis guineensis Jacq.) is a perennial vegetable and a high oil-yielding crop (4–6 t/ha). There is a large scope for increasing the oil yield by selecting elite planting material for breeding programme in germplasm evaluation, characterization and utilization. In the present study, a diverse range of 150 oil palm genotypes were characterized using 12 quantitative variables with 54 genomic microsatellite markers. A wide variation was observed in the morphological traits among indigenous populations. Highly significant and positive correlations were observed between vegetative dry matter (VDM) and total dry matter (TDM) (0.862), and height and height increment (0.838). The first two principal component analyses explained 67.7% of total variation among morphological traits. The genotypes IC0610001-59 (Pune-2) and IC0610001-60 (Pune-2) were found highly promising based on less height increment, more TDM with high yield. For the mapping study, general linear model (GLM) approach, quantitative-trait loci (QTL) for annual height increment, number of bunches, bunch yield and bunch index were linked to simple-sequence repeat (SSR) loci mEgCIR3649 with phenotypic variance of 15.08, 10.43, 11.74, 15.39. TDM and VDM were linked to mEgCIR0192 (27.34 and 24.19%), mEgCIR3684 (16.84 and 18.30%), SPSC00163 (18.8 and 15.39%) and mEgCIR0555 (16.47 and 18.81%), with at a significant threshold (P) level of B0.001 and by mixed linear model (MLM) approach. TDM was linked to mEgCIR0555 with phenotypic variance of 20.72%, bunch yield and bunch index were linked to mEgCIR2813 at phenotypic variance of 17.11% and 12.88%, respectively, at a significant threshold (P) level of B0.01.

Genome survey study of Alpinia katsumadai based on Illumina high throughput sequencing / 中草药

Objective: To analyze the genome survey of medicinal and edible plant Alpinia katsumadai and complete its genome genetic information. Methods: This study was based on high throughput sequencing platform Illumina, and K-mer analysis was applied to estimate the genome size and heterozygosity rate of A. katsumadai. Meanwhile, simple sequence repeat (SSR) loci that were suitable as markers were identified by MISA software. Results: The estimated genome size of A. katsumadai was 1.60 Gb, with a 0.44% heterozygosity rate and 72.72% repeats; In the genome sequence, 364 395 simple sequence repeats (SSRs) were detected by SSR molecular marker analysis, among which mono-nucleotide, di-nucleotide and tri-nucleotide repetitive motifs ranked the higher percentages of 64.25%, 24.05% and 10.31%, summed up to 98.61%; From the 350 bp library obtained by sequencing, 10 000 single-end reads were randomly selected and blasted with NT bank, the results showed that its genetically close species Alpinia zerumbet and Elettaria cardamomum were blasted with the reads of 12.89% and 12.36% in NT bank. Conclusion: The genome size, heterozygosity rate and SSR molecular marker analysis' genome survey study on A. katsumadai indicated that the genome of A. katsumadai species was a complex, highly repetitive and large genome, which provided genetic information support for the resource protection, genetic diversity analysis and variety breeding of A. katsumadai.

Mining lycodine-type alkaloid biosynthetic genes and genetic markers from transcriptome of Lycopodiastrum casuarinoides / 中草药·英文版

Objective: Lycopodiastrum casuarinoides, a fern of the Lycopodiaceae family, is a traditional Chinese medicine, which has similar efficacy to that of Huperzia serrata in treating rheumatoid arthritis (RA). However, they are different in the contents and compositions of lycopodium alkaloids. In this study, the biosynthesis related genes of lycopodium alkaloids and genetic markers are discovered in L. casuarinoides transcriptome. Methods: The plant of L. casuarinoides was collected and was subjected to the RNA isolation, cDNA library construction, high throughput RNA sequencing and bioinformatics analysis. Results: Totally 124, 524 high-quality unigenes were assembled from RNA sequencing reads, with an average sequence length of 601 bp. Among the L. casuarinoides transcripts, 61,304 shared the significant similarity (E-value < 10−5) with existing protein sequences in the public databases. From 124,524 unigenes, 47,538 open reading frames (ORFs) were predicted. Based on the bioinformatics analysis, all possible enzyme genes involved in the lycodine-type alkaloids biosynthetic pathway of L. casuarinoides were identified, including lysine decarboxylase (LDC), primary amine oxidase (PAO), malonyl-CoA decarboxylase, etc. Sixty-four putative cytochrome p450 (CYP) and 827 putative transcription factors were selected from the transcriptome unigenes as the candidates of lycodine-type alkaloids biosynthesis modifiers. Furthermore, 13,352 simple sequence repeats (SSRs) were identified from 124,524 unigenes, of which dinucleotide motifs AG/CT were the most abundant (50.1%). Meanwhile, we confirmed the amplification effectiveness of 25 PCR primer pairs for randomly selected SSRs. Conclusion: We obtained the comprehensive transcriptomic information from the high throughput RNA sequencing and bioinformatics analysis, which provided a valuable resource of transcript sequences of L. casuarinoides in public databases.

SSR-based Analysis of Genetic Diversity and Quality Trait-related Markers of 69 Picria felterrae Germplasm Samples / 中国实验方剂学杂志

Objective::Sixty-nine germplasm samples of Picria felterrae collected from the main producing areas in Guangxi were subject to genetic diversity and genetic relationship analyses using the simple seguence repeat(SSR) molecular marker technology and good germplasm genes associated with the content of picfeltarraenins were screened so as to provide references for germplasm resource evaluation, phylogenetic analysis, and molecular mark assisted breeding of that species. Method::20 pairs of randomly selected primers were amplified based on the transcriptome sequencing technology. The genetic diversity of and genetic relationship between the 69 samples were analyzed using the genetic polymorphic information for each marker locus, and one-variable linear regression and multiple stepwise regression analyses were performed to screen molecular markers associated with the content of picfeltarraenins. Result::The amplification using the 20 pairs of SSR primers produced 76 alleles, 3.8 alleles for each locus on average, higher than effective alleles (1.969 2), and the rare allele rate was 38.2%, suggesting that the alleles distributed unequally. The polymorphism rates of alleles varied between 0-59%, with an average of 38.24%, showing a great difference among loci. The polymorphic information content (PIC) varied between 0-0.621 1, with an average of 0.378 0.Shannon polymorphic information index varied between 0-1.240 1, with an average of 0.759.Nei's gene diversity index (Nei) varied between 0-0.682 3, with an average of 0.440 9.P21 had the highest level accompanied with the lowest P7 for the above three indexes, and significant genetic diversity differences were identified among the loci. For all loci, the mean observed heterozygosity was 0.382 4, lower than the average expected heterozygosity of 0.442 5, suggesting the loss of heterozygosity, the average genetic differentiation coefficient (Fst) was 0.365 9 and the average gene flow Nm was 0.433 2, suggesting a high genetic divergence and a low gene flow. The results of one-variable linear regression and multiple stepwise regression showed that there were 5 loci related to each of the picfeltarraenin IA and IB, and only 1 loci was associated with the content of both. Conclusion::There were significant differences in the genetic diversity of 20 SSR marker sites, and the 69 germplasm samples were greatly genetically differentiated and had low gene flow. From the selected 20 SSR markers 9 marker loci associated with the content of picfeltarraenin IA and IB were selected. The results can be used as a reference for phylogenetic analysis and selective breeding of Picria felterrae.

ISSR Analysis of Genetic Relationship and Population Structure of Turpinia arguta Fresh Leaves in Jiangxi Province / 中国实验方剂学杂志

Objective:To explore genetic relationship and population structure of Turpinia arguta in six locations of Jiangxi province by inter-simple sequence repeat （ISSR） molecular marker technique， and to provide theoretical basis for the protection and utilization of this medicinal material resource. Method:A total of 22 samples from six locations in four counties in Jiangxi province were collected， and genomic DNA was extracted by kit method. Polymerase chain reaction （PCR） amplification was performed using sixty-four universal ISSR molecular marker primers， and the products were detected with polyacrylamide gel electrophoresis （PAGE）. NTsys 2.10e software was selected to calculate the genetic similarity coefficient by unweighted pair group method with arithmetic mean （UPGMA） and cluster analysis. Population genetic structure was analyzed by Structure 2.1 software. Result:A total of forty-eight ISSR primers were amplified to obtain the product， the percent of polymorphic bands ranged from 45.45% to 100%. UPGMA cluster analysis showed that these plant individuals could not be clustered according to their respective executive locations. Analysis of population genetic structure showed that 22 samples of T. arguta could be divided into three populations. Conclusion:There is gene exchange among the populations of T. arguta in Jiangxi province， and it can affect the genetic structure of germplasm resources from different geographical sources.

Complete chloroplast genome of Salvia plebeia: organization, specific barcode and phylogenetic analysis / 中国天然药物

Salvia plebeia has been in use as traditional Chinese medicine (TCM) for more than 500 years. In this study, the complete chloroplast (cp) genome of S. plebeia was sequenced, assembled and compared to those of other five published Salvia cp genomes. It was found that the cp genome structure of S. plebeia was well conserved and had a total size of 151 062 bp. Four parameters were used to display the usage conditions of the codons of the amino acids in Salvia genus. Although the number of protein-coding genes in each species was the same, the total number of codons was different. Except for amino acids Trp and Met whose Relative Synonymous Codon Usage (RSCU) value of one condon was equal to 1, the remaining 19 amino acids had 1-3 preferred codons. The preferred codon names of each amino acid were coincident. The period size for the tandem repeats of six species ranged from 9 to 410 bp. Salvia cp genomes mainly possessed tandem repeats with a copy number less than or equal to 3. The sequence length of tandem repeats of the six species ranged from 25 to 824 bp. Highly viarable regions including four intergenic spacers and six partial genes were discovered as potential specific barcodes for Salvia species through cp genome-wide comparison. Finally, we performed phylogenetic analyses based on the complete cp genome and coding sequences respectively. These results provide information to help construct the cp genome library for Salvia, which may support studies of phylogenetics, DNA barcoding, population and transplastomics.

Establish and optimization of inter-simple sequence repeat PCR reaction system of Gnaphalium affine / 药学实践杂志

Objective To provide the experimental basis for the subsequent genetic diversity research through establishing and optimizing the inter-simple sequence repeat PCR (ISSR-PCR) reaction system of Gnaphalium affine. Methods The single-factor experimental method and full experimental method were used to optimize the ISSR-PCR reaction system of Gnaphalium affine. Under the optimal system, after screening primers and corresponding annealing temperatures, the systematic feasibility was verified. Results The optimal ISSR-PCR reaction system was consisted of 10 μl Premix Taq DNA polymerase, 0.3 μmol/L primer, 10 ng DNA template, and sterilized water added to 20 μl. Finally, 10 primers were screened from 100 universal primers, and verification results indicated the system had high stability, good reproducibility, and the selected primers had good polymorphism. Conclusion The ISSR-PCR amplification system of Gnaphalium affine was established for the first time and the primers with appropriate annealing temperatures were filtered out, which provided a reference for the subsequent genetic diversity research of Gnaphalium affine.