ABSTRACT
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><sub>0</sub>） values were determined to be 0.603 and 0.613， the expected heterozygosity （<italic>H</italic><sub>e</sub>）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><sub>st</sub>） was 0.123 and gene flow （<italic>N</italic><sub>m</sub>） 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>
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Campomanesia xanthocarpa is a native tree, of common occurrence in almost all Brazilian Forest formations, which has its fruits and timber with high commercial value. Using an enriched genomic library we isolated and characterized microsatellite loci for C. xanthocarpa (Myrtaceae), in order to estimate genetic diversity parameters for this and related species. Twenty-eight microsatellite loci were identified and ten of them successfully amplified and showed polymorphism in a sample of 96 individuals, from four natural populations. The number of alleles per locus ranged from two to eight, and the observed and expected heterozygosities varied from 0.042 to 1.000 and from 0.294 to 0.855, respectively. These markers were tested and validated in two related species (C. eugenioides and C. guazumifolia). The microsatellite markers will be used in further studies of population genetics of C. xanthocarpa, in order to understand the genetic variability and to define the strategies needed for the conservation of the species.
Subject(s)
Myrtaceae/genetics , Microsatellite RepeatsABSTRACT
Callicarpa nudiflora,which is a big brand of Li nationality medicine with Hainan characteristics,has the effects of dissolving stasis,hemostasis,anti-inflammatory and antibacterial. At present,there is a lack of information about the reference genome of C. nudiflora. The study of the genome size,heterozygosity rate and characteristics of SSR of C. nudiflora,can provide an effective basis for the formulation of the whole genome de novo sequencing strategy and development of SSR molecular markers of C. nudiflora. To realize this purpose,high throughput sequencing platform Illumina Hiseq was used to sequence the genome structure of C. nudiflora and K-mer analysis was applied to estimate genome size,repeat sequences and heterozygosity rate. Simple-sequence repeat( SSR) loci that are suitable as markers were identified by MISA software. The results showed the estimated genome size of C. nudiflora was 822. 43 Mb,with a 0. 85% heterozygosity rate and 71. 67% repeats,and the GC content of genome was about 49. 20%. Therefore,C. nudiflora belongs to a complex genome with high heterozygosity and repetition. SSR molecular genetic markers were analyzed in the genome sequence,and a total of 206 049 SSRs were identified,among which mono-nucleotide,di-nucleotide and tri-nucleotide repetitive motifs summed up to 198 993,accounting for 96. 57% of the total SSRs. Among the 2-6 nucleotide repeats,AT/AT,AAT/ATT,AGCC/CTGG,AAAAT/ATTTT and AGATAT/ATATCT have the largest number,respectively. This report represents the first genome-wide characterization of C. nudiflora,and provides a reference for the construction of the library for the fine sequencing of the genome,and a molecular basis for the development of SSR molecular markers as well as for the protection and utilization of gene resources.
Subject(s)
Callicarpa/genetics , Genetic Markers , Genome, Plant , Microsatellite Repeats , Polymorphism, GeneticABSTRACT
OBJECTIVE o facilitate the basic acquaintance with the bioactive lycodine-type alka-loids biosynthetic pathways, we conducted the transcriptome analysis of L. casuarinoides by illumina sequencing.METHODS The plant of L.casuarinoides was collected and was subjected to RNA isolation, cDNA library construction, and illumina sequencing before bioinformatics analysis. After sequencing, the clean reads were obtained for de novo assembly by using Trinity software,and then further processed with TGICL sequencing clustering software to generate unigenes,The unigenes are aligned by Blast X alignment to six public protein database. In addition, all unigenes are functionally annotated by GO, KEGG and characterized putative genes involved in lycopodium alkaloids biosynthesis. RESULTS In total, 124, 524 high-quality unigenes were obtained with an average sequence length of 601 bp. Among the L. casuarinoides transcripts, 61,304 showed significant similarity (E-value<1 e-5) to the known proteins in the public database.Among the total 124 524 unigenes,47,538 open reading frame (ORFs) were predicted. Based on the bioinformatics analysis, all possible enzymes involved in the Lycodine-type alkaloids biosynthetic pathway of L. casuarinoides were identified, including primary amine oxidase (PAO), and Malonly-CoA decarboxylase. In addition, a total of 64 putative cytochrome P450 (CYP450) and 827 transcription factors were selected as the candidates of Lycodine-type alka-loids modifiers. Furthermore, a total of 13 352 simple sequence repeats (SSRs) were identified from the 124, 524 unigenes, of which dinucleotide motifs AG/CT (50.1%), were the most abundant. CONCLU-SION This transcriptome analysis of L.casuarinoides,provides many valuable candidate genes involv-ing in the biosynthesis of novel secondary metabolites but also lays the foundation for genetic diversity analysis via SSRs markers in L.casuarinoides.
ABSTRACT
Tibouchina hatschbachii Wurdack (Melastomataceae) is an autogamous shrub restricted to granite (GO) and sandstone (SO) rock outcrops from subtropical Brazil. We designed primers for the amplification of microsatellite regions for T. hatschbachii, and characterized these primers to estimate genetic diversity parameters and contemporary genetic structure patterns. Eight loci were successfully amplified and were characterized using 70 individuals from three natural populations. Polymorphic information content ranged from 0.200 to 0.772 per locus. All loci were polymorphic, with allele numbers ranging from two to eight. The low degree of polymorphism may be explained by the fact that T. hatschbachii has disjunct populations and a recent genetic bottleneck, and also that it is self-pollinated. The observed and expected heterozygosities ranged from 0.115 to 1.000 and from 0.112 to 0.800, respectively. We observed private alleles in all loci. These are important features that enable us to identify population differentiation and help to us understand gene flow patterns for T. hatschbachii in subtropical Brazil. Eight microsatellite loci from other species of Tibouchina amplified positively in T. hatschbachii.
Tibouchina hatschbachii Wurdack (Melastomataceae) é um arbusto autógamo, com ocorrência restrita em afloramentos rochosos graníticos (GO) e areníticos (SO) na região subtropical do Brasil. Neste trabalho, foram desenvolvidos marcadores para a amplificação de regiões microssatélites para T. hatschbachii e caracterizados esses primers para estimar parâmetros de diversidade genética. Oito loci foram amplificados com sucesso e caracterizados, utilizando 70 indivíduos de três populações naturais. O conteúdo de informação polimórfica variou de 0,200 a 0,772 por locus. Todos os loci foram polimórficos, com números de alelos que variam de dois a oito. O baixo grau de polimorfismo pode ser explicado pelo fato de que T. hatschbachii possui populações disjuntas e uma história recente de gargalo genético populacional, e também pelo fato de apresentar um sistema reprodutivo de autopolinização, tendendo a favorecer a baixa variação. As heterozigosidades observadas e esperadas variaram entre 0,115-1,000 e 0,112-0,800, respectivamente. Também foi observada a presença de alelos privados em todos os loci. Estas são características importantes que nos permitirão identificar a diferenciação entre populações e poderão ajudar na compreensão dos padrões de fluxo gênico atual de T. hatschbachii na região subtropical do Brasil. Oito loci microssatélites de outras espécies de Tibouchina amplificaram
Subject(s)
Animals , Melastomataceae/growth & development , Melastomataceae/genetics , Microsatellite Repeats , Restriction Mapping/veterinaryABSTRACT
Tibouchina hatschbachii Wurdack (Melastomataceae) is an autogamous shrub restricted to granite (GO) and sandstone (SO) rock outcrops from subtropical Brazil. We designed primers for the amplification of microsatellite regions for T. hatschbachii, and characterized these primers to estimate genetic diversity parameters and contemporary genetic structure patterns. Eight loci were successfully amplified and were characterized using 70 individuals from three natural populations. Polymorphic information content ranged from 0.200 to 0.772 per locus. All loci were polymorphic, with allele numbers ranging from two to eight. The low degree of polymorphism may be explained by the fact that T. hatschbachii has disjunct populations and a recent genetic bottleneck, and also that it is self-pollinated. The observed and expected heterozygosities ranged from 0.115 to 1.000 and from 0.112 to 0.800, respectively. We observed private alleles in all loci. These are important features that enable us to identify population differentiation and help to us understand gene flow patterns for T. hatschbachii in subtropical Brazil. Eight microsatellite loci from other species of Tibouchina amplified positively in T. hatschbachii.
Tibouchina hatschbachii Wurdack (Melastomataceae) é um arbusto autógamo, com ocorrência restrita em afloramentos rochosos graníticos (GO) e areníticos (SO) na região subtropical do Brasil. Neste trabalho, foram desenvolvidos marcadores para a amplificação de regiões microssatélites para T. hatschbachii e caracterizados esses primers para estimar parâmetros de diversidade genética. Oito loci foram amplificados com sucesso e caracterizados, utilizando 70 indivíduos de três populações naturais. O conteúdo de informação polimórfica variou de 0,200 a 0,772 por locus. Todos os loci foram polimórficos, com números de alelos que variam de dois a oito. O baixo grau de polimorfismo pode ser explicado pelo fato de que T. hatschbachii possui populações disjuntas e uma história recente de gargalo genético populacional, e também pelo fato de apresentar um sistema reprodutivo de autopolinização, tendendo a favorecer a baixa variação. As heterozigosidades observadas e esperadas variaram entre 0,115-1,000 e 0,112-0,800, respectivamente. Também foi observada a presença de alelos privados em todos os loci. Estas são características importantes que nos permitirão identificar a diferenciação entre populações e poderão ajudar na compreensão dos padrões de fluxo gênico atual de T. hatschbachii na região subtropical do Brasil. Oito loci microssatélites de outras espécies de Tibouchina amplificaram positivamente em T. hatschbachii..
Subject(s)
Melastomataceae , Genetics, PopulationABSTRACT
Simple sequence repeats (SSRs) are ubiquitous short tandem duplications found within eukaryotic genomes. Their length variability and abundance throughout the genome has led them to be widely used as molecular markers for crop-breeding programs, facilitating the use of marker-assisted selection as well as estimation of genetic population structure. Here, we report a software application, "SSR-Primer Generator" for SSR discovery, SSR-primer design, and homology-based search of in silico amplicons from a DNA sequence dataset. On submission of multiple FASTA-format DNA sequences, those analyses are batch processed in a Java runtime environment (JRE) platform, in a pipeline, and the resulting data are visualized in HTML tabular format. This application will be a useful tool for reducing the time and costs associated with the development and application of SSR markers.