Development and testing of new gene-homologous EST-SSRs for Eucalyptus gomphocephala (Myrtaceae).
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ABSTRACT:
SUBMITTER: Bradbury D
PROVIDER: S-EPMC4103447 | biostudies-literature | 2013 Aug
REPOSITORIES: biostudies-literature
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Project description:Sorbus pohuashanensis is a native tree species of northern China that is used for a variety of ecological purposes. The species is often grown as an ornamental landscape tree because of its beautiful form, silver flowers in early summer, attractive pinnate leaves in summer, and red leaves and fruits in autumn. However, development and further utilization of the species are hindered by the lack of comprehensive genetic information, which impedes research into its genetics and molecular biology. Recent advances in de novo transcriptome sequencing (RNA-seq) technology have provided an effective means to obtain genomic information from non-model species. Here, we applied RNA-seq for sequencing S. pohuashanensis leaves and obtained a total of 137,506 clean reads. After assembly, 96,213 unigenes with an average length of 770 bp were obtained. We found that 64.5% of the unigenes could be annotated using bioinformatics tools to analyze gene function and alignment with the NCBI database. Overall, 59,089 unigenes were annotated using the Nr database(non-redundant protein database), 35,225 unigenes were annotated using the GO (Gene Ontology categories) database, and 33,168 unigenes were annotated using COG (Cluster of Orthologous Groups). Analysis of the unigenes using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database indicated that 13,953 unigenes were involved in 322 metabolic pathways. Finally, simple sequence repeat (SSR) site detection identified 6,604 unigenes that included EST-SSRs and a total of 7,473 EST-SSRs in the unigene sequences. Fifteen polymorphic SSRs were screened and found to be of use for future genetic research. These unigene sequences will provide important genetic resources for genetic improvement and investigation of biochemical processes in S. pohuashanensis.
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Project description:A set of 899 L. gmelinii expression sequence tags (ESTs), available at the National Center of Biotechnology Information (NCBI), was employed to address the feasibility on development of simple sequence repeat (SSR) markers for Larch species. Totally, 634 non-redundant unigenes including 145 contigs and 489 singletons were finally identified and mainly involved in biosynthetic, metabolic processes and response to stress according to BLASTX results, gene ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) maps. Approximately 11.7% (74) unigenes contained 90 candidate SSRs, which were mainly trinucleotides (29, 32.2%) and dinucleotides (26, 28.9%). A relatively high frequency of SSRs was respectively found in the Open Reading Frame (ORF, about 54.4%) and 5'-untranslated region (5'-UTR, 31.2%), while a low frequency was observed in the 3'-untranslated region (3'-UTR, about 14.4%). Of the 45 novel EST-SSRs markers, nine were found to be polymorphic at two L. gmelinii populations. The number of alleles per locus (Na) ranged from two to four, and the observed (Ho) and expected (He) heterozygosity values were 0.200-0.733 and 0.408-0.604, respectively. The inbreeding coefficients (FIS) for all loci were more than zero except Lg41. Most of these 9EST-SSR markers were transferable to its related species L. kaempferi, L. principis-rupprechtii and L. olgensis. These novel EST-SSRs will be useful for further research on comparative genomics, genetic resources conservation and molecular breeding in larch trees.
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Project description:BACKGROUND: Epimedium sagittatum (Sieb. Et Zucc.) Maxim, a traditional Chinese medicinal plant species, has been used extensively as genuine medicinal materials. Certain Epimedium species are endangered due to commercial overexploition, while sustainable application studies, conservation genetics, systematics, and marker-assisted selection (MAS) of Epimedium is less-studied due to the lack of molecular markers. Here, we report a set of expressed sequence tags (ESTs) and simple sequence repeats (SSRs) identified in these ESTs for E. sagittatum. RESULTS: cDNAs of E. sagittatum are sequenced using 454 GS-FLX pyrosequencing technology. The raw reads are cleaned and assembled into a total of 76,459 consensus sequences comprising of 17,231 contigs and 59,228 singlets. About 38.5% (29,466) of the consensus sequences significantly match to the non-redundant protein database (E-value < 1e-10), 22,295 of which are further annotated using Gene Ontology (GO) terms. A total of 2,810 EST-SSRs is identified from the Epimedium EST dataset. Trinucleotide SSR is the dominant repeat type (55.2%) followed by dinucleotide (30.4%), tetranuleotide (7.3%), hexanucleotide (4.9%), and pentanucleotide (2.2%) SSR. The dominant repeat motif is AAG/CTT (23.6%) followed by AG/CT (19.3%), ACC/GGT (11.1%), AT/AT (7.5%), and AAC/GTT (5.9%). Thirty-two SSR-ESTs are randomly selected and primer pairs are synthesized for testing the transferability across 52 Epimedium species. Eighteen primer pairs (85.7%) could be successfully transferred to Epimedium species and sixteen of those show high genetic diversity with 0.35 of observed heterozygosity (Ho) and 0.65 of expected heterozygosity (He) and high number of alleles per locus (11.9). CONCLUSION: A large EST dataset with a total of 76,459 consensus sequences is generated, aiming to provide sequence information for deciphering secondary metabolism, especially for flavonoid pathway in Epimedium. A total of 2,810 EST-SSRs is identified from EST dataset and approximately 1580 EST-SSR markers are transferable. E. sagittatum EST-SSR transferability to the major Epimedium germplasm is up to 85.7%. Therefore, this EST dataset and EST-SSRs will be a powerful resource for further studies such as taxonomy, molecular breeding, genetics, genomics, and secondary metabolism in Epimedium species.
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Project description:Salt stress adversely affects the global wheat production and productivity. To improve salinity tolerance of crops, identification of robust molecular markers is highly imperative for development of salt-tolerant cultivars to mimic yield losses under saline conditions. In this study, we mined 171 salt-responsive genes (including 10 miRNAs) from bread wheat genome using the sequence information of functionally validated salt-responsive rice genes. Salt-stress, tissue and developmental stage-specific expression analysis of RNA-seq datasets revealed the constitutive as well as the inductive response of salt-responsive genes in different tissues of wheat. Fifty-four genotypes were phenotyped for salt stress tolerance. The stress tolerance index of the genotypes ranged from 0.30 to 3.18. In order to understand the genetic diversity, candidate gene based SSRs (cg-SSRs) and MIR gene based SSRs (miR-SSRs) were mined from 171 members of salt-responsive genes of wheat and validated among the contrasting panels of 54 tolerant as well as susceptible wheat genotypes. Among 53 SSR markers screened, 10 cg-SSRs and 8 miR-SSRs were found to be polymorphic. Polymorphic information content between the wheat genotypes ranged from 0.07 to 0.67, indicating the extant of wide genetic variation among the salt tolerant and susceptible genotypes at the DNA level. The genetic diversity analysis based on the allelic data grouped the wheat genotypes into three separate clusters of which single group encompassing most of the salt susceptible genotypes and two of them containing salt tolerance and moderately salt tolerance wheat genotypes were in congruence with penotypic data. Our study showed that both salt-responsive genes and miRNAs based SSRs were more diverse and can be effectively used for diversity analysis. This study reports the first extensive survey on genome-wide analysis, identification, development and validation of salt-responsive cg-SSRs and miR-SSRs in wheat. The information generated in the present study on genetic divergence among genotypes having a differential response to salt will help in the selection of suitable lines as parents for developing salt tolerant cultivars in wheat.
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Project description:The western flower thrips, Frankliniella occidentalis (Pergande), is an invasive species and the most economically important pest within the insect order Thysanoptera. For a better understanding of the genetic makeup and migration patterns of F. occidentalis throughout the world, we characterized 18 novel polymorphic EST-derived microsatellites. The mutational mechanism of these EST-SSRs was also investigated to facilitate the selection of appropriate combinations of markers for population genetic studies. Genetic diversity of these novel markers was assessed in 96 individuals from three populations in China (Harbin, Dali, and Guiyang). The results showed that all these 18 loci were highly polymorphic; the number of alleles ranged from 2 to 15, with an average of 5.50 alleles per locus. The observed (H(O)) and expected (H(E)) heterozygosities ranged from 0.072 to 0.707 and 0.089 to 0.851, respectively. Furthermore, only two locus/population combinations (WFT144 in Dali and WFT50 in Guiyang) significantly deviated from Hardy-Weinberg equilibrium (HWE). Pairwise F(ST) analysis showed a low but significant differentiation (0.026 < F(ST) < 0.032) among all three pairwise population comparisons. Sequence analysis of alleles per locus revealed a complex mutational pattern of these EST-SSRs. Thus, these EST-SSRs are useful markers but greater attention should be paid to the mutational characteristics of these microsatellites when they are used in population genetic studies.
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Project description:BackgroundGenetic markers and linkage mapping are basic prerequisites for comparative genetic analyses, QTL detection and map-based cloning. A large number of mapping populations have been developed for oak, but few gene-based markers are available for constructing integrated genetic linkage maps and comparing gene order and QTL location across related species.ResultsWe developed a set of 573 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and located 397 markers (EST-SSRs and genomic SSRs) on the 12 oak chromosomes (2n = 2x = 24) on the basis of Mendelian segregation patterns in 5 full-sib mapping pedigrees of two species: Quercus robur (pedunculate oak) and Quercus petraea (sessile oak). Consensus maps for the two species were constructed and aligned. They showed a high degree of macrosynteny between these two sympatric European oaks. We assessed the transferability of EST-SSRs to other Fagaceae genera and a subset of these markers was mapped in Castanea sativa, the European chestnut. Reasonably high levels of macrosynteny were observed between oak and chestnut. We also obtained diversity statistics for a subset of EST-SSRs, to support further population genetic analyses with gene-based markers. Finally, based on the orthologous relationships between the oak, Arabidopsis, grape, poplar, Medicago, and soybean genomes and the paralogous relationships between the 12 oak chromosomes, we propose an evolutionary scenario of the 12 oak chromosomes from the eudicot ancestral karyotype.ConclusionsThis study provides map locations for a large set of EST-SSRs in two oak species of recognized biological importance in natural ecosystems. This first step toward the construction of a gene-based linkage map will facilitate the assignment of future genome scaffolds to pseudo-chromosomes. This study also provides an indication of the potential utility of new gene-based markers for population genetics and comparative mapping within and beyond the Fagaceae.
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Project description:Simple sequence repeats (SSRs) are among the most important markers for population analysis and have been widely used in plant genetic mapping and molecular breeding. Expressed sequence tag-SSR (EST-SSR) markers, located in the coding regions, are potentially more efficient for QTL mapping, gene targeting, and marker-assisted breeding. In this study, we investigated 51,694 nonredundant unigenes, assembled from clean reads from deep transcriptome sequencing with a Solexa/Illumina platform, for identification and development of EST-SSRs in Chinese cabbage. In total, 10,420 EST-SSRs with over 12 bp were identified and characterized, among which 2744 EST-SSRs are new and 2317 are known ones showing polymorphism with previously reported SSRs. A total of 7877 PCR primer pairs for 1561 EST-SSR loci were designed, and primer pairs for twenty-four EST-SSRs were selected for primer evaluation. In nineteen EST-SSR loci (79.2%), amplicons were successfully generated with high quality. Seventeen (89.5%) showed polymorphism in twenty-four cultivars of Chinese cabbage. The polymorphic alleles of each polymorphic locus were sequenced, and the results showed that most polymorphisms were due to variations of SSR repeat motifs. The EST-SSRs identified and characterized in this study have important implications for developing new tools for genetics and molecular breeding in Chinese cabbage.
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Project description:New source of molecular markers accelerate the efforts in improving cotton fiber traits and aid in developing high-density integrated genetic maps. We developed new markers based on candidate genes and G. arboreum EST sequences that were used for polymorphism detection followed by genetic and physical mapping. Nineteen gene-based markers were surveyed for polymorphism detection in 26 Gossypium species. Cluster analysis generated a phylogenetic tree with four major sub-clusters for 23 species while three species branched out individually. CAP method enhanced the rate of polymorphism of candidate gene-based markers between G. hirsutum and G. barbadense. Two hundred A-genome based SSR markers were designed after datamining of G. arboreum EST sequences (Mississippi Gossypium arboreum??EST-SSR: MGAES). Over 70% of MGAES markers successfully produced amplicons while 65 of them demonstrated polymorphism between the parents of G. hirsutum and G. barbadense RIL population and formed 14 linkage groups. Chromosomal localization of both candidate gene-based and MGAES markers was assisted by euploid and hypoaneuploid CS-B analysis. Gene-based and MGAES markers were highly informative as they were designed from candidate genes and fiber transcriptome with a potential to be integrated into the existing cotton genetic and physical maps.
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Project description:Bletilla striata is an endangered orchid that has been used for millennia as a medicinal herb, in cosmetics and as a horticultural plant. To construct the first nucleotide database for this species and to develop abundant EST-SSR markers for facilitating further studies, various tissues and organs of plants in the main developmental stages were harvested for mRNA isolation and subsequent RNA sequencing. A total of 106,054,784 clean reads were generated by using Illumina paired-end sequencing technology. The reads were assembled into 127,261 unigenes by the Trinity package; the unigenes had an average length of 612 bp and an N50 of 957 bp. Of these unigenes, 67,494 (51.86%) were annotated in a series of databases. Of these annotated unigenes, 41,818 and 24,615 were assigned to gene ontology categories and clusters of orthologous groups, respectively. Additionally, 20,764 (15.96%) unigenes were mapped onto 275 pathways using the KEGG database. In addition, 25,935 high-quality EST-SSR primer pairs were developed from the 15,433 unigenes by MISA mining. To validate the accuracy of the newly designed markers, 87 of 100 randomly selected primers were effectively amplified; 63 of those yielded PCR products of the expected size, and 25 yielded products with significant amounts of polymorphism among the 4 landraces. Furthermore, the transferability test of the 25 polymorphic markers was performed in 6 individuals of two closely related genus Phalaenopsis and dendrobium. Which results showed a total of 5 markers can successfully amplified among these populations. This research provides a comprehensive nucleotide database and lays a solid foundation for functional gene mining and genomic research in B. striata. The developed EST-SSR primers could facilitate phylogenetic studies and breeding.
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Project description:Eucalyptus robusta is a great tree of Myrtaceae. It is mainly distributed in Southeast provinces of China and Southeast Asian countries. There is no study on the genome of E. robusta far. Here we report the complete chloroplast genome of E. robusta, assembled from whole-genome high-throughput sequencing data, as a resource for future studies on the taxonomy and evolution of E. robusta. The complete chloroplast genome of E. robusta is 160,201 bp in length with a typical quadripartite structure, consisting of a large single-copy region (LSC, 88,905 bp), a single-copy region (SSC, 18,498 bp) and a pair of inverted repeats (IRs, 26,399 bp). It was predicted to contain a total of 128 genes, with an overall GC content of 36.86%. Phylogenetic analysis placed E. robusta closest to Eucalyptus globulus.
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