GinMicrosatDb: a genome-wide microsatellite markers database for sesame (Sesamum indicum L.).
ABSTRACT: Molecular breeding in sesame is still at infancy due to limited number of microsatellite markers available and the low level of polymorphism exhibited by them. Therefore, whole genome sequencing was used for development of microsatellite markers so as to ensure availability of substantial number of polymorphic markers for use in marker assisted breeding programs. Whole genome sequencing of sesame variety 'Swetha' was done using Illumina paired-end sequencing and Roche 454 shotgun sequencing technologies (GCA_000975565.1 in GenBank). 'GinMicrosatDb', a genome-wide microsatellite marker database has been developed using the whole genome sequence data of sesame variety 'Swetha'. The database consists of microsatellites localized on both linkage groups and scaffolds with their genomic co-ordinates. It provides five sets of forward and reverse primers for each of the microsatellite loci along with the flanking sequences, primer GC content, product size and melting temperature etc. The distribution of microsatellites can be viewed and selected through a genome browser as well as through a physical map. The newly identified microsatellite markers are expected to help sesame breeders in developing marker tags for traits of economic importance thereby bringing about greater efficiency in marker-assisted selection programs.
Project description:The sequencing of the full nuclear genome of sesame (<i>Sesamum indicum</i> L.) provides the platform for functional analyses of genome components and their application in breeding programs. Although the importance of microsatellites markers or simple sequence repeats (SSR) in crop genotyping, genetics, and breeding applications is well established, only a little information exist concerning SSRs at the whole genome level in sesame. In addition, SSRs represent a suitable marker type for sesame molecular breeding in developing countries where it is mainly grown. In this study, we identified 138,194 genome-wide SSRs of which 76.5% were physically mapped onto the 13 pseudo-chromosomes. Among these SSRs, up to three primers pairs were supplied for 101,930 SSRs and used to <i>in silico</i> amplify the reference genome together with two newly sequenced sesame accessions. A total of 79,957 SSRs (78%) were polymorphic between the three genomes thereby suggesting their promising use in different genomics-assisted breeding applications. From these polymorphic SSRs, 23 were selected and validated to have high polymorphic potential in 48 sesame accessions from different growing areas of Africa. Furthermore, we have developed an online user-friendly database, SisatBase (http://www.sesame-bioinfo.org/SisatBase/), which provides free access to SSRs data as well as an integrated platform for functional analyses. Altogether, the reference SSR and SisatBase would serve as useful resources for genetic assessment, genomic studies, and breeding advancement in sesame, especially in developing countries.
Project description:Microsatellite markers of Jasminum sambac (Oleaceae) were isolated to investigate wild germplasm resources and provide markers for breeding.Illumina sequencing was used to isolate microsatellite markers from the transcriptome of J. sambac. A total of 1322 microsatellites were identified from 49,772 assembled unigenes. One hundred primer pairs were randomly selected to verify primer amplification efficiency. Out of these tested primer pairs, 31 were successfully amplified: 18 primer pairs yielded a single allele, seven exhibited fixed heterozygosity with two alleles, and only six displayed polymorphisms.This study obtained the first set of microsatellite markers for J. sambac, which will be helpful for the assessment of wild germplasm resources and the development of molecular marker-assisted breeding.
Project description:Over the last few decades, the use of molecular markers has played an increasing role in rice breeding and genetics. Of the different types of molecular markers, microsatellites have been utilized most extensively, because they can be readily amplified by PCR and the large amount of allelic variation at each locus. Microsatellites are also known as simple sequence repeats (SSR), and they are typically composed of 1-6 nucleotide repeats. These markers are abundant, distributed throughout the genome and are highly polymorphic compared with other genetic markers, as well as being species-specific and co-dominant. For these reasons, they have become increasingly important genetic markers in rice breeding programs. The evolution of new biotypes of pests and diseases as well as the pressures of climate change pose serious challenges to rice breeders, who would like to increase rice production by introducing resistance to multiple biotic and abiotic stresses. Recent advances in rice genomics have now made it possible to identify and map a number of genes through linkage to existing DNA markers. Among the more noteworthy examples of genes that have been tightly linked to molecular markers in rice are those that confer resistance or tolerance to blast. Therefore, in combination with conventional breeding approaches, marker-assisted selection (MAS) can be used to monitor the presence or lack of these genes in breeding populations. For example, marker-assisted backcross breeding has been used to integrate important genes with significant biological effects into a number of commonly grown rice varieties. The use of cost-effective, finely mapped microsatellite markers and MAS strategies should provide opportunities for breeders to develop high-yield, blast resistance rice cultivars. The aim of this review is to summarize the current knowledge concerning the linkage of microsatellite markers to rice blast resistance genes, as well as to explore the use of MAS in rice breeding programs aimed at improving blast resistance in this species. We also discuss the various advantages, disadvantages and uses of microsatellite markers relative to other molecular marker types.
Project description:Sesame (Sesamum indicum), an important oil crop, is widely grown in tropical and subtropical regions. It provides part of the daily edible oil allowance for almost half of the world's population. A limited number of co-dominant markers has been developed and applied in sesame genetic diversity and germplasm identity studies. Here we report for the first time a whole genome survey used to develop simple sequence repeat (SSR) markers and to detect the genetic diversity of sesame germplasm. From the initial assembled sesame genome, 23,438 SSRs (?5 repeats) were identified. The most common repeat motif was dinucleotide with a frequency of 84.24%, followed by 13.53% trinucleotide, 1.65% tetranucleotide, 0.3% pentanucleotide and 0.28% hexanucleotide motifs. From 1500 designed and synthesised primer pairs, 218 polymorphic SSRs were developed and used to screen 31 sesame accessions that from 12 countries. STRUCTURE and phylogenetic analyses indicated that all sesame accessions could be divided into two groups: one mainly from China and another from other countries. Cluster analysis classified Chinese major sesame varieties into three groups. These novel SSR markers are a useful tool for genetic linkage map construction, genetic diversity detection, and marker-assisted selective sesame breeding.
Project description:Although much research has been conducted to characterize microsatellites and develop markers, the distribution of microsatellites remains ambiguous and the use of microsatellite markers in genomic studies and marker-assisted selection is limited. To identify microsatellites for cotton research, we mined 100,290, 83,160, and 56,937 microsatellites with frequencies of 41.2, 49.1, and 74.8 microsatellites per Mb in the recently sequenced Gossypium species: G. hirsutum, G. arboreum, and G. raimondii, respectively. The distributions of microsatellites in their genomes were non-random and were positively and negatively correlated with genes and transposable elements, respectively. Of the 77,996 developed microsatellite markers, 65,498 were physically anchored to the 26 chromosomes of G. hirsutum with an average marker density of 34 markers per Mb. We confirmed 67,880 (87%) universal and 7,705 (9.9%) new genic microsatellite markers. The polymorphism was estimated in above three species by in silico PCR and validated with 505 markers in G. hirsutum. We further predicted 8,825 polymorphic microsatellite markers within G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124. In our study, genome-wide mining and characterization of microsatellites, and marker development were very useful for the saturation of the allotetraploid genetic linkage map, genome evolution studies and comparative genome mapping.
Project description:Sesame is prized for its oil. Genetic improvement of sesame can be enhanced through marker-assisted breeding. However, few simple sequence repeat (SSR) markers and SSR-based genetic maps were available in sesame. In this study, 7,357 SSR markers were developed from the sesame genome and transcriptomes, and a genetic map was constructed by generating 424 novel polymorphic markers and using a cross population with 548 recombinant inbred lines (RIL). The genetic map had 13 linkage groups, equalling the number of sesame chromosomes. The linkage groups ranged in size from 113.6 to 179.9 centimorgans (cM), with a mean value of 143.8?cM over a total length of 1869.8?cM. Fourteen quantitative trait loci (QTL) for sesame charcoal rot disease resistance were detected, with contribution rates of 3-14.16% in four field environments; ~60% of the QTL were located within 5?cM at 95% confidence interval. The QTL with the highest phenotype contribution rate (qCRR12.2) and those detected in different environments (qCRR8.2 and qCRR8.3) were used to predict candidate disease response genes. The new SSR-based genetic map and 14 novel QTLs for charcoal rot disease resistance will facilitate the mapping of agronomic traits and marker-assisted selection breeding in sesame.
Project description:The development and validation of different types of molecular markers is crucial to conducting marker-assisted sesame breeding. Insertion-deletion (InDel) markers are highly polymorphic and suitable for low-cost gel-based genotyping. From this perspective, this study aimed to discover and develop InDel markers through bioinformatic analysis of double digest restriction site-associated DNA sequencing (ddRADSeq) data from 95 accessions belonging to the Mediterranean sesame core collection. Bioinformatic analysis indicated the presence of 7477 InDel positions genome wide. Deletions accounted for 61% of the InDels and short deletions (1-2 bp) were the most abundant type (94.9%). On average, InDels of at least 2 bp in length had a frequency of 2.99 InDels/Mb. The 86 InDel sites having length ?8 bp were detected in genome-wide analysis. These regions can be used for the development of InDel markers considering low-cost genotyping with agarose gels. In order to validate these InDels, a total of 38 InDel regions were selected and primers were successfully amplified. About 13% of these InDels were in the coding sequences (CDSs) and in the 3'- and 5'- untranslated regions (UTRs). Furthermore, the efficiencies of these 16 InDel markers were assessed on 32 sesame accessions. The polymorphic information content (PIC) of these 16 markers ranged from 0.06 to 0.62 (average: 0.33). These results demonstrated the success of InDel identification and marker development for sesame with the use of ddRADSeq data. These agarose-resolvable InDel markers are expected to be useful for sesame breeders.
Project description:Molecular marker-assisted breeding provides an efficient tool to develop improved crop varieties. A major challenge for the broad application of markers in marker-assisted selection is that the marker phenotypes must match plant phenotypes in a wide range of breeding germplasm. In this study, we used the legume crop species Lupinus angustifolius (lupin) to demonstrate the utility of whole genome sequencing and re-sequencing on the development of diagnostic markers for molecular plant breeding.Nine lupin cultivars released in Australia from 1973 to 2007 were subjected to whole genome re-sequencing. The re-sequencing data together with the reference genome sequence data were used in marker development, which revealed 180,596 to 795,735 SNP markers from pairwise comparisons among the cultivars. A total of 207,887 markers were anchored on the lupin genetic linkage map. Marker mining obtained an average of 387 SNP markers and 87 InDel markers for each of the 24 genome sequence assembly scaffolds bearing markers linked to 11 genes of agronomic interest. Using the R gene PhtjR conferring resistance to phomopsis stem blight disease as a test case, we discovered 17 candidate diagnostic markers by genotyping and selecting markers on a genetic linkage map. A further 243 candidate diagnostic markers were discovered by marker mining on a scaffold bearing non-diagnostic markers linked to the PhtjR gene. Nine out from the ten tested candidate diagnostic markers were confirmed as truly diagnostic on a broad range of commercial cultivars. Markers developed using these strategies meet the requirements for broad application in molecular plant breeding.We demonstrated that low-cost genome sequencing and re-sequencing data were sufficient and very effective in the development of diagnostic markers for marker-assisted selection. The strategies used in this study may be applied to any trait or plant species. Whole genome sequencing and re-sequencing provides a powerful tool to overcome current limitations in molecular plant breeding, which will enable plant breeders to precisely pyramid favourable genes to develop super crop varieties to meet future food demands.
Project description:Flax (Linum usitatissimum L.) is a major fiber and oil yielding crop grown in northeastern China. Identification of flax molecular markers is a key step toward improving flax yield and quality via marker-assisted breeding. Simple sequence repeat (SSR) markers, which are based on genomic structural variation, are considered the most valuable type of genetic marker for this purpose. In this study, we screened 1574 microsatellites from Linum usitatissimum L. obtained using reduced representation genome sequencing (RRGS) to systematically identify SSR markers. The resulting set of microsatellites consisted mainly of trinucleotide (56.10%) and dinucleotide (35.23%) repeats, with each motif consisting of 5-8 repeats. We then evaluated marker sensitivity and specificity based on samples of 48 flax isolates obtained from northeastern China. Using the new SSR panel, the results demonstrated that fiber flax and oilseed flax varieties clustered into two well separated groups. The novel SSR markers developed in this study show potential value for selection of varieties for use in flax breeding programs.
Project description:Morphology-based taxonomy via exiguously reproductive organ has severely limitation on bamboo taxonomy, mainly owing to infrequent and unpredictable flowering events of bamboo. Here, we present the first genome-wide analysis and application of microsatellites based on the genome of moso bamboo (Phyllostachys edulis) to assist bamboo taxonomy. Of identified 127,593 microsatellite repeat-motifs, the primers of 1,451 microsatellites were designed and 1,098 markers were physically mapped on the genome of moso bamboo. A total of 917 markers were successfully validated in 9 accessions with ~39.8% polymorphic potential. Retrieved from validated microsatellite markers, 23 markers were selected for polymorphic analysis among 78 accessions and 64 alleles were detected with an average of 2.78 alleles per primers. The cluster result indicated the majority of the accessions were consistent with their current taxonomic classification, confirming the suitability and effectiveness of the developed microsatellite markers. The variations of microsatellite marker in different species were confirmed by sequencing and in silico comparative genome mapping were investigated. Lastly, a bamboo microsatellites database (http://www.bamboogdb.org/ssr) was implemented to browse and search large information of bamboo microsatellites. Consequently, our results of microsatellite marker development are valuable for assisting bamboo taxonomy and investigating genomic studies in bamboo and related grass species.