Gene Based SSR Markers Research Articles

Assessment of the genetic variability and population structure in boro rice cultivars of Assam, India using candidate gene based SSR markers

Assessment of the genetic variability and population structure in boro rice cultivars of Assam, India using candidate gene based SSR markers

Development and validation of heat-responsive candidate gene and miRNA gene based SSR markers to analysis genetic diversity in wheat for heat tolerance breeding

Being a major staple food crop of the world, wheat provides nutritional food security to the global populations. Heat stress is a major abiotic stress that adversely affects wheat production throughout the world including Indo-Gangatic Plains (IGP) where four wheat growing countries viz., India, Bangladesh, Nepal and Pakistan produce 42% of the total wheat production. Therefore, identification of heat stress responsive molecular markers is imperative to marker assisted breeding programs. Information about trait specific gene based SSRs is available but there is lack of information on SSRs from non-coding regions. In the present study, we developed 177 heat-responsive gene-based SSRs (cg-SSR) and MIRgene-based SSR (miRNA-SSR) markers from wheat genome for assessing genetic diversity analysis of thirty- six contrasting wheat genotypes for heat tolerance. Of the 177 SSR loci, 144 yielded unambiguous and repeatable amplicons, however, thirty-seven were found polymorphic among the 36 wheat genotypes. The polymorphism information content (PIC) of primers used in this study ranged from 0.03-0.73, with a mean of 0.35. Number of alleles produced per primer varied from 2 to 6, with a mean of 2.58. The UPGMA dendrogram analysis grouped allwheat genotypes into four clusters. The markers developed in this study has potential application in the MAS based breeding programs for developing heat tolerant wheat cultivars and genetic diversity analysis of wheat germplasm. Identification of noncoding region based SSRs will be fruitful for identification of trait specific wheat germplasm.

Development of SSR markers based on transcriptome data and association mapping analysis for fruit shell thickness associated traits in oil palm (Elaeis guineensis Jacq.).

Present study mainly aimed to ascertain the distribution characteristics of gene-based microsatellite loci and to develop polymorphic SSR markers from the already available transcriptome data of Elaeis guineensis Jacq, an important oil crop. From this study, we identified the sum of 5791 SSRs across 51,425 unigenes from the transcripts of oil palm. We were able to evaluate 331primer pairs and characterized 183 polymorphic gene-based SSR markers. We identified a total of 506 alleles from the 183 polymorphic SSR loci, with an average of 2.77 alleles per locus. The characterized gene-based SSR markers from the transcriptome data of oil palm exhibited moderate levels of polymorphism with a significant level of heterozygosity ranges from 0.096 to 0.594 (mean = 0.336 ± 0.11). Among the identified SSR markers, sixty polymorphic markers were used to analyze genotypes of 55 oil palm accessions selected from three different provinces of China. Association mapping analysis provided the information of four markers that are associated with fruit shell thickness trait of oil palm. Among the four markers identified from association analysis, one SSR marker obtained from Unigene17150 is strictly associated with the oil palm fruit shell thickness trait.

Identification, analysis and development of salt responsive candidate gene based SSR markers in wheat

BackgroundSalinity severely limits wheat production in many parts of the world. Development of salt tolerant varieties represents the most practical option for enhancing wheat production from these areas. Application of marker assisted selection may assist in fast tracking development of salt tolerant wheat varieties. However, SSR markers available in the public domain are not specifically targeted to functional regions of wheat genome, therefore large numbers of these need to be analysed for identification of markers associated with traits of interest. With the availability of a fully annotated wheat genome assembly, it is possible to develop SSR markers specifically targeted to genic regions. We performed extensive analysis to identify candidate gene based SSRs and assessed their utility in characterizing molecular diversity in a panel of wheat genotypes.ResultsOur analysis revealed, 161 SSR motifs in 94 salt tolerance candidate genes of wheat. These SSR motifs were nearly equally distributed on the three wheat sub-genomes; 29.8% in A, 35.7% in B and 34.4% in D sub-genome. The maximum number of SSR motifs was present in exons (31.1%) followed by promoters (29.8%), 5’UTRs (21.1%), introns (14.3%) and 3’UTRs (3.7%). Out of the 65 candidate gene based SSR markers selected for validation, 30 were found polymorphic based on initial screening and employed for characterizing genetic diversity in a panel of wheat genotypes including salt tolerant and susceptible lines. These markers generated an average of 2.83 alleles/locus. Phylogenetic analysis revealed four clusters. Salt susceptible genotypes were mainly represented in clusters I and III, whereas high and moderate salt tolerant genotypes were distributed in the remaining two clusters. Population structure analysis revealed two sub-populations, sub-population 1 contained the majority of salt tolerant whereas sub-population 2 contained majority of susceptible genotypes. Moreover, we observed reasonably higher transferability of SSR markers to related wheat species.ConclusionWe have developed salt responsive gene based SSRs in wheat for the first time. These were highly useful in unravelling functional diversity among wheat genotypes with varying responses to salt stress. The identified gene based SSR markers will be valuable genomic resources for genetic/association mapping of salinity tolerance in wheat.

Candidate gene based characterization of common bean genotypes

A set of 96 common bean genotypes were characterized using 25 candidate gene based SSR markers associated with yield traits. Twenty three SSR markers were found polymorphic and their discriminatory power estimation was performed on the basis of PIC, resolving power and marker index that mainly explain ability of molecular markers to distinguish genotypes. An average of 0.33 PIC was observed where highest value was exhibited by the primer BM184. The primers BM170 and Bmd8 generated four alleles whereas BM170 showed maximum marker index of 1.28 and highest resolving power of 1.89 was found in case of primer BM180. Further, the genotypes were grouped in three major clusters based on DARwin5 software. High level of genetic diversity observed within the genotypes could be used to accelerate genetic improvement of germplasm in common bean targeting yield attributing traits.

Insights into Musa balbisiana and Musa acuminata species divergence and development of genic microsatellites by transcriptomics approach

The banana species, Musa balbisiana Colla, and Musa acuminata are progenitors of cultivated banana and belong to the family Musaceae. M. balbisiana, a wild banana species harbors many useful important traits such as disease resistance and abiotic stress tolerance and represents an important genetic resource for banana improvement programs. Therefore, in our study, we for the first time have produced transcriptome data from 12 different tissues of Musa balbisiana collection ‘Bee Hee Kela’ and Musa acuminata ssp. burmannicoides — ‘Calcutta-4’ separately using Illumina GA II X technology. We have done a comparative analysis of the transcriptome of these two species. The high quality reads were assembled and mapped to the DH-Pahang reference genome. A total of 9857 sequences from BB genome and a total of 4424 sequences from the AA genome had SSRs and validation was done for a few selected SSRs. We have analyzed effective number of codons, calculated Ka/Ks ratio, and overall GC and GC3 contents in the two species. In the present study a large number of gene based SSR markers developed for both Musa species will facilitate molecular marker breeding strategies, development of a genetic linkage map and QTL analysis.

Exploiting transcriptome data for the development and characterization of gene-based SSR markers related to cold tolerance in oil palm (Elaeis guineensis).

BackgroundThe oil palm (Elaeis guineensis, 2n = 32) has the highest oil yield of any crop species, as well as comprising the richest dietary source of provitamin A. For the tropical species, the best mean growth temperature is about 27°C, with a minimal growth temperature of 15°C. Hence, the plantation area is limited into the geographical ranges of 10°N to 10°S. Enhancing cold tolerance capability will increase the total cultivation area and subsequently oil productivity of this tropical species. Developing molecular markers related to cold tolerance would be helpful for molecular breeding of cold tolerant Elaeis guineensis.ResultsIn total, 5791 gene-based SSRs were identified in 51,452 expressed sequences from Elaeis guineensis transcriptome data: approximately one SSR was detected per 10 expressed sequences. Of these 5791 gene-based SSRs, 916 were derived from expressed sequences up- or down-regulated at least two-fold in response to cold stress. A total of 182 polymorphic markers were developed and characterized from 442 primer pairs flanking these cold-responsive SSR repeats. The polymorphic information content (PIC) of these polymorphic SSR markers across 24 lines of Elaeis guineensis varied from 0.08 to 0.65 (mean = 0.31 ± 0.12). Using in-silico mapping, 137 (75.3%) of the 182 polymorphic SSR markers were located onto the 16 Elaeis guineensis chromosomes. Total coverage of 473 Mbp was achieved, with an average physical distance of 3.4 Mbp between adjacent markers (range 96 bp - 20.8 Mbp). Meanwhile, Comparative analysis of transcriptome under cold stress revealed that one ICE1 putative ortholog, five CBF putative orthologs, 19 NAC transcription factors and four cold-induced orhologs were up-regulated at least two fold in response to cold stress. Interestingly, 5′ untranslated region of both Unigene21287 (ICE1) and CL2628.Contig1 (NAC) both contained an SSR markers.ConclusionsIn the present study, a series of SSR markers were developed based on sequences differentially expressed in response to cold stress. These EST-SSR markers would be particularly useful for gene mapping and population structure analysis in Elaeis guineensis. Meanwhile, the EST-SSR loci were inducible expressed in response to low temperature, which may have potential application in identifying trait-associated markers in oil palm in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0384-2) contains supplementary material, which is available to authorized users.

Development of an expressed gene catalogue and molecular markers from the de novo assembly of short sequence reads of the lentil (Lens culinaris Medik.) transcriptome

Genomic resources such as ESTs, molecular markers and linkage maps are essential for crop improvement. However, these resources are still limited in important legumes such as lentil (Lens culinaris Medik.), which is valued world wide as a rich source of dietary protein. In this study, the de novo transcriptome assembly of 119,855,798 short reads, generated by Illumina paired-end sequencing, was performed using various assembly programs. This resulted in 42,196 nonredundant high-quality transcripts of average length 810 bases, N50 value of 1,432 and an average expression per transcript of 26.21 rpkm reads per kilobase per million(RPKM). Similarity search with the unigenes and protein sequences of other plants resulted in maximum similarity with soybean. A total of 20,009 nonredundant transcripts showed similarity with the UniProtKB database and of these, 18,064 transcripts were grouped into three main GO categories, that is, biological process (15,126), molecular function (15,505) and cellular component (9,434). Annotated transcripts were mapped to 289 predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 8,893 transcripts were classified into 24 functional categories based on Cluster of Orthologous Groups (COG) of proteins. Mining the data set for the presence of SSRs resulted in 8,722 SSRs with a frequency occurrence of one SSR per 3.92 kb. From these, 5,673 SSR primer pairs were designed, and a subset of these were utilized for diversity analysis. This study, which provides a large data set of annotated transcripts and gene-based SSR markers, would serve as a foundation for various applications in lentil breeding and genetics.

Identification of candidate gene–based SSR markers for lysine and tryptophan metabolic pathways in maize (Zea mays)

With 3 figures and 5 tablesAbstractIdentification of candidate gene–based SSR markers is extremely useful in genetic enhancement of crop plants. A total of twenty‐four SSR loci were developed for the candidate genes of lysine and tryptophan metabolic pathways and for opaque2 (o2) modifiers. Of the 24 SSR loci, 23 yielded amplicons of expected size; however, fifteen were found polymorphic among the 22 maize genotypes including normal and quality protein maize inbreds from India and exotic inbreds from CIMMYT, Mexico. The polymorphism information content of primers was in the range of 0.16–0.72, with a mean of 0.49. The gene diversity ranged from 0.17 to 0.75, and the inbreeding coefficient was in the range of 0.48–1.0. The candidate genes of lysine metabolism showed higher polymorphism levels and genetic diversity than other candidate genes. The SSR loci developed from the candidate genes of aspartate kinase 2 and homoserine dehydrogenase were found to have high potential. Thus, the SSR loci identified in this study added useful markers for fine mapping and high‐density mapping of opaque2 modifiers.