Development Of Gene-based Markers Research Articles

Development of Gene-based Markers for capsanthin-capsorubin synthase and zeaxanthin epoxidase Genes to Distinguish Fruit Colors in Paprika (Capsicum annuum L.)

Development of Gene-based Markers for capsanthin-capsorubin synthase and zeaxanthin epoxidase Genes to Distinguish Fruit Colors in Paprika (Capsicum annuum L.)

Transcriptome analysis of two recombinant inbred lines of common bean contrasting for symbiotic nitrogen fixation.

Common bean (Phaseolus vulgaris L.) fixes atmospheric nitrogen (N2) through symbiotic nitrogen fixation (SNF) at levels lower than other grain legume crops. An understanding of the genes and molecular mechanisms underlying SNF will enable more effective strategies for the genetic improvement of SNF traits in common bean. In this study, transcriptome profiling was used to identify genes and molecular mechanisms underlying SNF differences between two common bean recombinant inbred lines that differed in their N-fixing abilities. Differential gene expression and functional enrichment analyses were performed on leaves, nodules and roots of the two lines when grown under N-fixing and non-fixing conditions. Receptor kinases, transmembrane transporters, and transcription factors were among the differentially expressed genes identified under N-fixing conditions, but not under non-fixing conditions. Genes up-regulated in the stronger nitrogen fixer, SA36, included those involved in molecular functions such as purine nucleoside binding, oxidoreductase and transmembrane receptor activities in nodules, and transport activity in roots. Transcription factors identified in this study are candidates for future work aimed at understanding the functional role of these genes in SNF. Information generated in this study will support the development of gene-based markers to accelerate genetic improvement of SNF in common bean.

Generation and Characterisation of a Reference Transcriptome for Lentil (Lens culinaris Medik.).

RNA-Seq using second-generation sequencing technologies permits generation of a reference unigene set for a given species, in the absence of a well-annotated genome sequence, supporting functional genomics studies, gene characterisation and detailed expression analysis for specific morphophysiological or environmental stress response traits. A reference unigene set for lentil has been developed, consisting of 58,986 contigs and scaffolds with an N50 length of 1719 bp. Comparison to gene complements from related species, reference protein databases, previously published lentil transcriptomes and a draft genome sequence validated the current dataset in terms of degree of completeness and utility. A large proportion (98%) of unigenes were expressed in more than one tissue, at varying levels. Candidate genes associated with mechanisms of tolerance to both boron toxicity and time of flowering were identified, which can eventually be used for the development of gene-based markers. This study has provided a comprehensive, assembled and annotated reference gene set for lentil that can be used for multiple applications, permitting identification of genes for pathway-specific expression analysis, genetic modification approaches, development of resources for genotypic analysis, and assistance in the annotation of a future lentil genome sequence.

Development of gene-based markers for use in construction of the chickpea (Cicer arietinum L.) genetic linkage map and identification of QTLs associated with seed weight and plant height.

Seed weight and plant height are important agronomic traits and contribute to seed yield. The objective of this study was to identify QTLs underlying these traits using an intra-specific mapping population of chickpea. A F11 population of 177 recombinant inbred lines derived from a cross between SBD377 (100-seed weight--48 g and plant height--53 cm) and BGD112 (100-seed weight--15 g and plant height--65 cm) was used. A total of 367 novel EST-derived functional markers were developed which included 187 EST-SSRs, 130 potential intron polymorphisms (PIPs) and 50 expressed sequence tag polymorphisms (ESTPs). Along with these, 590 previously published markers including 385 EST-based markers and 205 genomic SSRs were utilized. Of the 957 markers tested for analysis of parental polymorphism between the two parents of the mapping population, 135 (14.64%) were found to be polymorphic. Of these, 131 polymorphic markers could be mapped to the 8 linkage groups. The linkage map had a total length of 1140.54 cM with an average marker density of 8.7 cM. The map was further used for QTL identification using composite interval mapping method (CIM). Two QTLs each for seed weight, qSW-1 and qSW-2 (explaining 11.54 and 19.24% of phenotypic variance, respectively) and plant height, qPH-1 and qPH-2 (explaining 13.98 and 12.17% of phenotypic variance, respectively) were detected. The novel set of genic markers, the intra-specific linkage map and the QTLs identified in the present study will serve as valuable genomic resources in improving the chickpea seed yield using marker-assisted selection (MAS) strategies.

Development of gene-based markers for the identification of wheat-rye translocations possessing 1RS

Wheat-rye translocations carry valuable genes for pest resistance; this has led to their incorporation into wheat varieties. To develop gene-based 1RS specific markers, expressed sequence tags (ESTs) mapped to the short arm of wheat homoeologous group 1 were downloaded from a public database. ESTs and mature transcript sequences of nine species from the Poaceae family were BLASTed to perform comparative analysis. ESTs selected using the BLAST tool were used for primer design. The target sequences of primers were expected to exist in most species of the Poaceae family, and therefore, cross-species primers were designed. Amplicons obtained using these cross-species primer pairs were sequenced in the 1BL.1RS near-isolines (‘OK91G113’ [1BL.1RS] and ‘OK91G114’ [non-translocation]), ‘Chinese Spring’ and ‘Petkus’ rye (1RS donor for 1BL.1RS translocations). Through in silico analysis and primer validation, we were able to develop four 1RS-specific markers and these markers were confirmed with analysis involving 15 1RS translocation lines.

Development of gene based markers for mapping analysis in oil palm

Gene based SNP markers for oil palm were developed from cloned candidate genes that are involved in fatty acid (FA), triacylglycerol and pyruvate-acetyl CoA synthesis pathways of oil palm. Based on exon/introns splice junctions, a total of 28 specific primer pairs corresponding to 22 gene loci were designed and tested for polymorphism between 23 parents (13 dura and 10 pisifera ) used in an oil palm breeding programme. Single strand conformation polymorphism polyacrylamide gel electrophoresis (SSCP) was used to detect the polymorphism. More than 80% of the gene loci showed clear polymorphic patterns in target parents which progenies of at least 3, up to all 14 crosses would display segregation for the markers. The gene based SNP markers developed in this study do not only provide potential marker tools for further QTL mapping analysis in multiple crosses but can also be used as anchor points for further comparing and integrating oil palm linkage maps developed from individual progenies.

Molecular physiology and breeding at the crossroads of cold hardiness improvement

Alfalfa (Medicago sativa L.) is a major forage legume grown extensively worldwide with important agronomic and environmental attributes. Insufficient cold hardiness is a major impediment to its reliable production in northern climates. Improvement of freezing tolerance using conventional breeding approaches is slowed by the quantitative nature of inheritance and strong interactions with the environment. The development of gene-based markers would facilitate the identification of genotypes with superior stress tolerance. Successive cycles of recurrent selection were applied using an indoor screening method to develop populations with significantly higher tolerance to freezing (TF). Bulk segregant analysis of heterogeneous TF populations identified DNA variations that are progressively enriched in frequency in response to selection. Polymorphisms resulting from intragenic variations within a dehydrin gene were identified and could potentially lead to the development of robust selection tools. Our results illustrate the benefits of feedback interactions between germplasm development programs and molecular physiology for a deeper understanding of the molecular and genetic bases of cold hardiness.

Development of gene-based markers and construction of an integrated linkage map in eggplant by using Solanum orthologous (SOL) gene sets

We constructed an integrated DNA marker linkage map of eggplant (Solanum melongena L.) using DNA marker segregation data sets obtained from two independent intraspecific F(2) populations. The linkage map consisted of 12 linkage groups and encompassed 1,285.5 cM in total. We mapped 952 DNA markers, including 313 genomic SSR markers developed by random sequencing of simple sequence repeat (SSR)-enriched genomic libraries, and 623 single-nucleotide polymorphisms (SNP) and insertion/deletion polymorphisms (InDels) found in eggplant-expressed sequence tags (ESTs) and related genomic sequences [introns and untranslated regions (UTRs)]. Because of their co-dominant inheritance and their highly polymorphic and multi-allelic nature, the SSR markers may be more versatile than the SNP and InDel markers for map-based genetic analysis of any traits of interest using segregating populations derived from any intraspecific crosses of practical breeding materials. However, we found that the distribution of microsatellites in the genome was biased to some extent, and therefore a considerable part of the eggplant genome was first detected when gene-derived SNP and InDel markers were mapped. Of the 623 SNP and InDel markers mapped onto the eggplant integrated map, 469 were derived from eggplant unigenes contained within Solanum orthologous (SOL) gene sets (i.e., sets of orthologous unigenes from eggplant, tomato, and potato). Out of the 469 markers, 326 could also be mapped onto the tomato map. These common markers will be informative landmarks for the transfer of tomato's more saturated genomic information to eggplant and will also provide comparative information on the genome organization of the two solanaceous species. The data are available from the DNA marker database of vegetables, VegMarks (http://vegmarks.nivot.affrc.go.jp).

Development of gene-based markers from functional Arabidopsis thaliana genes involved in phosphorus homeostasis and mapping in Brassica napus

Brassica napus is an important oilseed and fodder crop grown throughout the world. Although it is widely grown, little is known about the molecular basis of phosphorus (P) homeostasis for this species. In this research, a population of 124 recombinant inbred lines (RILs) (designated as BE-RIL) derived from a cross between P-inefficient cv. ‘B104-2’ and P-efficient cv. ‘Eyou Changjia’ was used to construct a genetic map of P homeostasis genes. A set of gene-based markers (GBMs) was developed from functional genes involved in Arabidopsis thaliana P homeostasis. In total, 46 GBMs corresponding to 26 genes, assigned to eight functional categories, were integrated into the BE-RIL map. A total of 243 simple sequence repeat (SSR) markers were developed from 171 bacterial artificial chromosome (BAC) end sequences and/or B. rapa seed BAC sequences. Of these SSR markers, 74 were added to the BE-RIL map. Based on the newly constructed genetic map, comparative genetic analysis between A. thaliana and B. napus was performed. A total of 90 conserved genomic blocks were aligned between A. thaliana pseudochromosomes and the BE-RIL linkage groups. According to physical positions on the Arabidopsis genome, 1223 orthologs of 356 genes involved in Arabidopsis P homeostasis were mapped onto syntenic blocks and insertion segments. This high-density genetic map will be useful for identifying quantitative trait loci (QTL) that control P homeostasis and putative candidate genes for the efficient use of P in B. napus.

Development of gene-based markers for the Bs2 bacterial spot resistance gene for marker-assisted selection in pepper (Capsicum spp.)

The Bs2 gene of pepper (Capsicum spp.) confers resistance to the most commonly detected races of Xanthomonas campestris pv vesicatora (Xcv). Using information from a published database, we developed gene-based markers for selection of Bs2 in pepper. High polymorphism was detected in exon 3, intron 2 and 3’-UTR of the Bs2 gene. The three regions were sequenced and numerous SNPs and In/Dels were identified between resistant and susceptible inbred lines. Seven SNPs were selected to develop SNP markers using the tetra-primer amplification refractory mutation system-PCR methodology (Tetra-primer ARMS-PCR). One SNP (G/C) was used to develop a molecular marker that can perfectly identify the presence of the bacterial spot resistance. The gene-based markers were further validated to test association with resistance in 80 lines and cultivars. The markers successfully identified a bell pepper commercial cultivar 62-2 that harbors the Bs2 gene. The successful development of the markers in this study provides a useful tool for MAS to screen the Bs2 gene in pepper.

Identification of a deletion in tms2 and development of gene-based markers for selection

Rice is one of the most important food crops. The temperature-sensitive genic male sterility (TGMS) system provides a great potential for improving food production by hybrids. The use of TGMS system is simple, inexpensive, effective, and eliminates the limitations of the conventional three-line system. A rice gene, tms2, generated by irradiation of a japonica variety has been reported to control TGMS in several rice lines. Previous studies reported genetic markers linked to this gene, and the gene was transferred to an aromatic Thai cultivar. Using information obtained from published databases, we located positions of the reported genetic markers flanking the gene in rice genomic sequences, and developed gene-based markers located inside the flanking markers for polymorphism detection. We found that inbred indica tms2 mutant plants contain about 1 Mb of japonica DNA, in which at least 70 kb was deleted. Using RT-PCR for expression analysis, four genes out of seven genes annotated as expressed proteins located inside the deletion showed expression in panicles. These genes could be responsible for TGMS phenotypes of tms2. In addition, we developed gene-based markers flanking and inside the deletion for selecting the tms2 gene in breeding populations. By genotyping 102 diverse rice lines including 38 Thai rice lines, 5 species of wild rice, and 59 exotic rice lines including TGMS lines and cultivars with desirable traits, a gene-based marker located inside the deletion and one flanking marker were shown to be highly specific for the tms2 mutant.

Development of ESTs from chickpea roots and their use in diversity analysis of the Cicer genus

BackgroundChickpea is a major crop in many drier regions of the world where it is an important protein-rich food and an increasingly valuable traded commodity. The wild annual Cicer species are known to possess unique sources of resistance to pests and diseases, and tolerance to environmental stresses. However, there has been limited utilization of these wild species by chickpea breeding programs due to interspecific crossing barriers and deleterious linkage drag. Molecular genetic diversity analysis may help predict which accessions are most likely to produce fertile progeny when crossed with chickpea cultivars. While, trait-markers may provide an effective tool for breaking linkage drag. Although SSR markers are the assay of choice for marker-assisted selection of specific traits in conventional breeding populations, they may not provide reliable estimates of interspecific diversity, and may lose selective power in backcross programs based on interspecific introgressions. Thus, we have pursued the development of gene-based markers to resolve these problems and to provide candidate gene markers for QTL mapping of important agronomic traits.ResultsAn EST library was constructed after subtractive suppressive hybridization (SSH) of root tissue from two very closely related chickpea genotypes (Cicer arietinum). A total of 106 EST-based markers were designed from 477 sequences with functional annotations and these were tested on C. arietinum. Forty-four EST markers were polymorphic when screened across nine Cicer species (including the cultigen). Parsimony and PCoA analysis of the resultant EST-marker dataset indicated that most accessions cluster in accordance with the previously defined classification of primary (C. arietinum, C. echinospermum and C. reticulatum), secondary (C. pinnatifidum, C. bijugum and C. judaicum), and tertiary (C. yamashitae, C. chrossanicum and C. cuneatum) gene-pools. A large proportion of EST alleles (45%) were only present in one or two of the accessions tested whilst the others were represented in up to twelve of the accessions tested.ConclusionGene-based markers have proven to be effective tools for diversity analysis in Cicer and EST diversity analysis may be useful in identifying promising candidates for interspecific hybridization programs. The EST markers generated in this study have detected high levels of polymorphism amongst both common and rare alleles. This suggests that they would be useful for allele-mining of germplasm collections for identification of candidate accessions in the search for new sources of resistance to pests / diseases, and tolerance to abiotic stresses.