Search

Key message We identified markers associated with GRD resistance after screening an Africa-wide core collection across three seasons in Uganda Abstract Groundnut is cultivated in several African countries where it is a major source of food, feed and income. One of the major constraints to groundnut production in Africa is groundnut rosette disease (GRD), which is caused by a complex of three agents: groundnut rosette assistor luteovirus, groundnut rosette umbravirus and its satellite RNA. Despite several years of breeding for GRD resistance, the genetics of the disease is not fully understood. The objective of the current study was to use the African core collection to establish the level of genetic variation in their response to GRD, and to map genomic regions responsible for the observed resistance. The African groundnut core genotypes were screened across two GRD hotspot locations in Uganda (Nakabango and Serere) for 3 seasons. The Area Under Disease Progress Curve combined with 7523 high quality SNPs were analyzed to establish marker-trait associations (MTAs). Genome-Wide Association Studies based on Enriched Compressed Mixed Linear Model detected 32 MTAs at Nakabango: 21 on chromosome A04, 10 on B04 and 1 on B08. Two of the significant markers were localised on the exons of a putative TIR-NBS-LRR disease resistance gene on chromosome A04. Our results suggest the likely involvement of major genes in the resistance to GRD but will need to be further validated with more comprehensive phenotypic and genotypic datasets. The markers identified in the current study will be developed into routine assays and validated for future genomics-assisted selection for GRD resistance in groundnut.

Groundnut (Arachis hypogaea L.) is an important food and cash crop grown mainly in semi-arid tropics (SAT) regions of the world where drought is the major constraint on productivity. With the aim of understanding the genetic basis and identification of quantitative trait loci (QTL) for drought tolerance, two new recombinant inbred line (RIL) mapping populations, namely ICGS 76 × CSMG 84-1 (RIL-2) and ICGS 44 × ICGS 76 (RIL-3), were used. After screening of 3,215 simple sequence repeat (SSR) markers on the parental genotypes of these populations, two new genetic maps were developed with 119 (RIL-2) and 82 (RIL-3) SSR loci. Together with these maps and the reference map with 191 SSR loci based on TAG 24 × ICGV 86031 (RIL-1), a consensus map was constructed with 293 SSR loci distributed over 20 linkage groups, spanning 2,840.8 cM. As all these three populations segregate for drought-tolerance-related traits, a comprehensive QTL analysis identified 153 main effect QTL (M-QTL) and 25 epistatic QTL (E-QTL) for drought-tolerance-related traits. Localization of these QTL on the consensus map provided 16 genomic regions that contained 125 QTL. A few key genomic regions were selected on the basis of the QTL identified in each region, and their expected role in drought adaptation is also discussed. Given that no major QTL for drought adaptation were identified, novel breeding approaches such as marker-assisted recurrent selection (MARS) and genomic selection (GS) approaches are likely to be the preferred approaches for introgression of a larger number of QTL in order to breed drought-tolerant groundnut genotypes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9660-0) contains supplementary material, which is available to authorized users.

A chickpea simple sequence repeat (SSR) marker reference kit has been developed based on the genotyping of the global chickpea composite collection (3,000 accessions) with 35 SSR markers. The kit consists of three pools of chickpea accessions along with supporting documentation on the SSR markers, polymerase chain reaction and detection conditions, and the expected allele sizes for each of the 35 SSR loci. These markers were selected based on quality criteria, genome coverage and locus-specific information content. Other important SSR selection criteria were quality of amplification products, locus complexity, polymorphism information content and well-dispersed location on a chickpea genetic map. The developed SSR kit has a wide range of applications, especially for genetic diversity studies in chickpea. Using the markers and reference accessions in the kit, scientists in other laboratories will be able to compare the genotypic data that they obtain for their germplasm with that obtained using the global composite collection.

With an objective to expand the repertoire of molecular markers in pigeonpea (Cajanus cajan), 36 microsatellite or simple sequence repeat (SSR) loci were isolated from a SSR-enriched genomic library. Primer pairs were designed for 23 SSR loci, of which 16 yielded amplicons of expected size. Thirteen SSR markers were polymorphic amongst 32 cultivated and eight wild pigeonpea genotypes representing six Cajanus species. These markers amplified a total of 72 alleles ranging from two to eight alleles with an average of 5.5 alleles per locus. The polymorphic information content for these markers ranged from 0.05 to 0.55 with an average of 0.32 per marker. Phenetic analysis clearly distinguished all wild species genotypes from each other and from the cultivated pigeonpea genotypes. These markers should be useful for genome mapping, trait mapping, diversity studies and assessment of gene flow between populations in pigeonpea.

In order to maximize polymorphism in the mapping populations for mapping loci for Fusarium wilt (FW) and sterility mosaic disease (SMD) resistance in pigeonpea, a set of 32 pigeonpea lines were screened for polymorphism with 30 microsatellite or simple sequence repeat markers. A total of 23 marker loci showed polymorphism with 2–4 alleles and the polymorphism information content for these markers ranged from 0.12 to 0.65 with an average of 0.43 per marker. High number of polymorphic markers, higher genetic dissimilarity coefficient and contrasting phenotypic data taken into consideration and five parental combinations were identified and crosses initiated for developing five genetically diverse mapping populations. Of these crosses, one cross segregates for FW resistance, two for SMD resistance and the remaining two crosses segregate for resistance to both FW and SMD. Development of mapping populations is in progress for mapping loci for resistance to FW and SMD in pigeonpea.

A set of 22 pearl millet inbred lines including the parents of eleven mapping populations, was screened with 627 markers including 100 pearl millet genomic SSRs (gSSRs), 60 pearl millet EST-SSRs (eSSRs), 410 intron sequence haplotypes (ISHs), and 57 exon sequence haplotypes (ESHs). In all, 267 (59%) of the markers were informative for at least one of the 11 mapping populations, which segregate for traits like drought and salinity tolerance; host plant resistance to downy mildew, rust and blast; fertility restoration and sterility and maintenance of cytoplasmic male sterility etc. An average of 116 polymorphic markers was identified per mapping population. The average PIC values and number of profiles (P) per polymorphic marker were: gSSRs (PIC = 0.62, P = 6.1), ISHs (PIC = 0.39, P = 2.6), eSSRs (PIC = 0.36, P = 3.1) and ESHs (PIC = 0.35, P = 3.1). A high correlation (r > 0.97, P

Contact: fax: +91 40 30713074/30713075. E-mail address: r.k.varshney@cgiar.org; International audience; The transcription factor, DREB2A, is one of the promising candidate genes involved in dehydration tolerance in crop plants. In order to isolate DREB2A homologues across cereals (rice, barley and sorghum) and legumes (common bean and chickpea), specific or degenerate primers were used. Gene/phylogenetic trees were constructed using a non-redundant set of 19 DREB1A and 27 DREB2A amino acid sequences and were combined with taxonomic/species tree to prepare reconciled phylogenetic trees. In total, 86 degenerate primers were designed for different clades and 295 degenerate primer combinations were used to amplify DREB homologues in targeted crop species. Successful amplification of DREB2A was obtained in case of sorghum. In parallel, gene-specific primers were used to amplify DREB2A homologues in rice, barley, common bean and chickpea. Seven to eight diverse genotypes from targeted species were used for sequence analysis at DREB2A locus identified/isolated. A maximum of eight SNPs were found in the common bean DREB2A, indicating two distinct haplotypes, three SNPs with five haplotypes were observed in barley whereas a single SNP was observed in rice, sorghum and chickpea. Parsimony based phylogenetic tree revealed distinct clustering of cereals and legumes. Furthermore, alignment of corresponding amino acid sequences showed conservation of AP2 domain across the targeted species.

The ability to identify genetic variation is indispensable for effective management and use of genetic resources in crop breeding. Genetic variation among 189 groundnut (Arachis hypogaea L.) accessions comprising landraces, cultivars, a mutant, advanced breeding lines and others (unknown genetic background) representing 29 countries and 10 geographical regions was assessed at 25 microsatellite or simple sequence repeat loci. A high number of alleles (265) were detected in the range of 3 (Ah1TC6G09) to 20 (Ah1TC11H06) with an average of 10.6 alleles per locus. The polymorphism information content value at these loci varied from 0.38 (Ah1TC6G09) to 0.88 (Ah1TC11H06) with an average of 0.70. A total of 59 unique alleles and 127 rare alleles were detected at almost all the loci assayed. Cluster analysis grouped 189 accessions into four clusters. In general, genotypes of South America and South Asia showed high level of diversity. Extraordinary level of natural genetic variation reported here provides opportunities to the groundnut community to make better decisions and define suitable strategies for harnessing the genetic variation in groundnut breeding.

The International Maize and Wheat Improvement Center (CIMMYT) aims to genetically enhance both crops and generate public sector-provided products for the resource poor, e.g., drought tolerant wheat and insect resistant maize, and through international-national partnerships facilitate the acquisition of improved germplasm for non-mandate crops in the cropping systems where maize and wheat thrives; e.g., GM-papaya through a national food security undertaking in Bangladesh. The Center also engages in public awareness campaigns in projects such as Insect Resistance Maize for Africa (IRMA), which includes food, feed and environmental safety, monitoring of resistance and establishment of refugia, non-target effects and gene flow. Monitoring of genetic resources is a wide concern among the centers of the Consultative Group on International Agricultural Research (CGIAR), with an emphasis on the quality of gene banks. Decisions, policies and procedures about monitoring should be science-based, and this requires education, an area where CIMMYT and other CGIAR centers can play an important role. There will be a need to continue to evaluate the need for, and type of monitoring, as new (and unique) products are developed and released in the emergent economies of the world.

Pigeonpea is a major legume of the semi-arid tropics that has been neglected in terms of molecular breeding. The objectives of this study were to develop microsatellite markers and evaluate their potential for use in pigeonpea genetics and breeding. Two hundred and eight microsatellite loci were isolated by screening a non-enriched partial genomic library. Primers were designed for 39 microsatellite loci, 20 of which amplified polymerase chain reaction products of the expected size. Nineteen of the primer pairs were polymorphic amongst 15 cultivated and nine wild pigeonpea accessions providing evidence for cross-species transferability within the genus Cajanus. A total of 98 alleles were detected at the 19 polymorphic loci with an average of 4.9 alleles per locus. The observed heterozygosity ranged from 0.17 to 0.80 with a mean of 0.42 per locus. Less allelic variation (31 alleles) was observed within the cultivated species than across the wild species (92 alleles). The diversity analysis readily distinguished all wild relatives from each other and from the cultivated germplasm. Development of more microsatellites is recommended for future genomic studies in pigeonpea.

Recent advances in cereal genomics have made it possible to analyse the architecture of cereal genomes and their expressed components, leading to an increase in our knowledge of the genes that are linked to key agronomically important traits. These studies have used molecular genetic mapping of quantitative trait loci (QTL) of several complex traits that are important in breeding. The identification and molecular cloning of genes underlying QTLs offers the possibility to examine the naturally occurring allelic variation for respective complex traits. Novel alleles, identified by functional genomics or haplotype analysis, can enrich the genetic basis of cultivated crops to improve productivity. Advances made in cereal genomics research in recent years thus offer the opportunities to enhance the prediction of phenotypes from genotypes for cereal breeding.

Leaf and stripe rusts, caused by Puccinia triticina and P. striiformis, respectively, are globally important fungal diseases of wheat that cause significant annual yield losses. A gene that confers slow rusting resistance to leaf rust, designated as Lr46, has recently been located on wheat chromosome 1B. The objectives of our study were to establish the precise genomic location of gene Lr46 using molecular approaches and to determine if there was an association of this locus with adult plant resistance to stripe rust. A population of 146 F5 and F6 lines produced from the cross of susceptible ‘Avocet S’ with resistant ‘Pavon 76’ was developed and classified for leaf rust and stripe rust severity for three seasons. Using patterns of segregation for the two diseases, we estimated that at least two genes with additive effects conferred resistance to leaf rust and three to four genes conferred resistance to stripe rust. Bulked segregant analysis and linkage mapping using amplified fragment length polymorphisms with the ‘Avocet’ × ‘Pavon 76’ population, F3 progeny lines of a single chromosome recombinant line population from the cross ‘Lalbahadur’ × ‘Lalbahadur (Pavon 1B)’, and the International Triticeae Mapping Initiative population established the genomic location of Lr46 at the distal end of the long arm of wheat chromosome 1B. A gene that is closely linked to Lr46 and confers moderate levels of adult plant resistance to stripe rust is identified and designated as Yr29.

Amplified fragment length polymorphism (AFLP) was employed to assess the diversity in the elite germplasm collection of Jatropha curcas, which has gained tremendous significance as a biofuel plant in India and many other countries recently. Forty-eight accessions, collected from six different states of India, were used with seven AFLP primer combinations that generated a total of 770 fragments with an average of 110 fragments per primer combination. A total of 680 (88%) fragments showed polymorphism in the germplasm analyzed, of which 59 (8.7%) fragments were unique (accession specific) and 108 (15.9%) fragments were rare (present in less than 10% accessions). In order to assess the discriminatory power of seven primer combinations used, a variety of marker attributes like polymorphism information content (PIC), marker index (MI) and resolving power (RP) values were calculated. Although the PIC values ranged from 0.20 (E-ACA/M-CAA) to 0.34 (E-ACT/M-CTT) with an average of 0.26 per primer combination and the MI values were observed in the range of 17.60 (E-ACA/M-CAA) to 32.30 (E-ACT/M-CTT) with an average of 25.13 per primer combination, the RP was recognized the real attribute for AFLP to determine the discriminatory power of the primer combination. The RP values for different primer combinations varied from 23.11 (E-ACA/M-CAA) to 46.82 (E-ACT/M-CTT) with an average of 35.21. Genotyping data obtained for all 680 polymorphic fragments were used to group the accessions analyzed using the UPGMA-phenogram and principal component analysis (PCA). Majority of groups obtained in phenogram and PCA contained accessions as per geographical locations. In general, accessions coming from Andhra Pradesh were found diverse as these were scattered in different groups, whereas accessions coming from Chhattisgarh showed occurrence of higher number of unique/rare fragments. Molecular diversity estimated in the present study combined with the datasets on other morphological/agronomic traits will be very useful for selecting the appropriate accessions for plant improvement through conventional as well as molecular breeding approaches.

The large amounts of EST sequence data available from a single species of an organism as well as for several species within a genus provide an easy source of identification of intra- and interspecies single nucleotide polymorphisms (SNPs). In the case of model organisms, the data available are numerous, given the degree of redundancy in the deposited EST data. There are several available bioinformatics tools that can be used to mine this data; however, using them requires a certain level of expertise: the tools have to be used sequentially with accompanying format conversion and steps like clustering and assembly of sequences become time-intensive jobs even for moderately sized datasets. We report here a pipeline of open source software extended to run on multiple CPU architectures that can be used to mine large EST datasets for SNPs and identify restriction sites for assaying the SNPs so that cost-effective CAPS assays can be developed for SNP genotyping in genetics and breeding applications. At the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), the pipeline has been implemented to run on a Paracel high-performance system consisting of four dual AMD Opteron processors running Linux with MPICH. The pipeline can be accessed through user-friendly web interfaces at http://hpc.icrisat.cgiar.org/PBSWeb and is available on request for academic use. We have validated the developed pipeline by mining chickpea ESTs for interspecies SNPs, development of CAPS assays for SNP genotyping, and confirmation of restriction digestion pattern at the sequence level.

Molecular markers and genetic linkage maps are pre-requisites for molecular breeding in any crop species. In case of peanut or groundnut (Arachis hypogaea L.), an amphidiploid (4X) species, not a single genetic map is, however, available based on a mapping population derived from cultivated genotypes. In order to develop a genetic linkage map for tetraploid cultivated groundnut, a total of 1,145 microsatellite or simple sequence repeat (SSR) markers available in public domain as well as unpublished markers from several sources were screened on two genotypes, TAG 24 and ICGV 86031 that are parents of a recombinant inbred line mapping population. As a result, 144 (12.6%) polymorphic markers were identified and these amplified a total of 150 loci. A total of 135 SSR loci could be mapped into 22 linkage groups (LGs). While six LGs had only two SSR loci, the other LGs contained 3 (LG_AhXV) to 15 (LG_AhVIII) loci. As the mapping population used for developing the genetic map segregates for drought tolerance traits, phenotyping data obtained for transpiration, transpiration efficiency, specific leaf area and SPAD chlorophyll meter reading (SCMR) for 2 years were analyzed together with genotyping data. Although, 2-5 QTLs for each trait mentioned above were identified, the phenotypic variation explained by these QTLs was in the range of 3.5-14.1%. In addition, alignment of two linkage groups (LGs) (LG_AhIII and LG_AhVI) of the developed genetic map was shown with available genetic maps of AA diploid genome of groundnut and Lotus and Medicago. The present study reports the construction of the first genetic map for cultivated groundnut and demonstrates its utility for molecular mapping of QTLs controlling drought tolerance related traits as well as establishing relationships with diploid AA genome of groundnut and model legume genome species. Therefore, the map should be useful for the community for a variety of applications.

Background Chickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. However, very limited number of molecular markers and candidate genes are available for undertaking molecular breeding in chickpea to tackle these stresses. This study reports generation and analysis of comprehensive resource of drought- and salinity-responsive expressed sequence tags (ESTs) and gene-based markers. Results A total of 20,162 (18,435 high quality) drought- and salinity- responsive ESTs were generated from ten different root tissue cDNA libraries of chickpea. Sequence editing, clustering and assembly analysis resulted in 6,404 unigenes (1,590 contigs and 4,814 singletons). Functional annotation of unigenes based on BLASTX analysis showed that 46.3% (2,965) had significant similarity (≤1E-05) to sequences in the non-redundant UniProt database. BLASTN analysis of unique sequences with ESTs of four legume species (Medicago, Lotus, soybean and groundnut) and three model plant species (rice, Arabidopsis and poplar) provided insights on conserved genes across legumes as well as novel transcripts for chickpea. Of 2,965 (46.3%) significant unigenes, only 2,071 (32.3%) unigenes could be functionally categorised according to Gene Ontology (GO) descriptions. A total of 2,029 sequences containing 3,728 simple sequence repeats (SSRs) were identified and 177 new EST-SSR markers were developed. Experimental validation of a set of 77 SSR markers on 24 genotypes revealed 230 alleles with an average of 4.6 alleles per marker and average polymorphism information content (PIC) value of 0.43. Besides SSR markers, 21,405 high confidence single nucleotide polymorphisms (SNPs) in 742 contigs (with ≥ 5 ESTs) were also identified. Recognition sites for restriction enzymes were identified for 7,884 SNPs in 240 contigs. Hierarchical clustering of 105 selected contigs provided clues about stress- responsive candidate genes and their expression profile showed predominance in specific stress-challenged libraries. Conclusion Generated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding. Mapping of gene-based markers in chickpea will also add more anchoring points to align genomes of chickpea and other legume species.