David A. Hoisington

AFLP-based molecular characterization of an elite germplasm collection of Jatropha curcas L., a biofuel plant.

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.

A comprehensive resource of drought- and salinity- responsive ESTs for gene discovery and marker development in chickpea (Cicer arietinum L.)

BackgroundChickpea (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.ResultsA 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.ConclusionGenerated 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.

Genetic relationships among seven sections of genus Arachis studied by using SSR markers

BackgroundThe genus Arachis, originated in South America, is divided into nine taxonomical sections comprising of 80 species. Most of the Arachis species are diploids (2n = 2x = 20) and the tetraploid species (2n = 2x = 40) are found in sections Arachis, Extranervosae and Rhizomatosae. Diploid species have great potential to be used as resistance sources for agronomic traits like pests and diseases, drought related traits and different life cycle spans. Understanding of genetic relationships among wild species and between wild and cultivated species will be useful for enhanced utilization of wild species in improving cultivated germplasm. The present study was undertaken to evaluate genetic relationships among species (96 accessions) belonging to seven sections of Arachis by using simple sequence repeat (SSR) markers developed from Arachis hypogaea genomic library and gene sequences from related genera of Arachis.ResultsThe average transferability rate of 101 SSR markers tested to section Arachis and six other sections was 81% and 59% respectively. Five markers (IPAHM 164, IPAHM 165, IPAHM 407a, IPAHM 409, and IPAHM 659) showed 100% transferability. Cluster analysis of allelic data from a subset of 32 SSR markers on 85 wild and 11 cultivated accessions grouped accessions according to their genome composition, sections and species to which they belong. A total of 109 species specific alleles were detected in different wild species, Arachis pusilla exhibited largest number of species specific alleles (15). Based on genetic distance analysis, the A-genome accession ICG 8200 (A. duranensis) and the B-genome accession ICG 8206 (A. ipaënsis) were found most closely related to A. hypogaea.ConclusionA set of cross species and cross section transferable SSR markers has been identified that will be useful for genetic studies of wild species of Arachis, including comparative genome mapping, germplasm analysis, population genetic structure and phylogenetic inferences among species. The present study provides strong support based on both genomic and genic markers, probably for the first time, on relationships of A. monticola and A. hypogaea as well as on the most probable donor of A and B-genomes of cultivated groundnut.

Molecular Genetics and Breeding of Grain Legume Crops for the Semi-Arid Tropics

Grain legumes are important crops for providing key components in the diets of resource-poor people of the semi-arid tropic (SAT) regions of the world. Although there are several grain legume crops grown in SAT, the present chapter deals with three important legumes i.e. groundnut or peanut (Arachis hypogaea), chickpea (Cicer arietinum) and pigeonpea (Cajanus cajan). Production of these legume crops are challenged by serious abiotic stresses e.g. drought, salinity as well as several fungal, viral and nematode diseases. To tackle these constraints through molecular breeding, some efforts have been initiated to develop genomic resources e.g. molecular markers, molecular genetic maps, expressed sequence tags (ESTs), macro-/micro- arrays, bacterial artificial chromosomes (BACs), etc. These genomic resources together with recently developed genetic and genomics strategies e.g. functional molecular markers, linkage-disequilibrium (LD) based association mapping, functional and comparative genomics offer the possibility of accelerating molecular breeding for abiotic and biotic stress tolerances in the legume crops. However, low level of polymorphism present in the cultivated genepools of these legume crops, imprecise phenotyping of the germplasm and the higher costs of development and application of genomic tools are critical factors in utilizing genomics in breeding of these legume crops.

An integrated agro-ecosystem and livelihood systems approach for the poor and vulnerable in dry areas

More than 400 million people in the developing world depend on dryland agriculture for their livelihoods. Dryland agriculture involves a complex combination of productive components: staple crops, vegetables, livestock, trees and fish interacting principally with rangeland, cultivated areas and watercourses. Managing risk and enhancing productivity through diversification and sustainable intensification is critical to securing and improving rural livelihoods. The main biophysical constraints are natural resource limitations and degradation, particularly water scarcity and encroaching desertification. Social and economic limitations, such as poor access to markets and inputs, weak governance and lack of information about alternative production technologies also limit the options available to farmers. Past efforts to address these constraints by focusing on individual components have either not been successful or are now facing a declining rate of impact, indicating the need for new integrated approaches to research for development of dryland systems. This article outlines the characteristics of such an approach, integrating agro-ecosystem and livelihoods approaches and presents a range of empirical examples of its application in dryland contexts. The authors draw attention to new insights about the design of research required to accelerate impact by integrating across disciplines and scales.

Laboratory Information Management Software for genotyping workflows: applications in high throughput crop genotyping

BackgroundWith the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow.ResultsA laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics.ConclusionA laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from http://www.icrisat.org/gt-bt/lims/lims.asp.

Analysis of Genetic Diversity in Pongamia Pongamia pinnata (L) Pierrre using AFLP Markers

In recent years, Pongamia has been considered as important renewable source of biodiesel, however not much molecular information is available in this species. Molecular characterization of this legume tree will enhance our understanding in improving the optimal yields of oil through breeding and enable us to meet the future demands for biodiesel. To assess the molecular genetic diversity in 46 Pongamia pinnata accessions collected from six different states of India, amplified fragment length polymorphism (AFLP) marker system was employed. Five AFLP primer combinations produced 520 discernible fragments, of which 502 (96.5%) were polymorphic. AFLP primer informativeness was estimated evaluating four parameters namely polymorphism information content (PIC), effective multiplex ratio (EMR), marker index (MI) and resolving power (RP). In total, 51 unique fragments were detected of which 19 unique fragments were observed with primer combination E-ACG / M-CTA. Although neighbour joining (NJ) method did not group accessions strictly according to their region of collection, a good level of genetic diversity was observed in examined germplasm. However, accessions collected from Karnataka showed comparatively higher diversity than accessions from other states. The diverse accessions identified in this study may be useful in Pongamia pinnata improvement to meet the future demands of biodiesel.

Research and field monitoring on transgenic crops by the Centro Internacional de Mejoramiento de Maiz y Trigo (CIMMYT)

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.

Peanut improvement: production of fertile hybrids and backcross progeny between Arachis hypogaea and A. kretschmeri

There are only a few reports of successful crosses between cultivated peanut (Arachis hypogaea L., section Arachis) and wild species from sections other than section Arachis. Many of the wild Arachis species harbor important traits necessary for the improvement of peanut. For example, Arachis kretschmeri Krapov., W.C. Gregory & C.E. Simpson (section Procumbentes) can grow under water-logged conditions and has been identified as one of the few wild species of Arachis with resistance to late leaf spot (LLS) and peanut rosette disease. Peanut rosette, caused by a combination of viruses, is an economically important disease only in Africa, while LLS, caused by Cercosporidium personatum, is an important fungal disease in Asia and the Americas as well as Africa. Interspecific hybrids between A. hypogaea and A. kretschmeri were produced by applying growth regulators to pollinated pistils and hybrid plants were obtained by germinating embryos in vitro. A total of seven hybrids were produced and confirmed by Simple Sequence Repeat (SSR) analysis. All hybrids were fertile, although initially slow growing. F1 hybrids were backcrossed to A. hypogaea and all plants in the F1BC1 generation were single-seeded with a prominent beak, characteristic of A. kretschmeri, but many of the F1BC2 pods were double-seeded resembling A. hypogaea. F1BC2 plants were moderately resistant to LLS. When a large number of seeds are obtained, the progeny will be screened for resistance to both LLS and rosette disease. Thus crosses with species outside the section Arachis may not only confer disease resistance but will also broaden the genetic base of cultivated peanut.

An integrated pipeline of open source software adapted for multi-CPU architectures: use in the large-scale identification of single nucleotide polymorphisms.

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.