Applications of Molecular Biology and Genomics to Genetic Enhancement of Crop Tolerance to Abiotic Stress : a Discussion Document

The discussions on abiotic stress genomics were initiated at TAC 80 in March 2000 at ICARDA, Aleppo, Syria. Under the agenda item Trends in Science: Implications for CGIAR, TAC discussed the opportunities offered by the new sciences in improving the relevance, quality and impact of research in the CGIAR. In the area of biological sciences, TAC considered that the advances in molecular biology had important long-term implications for CGIAR’s work on genetic enhancement and how that work could be organized in the future. This paper contains three reports: 1. The report by Mike Gale dated August 2002 where he states that it is an appropriate time to tackle abiotic stress head-on, given the motivation already in place, the experience of the ARIs in technology and model systems, the knowledge among the NARS plant breeders on stressed agricultural environments, and the CGIAR Centres’ comparative advantage over mandate crops along with their links to the developing world as well as to industry.2. The report entitled "Status of Breeding for Telerance of Abiotic Stresses and Prospects for Use of Molecular Techniques" by John Bennett dated March 2001. He states that the CGIAR Centers have a comparative advantage in many aspects of abiotic stress research because of their germplasm collections, their new capacity for genetic and molecular dissection of complex traits, and their ability to conduct multidisciplinary plant improvement programs in target environments. The combined resources of the CGIAR for this work are immense but are underutilized. Investment by the CGIAR in the new tools for gene discovery will produce breakthroughs in our understanding of abiotic stress tolerance that will benefit all the mandated crops.3. The report by Hirofumi Uchimiya dated September 2001 entitled "Genetic Engineering for Abiotic Stress Tolerance in Plants"

Identification of Drought-Responsive Universal Stress Proteins in Viridiplantae

Genes encoding proteins that contain the universal stress protein (USP) domain are known to provide bacteria, archaea, fungi, protozoa, and plants with the ability to respond to a plethora of environmental stresses. Specifically in plants, drought tolerance is a desirable phenotype. However, limited focused and organized functional genomic datasets exist on drought-responsive plant USP genes to facilitate their characterization. The overall objective of the investigation was to identify diverse plant universal stress proteins and Expressed Sequence Tags (ESTs) responsive to water-deficit stress. We hypothesize that cross-database mining of functional annotations in protein and gene transcript bioinformatics resources would help identify candidate drought-responsive universal stress proteins and transcripts from multiple plant species. Our bioinformatics approach retrieved, mined and integrated comprehensive functional annotation data on 511 protein and 1561 ESTs sequences from 161 viridiplantae taxa. A total of 32 drought-responsive ESTs from 7 plant genera Glycine, Hordeum, Manihot, Medicago, Oryza, Pinus and Triticum were identified. Two Arabidopsis USP genes At3g62550 and At3g53990 that encode ATP-binding motif were up-regulated in a drought microarray dataset. Further, a dataset of 80 simple sequence repeats (SSRs) linked to 20 singletons and 47 transcript assembles was constructed. Integrating the datasets on SSRs and drought-responsive ESTs identified three drought-responsive ESTs from bread wheat (BE604157), soybean (BM887317) and maritime pine (BX682209). The SSR sequence types were CAG, ATA and AT respectively. The datasets from cross-database mining provide organized resources for the characterization of USP genes as useful targets for engineering plant varieties tolerant to unfavorable environmental conditions

Gene Discovery and Advances in Finger Millet [Eleusine coracana (L.) Gaertn.] Genomics—An Important Nutri-Cereal of Future

The rapid strides in molecular marker technologies followed by genomics, and next generation sequencing advancements in three major crops (rice, maize and wheat) of the world have given opportunities for their use in the orphan, but highly valuable future crops, including finger millet [Eleusine coracana (L.) Gaertn.]. Finger millet has many special agronomic and nutritional characteristics, which make it an indispensable crop in arid, semi-arid, hilly and tribal areas of India and Africa. The crop has proven its adaptability in harsh conditions and has shown resilience to climate change. The adaptability traits of finger millet have shown the advantage over major cereal grains under stress conditions, revealing it as a storehouse of important genomic resources for crop improvement. Although new technologies for genomic studies are now available, progress in identifying and tapping these important alleles or genes is lacking. RAPDs were the default choice for genetic diversity studies in the crop until the last decade, but the subsequent development of SSRs and comparative genomics paved the way for the marker assisted selection in finger millet. Resistance gene homologues from NBS-LRR region of finger millet for blast and sequence variants for nutritional traits from other cereals have been developed and used invariably. Population structure analysis studies exhibit 2-4 sub-populations in the finger millet gene pool with separate grouping of Indian and exotic genotypes. Recently, the omics technologies have been efficiently applied to understand the nutritional variation, drought tolerance and gene mining. Progress has also occurred with respect to transgenics development. This review presents the current biotechnological advancements along with research gaps and future perspective of genomic research in finger millet

Native Plants to Arid Areas: A Genetic Reservoir for Drought-Tolerant Crops

Droughts are common in arid areas. These cause important losses in crop production, while the increasing population demands more food and goods. Cultivars able to produce under drought conditions are required to avoid or reduce production losses. Plants have evolved different mechanisms to face drought, and many genes have been already discovered in model and cultivated plants that are involved in this trait. Some of these genes have been successfully transformed into cultivated plants for drought tolerance. Plants native to arid lands may possess variants of drought tolerance mechanisms as compared to mesophytic or model plants. Also, different drought-related genes can be revealed. Studies using high-throughput and bioinformatic tools may allow to discover new genes and give new insights on the mechanisms involved in drought tolerance. However, still scarce studies in plants native to arid lands show that there are many drought-related genes that have not been already characterized and potentially they may be novel genes. These novel genes may be used to improve crops for drought tolerance. Therefore, more physiological, transcriptomic, proteomic, and metabolomic studies are needed on plants native to the deserts

Development and utilization of molecular markers to study genetic diversity of smooth cordgrass and cold tolerance sugarcane

Smooth cordgrass (Spartina alterniflora Loisel) and sugarcane (Saccharum spp. hybrids) are both grasses that belong to the family Poaceae and have significant impact on Louisiana environment and economy. Coastal Louisiana accounts to ~80% of the national wetland loss through natural and human interventions; (re)vegetation is considered an important component of coastal restoration efforts. Smooth cordgrass, a warm season marsh grass, is used as an ecosystem engineer to stabilize and restore Louisiana coastal marshes. In this study, genetic diversity was determined among 13 elite smooth cordgrass accessions of Louisiana through 276 polymorphic markers generated by 23 SSR and 32 RAPD primers. AMOVA results showed that sufficient variation existed among smooth cordgrass accessions for further exploitation in breeding program. A few genetic markers unique to specific smooth cordgrass accession(s) were also developed. Sugarcane, the number one row crop in Louisiana, is sensitive to cold; so cold tolerance is desirable to expand its cultivation in more northern environments. Cold tolerance alleles of Saccharum spontaneum can be exploited to develop sugarcane clones tolerant to low temperatures. To this end, forty differentially expressed genes (DEGs; 29 up-regulated and 11 down-regulated) were identified in a cold-tolerant clone Ho02-144 and a cold-sensitive clone L79-1002 under cold stress, using annealing control primer system. Reverse-transcription PCR (RT-PCR) analysis of the cold-responsive DEGs revealed distinctive expression profiles in the tolerant and sensitive clone. Mining of the cold-responsive DEGs yielded 12 sequences with simple sequence repeats (SSRs), which were used to design eSSR primers. Genotyping of 48 Louisiana sugarcane clones and 16 ancestral parents with these eSSRs generated 170 polymorphic markers that could classify cultivated sugarcane clones from their ancestral S. spontaneum clones at 0.23 similarity coefficient. The cold responsive genes will be useful for breeding cold tolerant sugarcane either through genetic engineering or selection through the use of gene-based markers

Integration of gene-based markers in a pearl millet genetic map for identification of candidate genes underlying drought tolerance QTLs

BACKGROUND: Identification of genes underlying drought tolerance (DT) quantitative trait loci (QTLs) will facilitate understanding of molecular mechanisms of drought tolerance, and also will accelerate genetic improvement of pearl millet through marker-assisted selection. We report a map based on genes with assigned functional roles in plant adaptation to drought and other abiotic stresses and demonstrate its use in identifying candidate genes underlying a major DT-QTL. RESULTS: Seventy five single nucleotide polymorphism (SNP) and conserved intron spanning primer (CISP) markers were developed from available expressed sequence tags (ESTs) using four genotypes, H 77/833-2, PRLT 2/89-33, ICMR 01029 and ICMR 01004, representing parents of two mapping populations. A total of 228 SNPs were obtained from 30.5 kb sequenced region resulting in a SNP frequency of 1/134 bp. The positions of major pearl millet linkage group (LG) 2 DT-QTLs (reported from crosses H 77/833-2 × PRLT 2/89-33 and 841B × 863B) were added to the present consensus function map which identified 18 genes, coding for PSI reaction center subunit III, PHYC, actin, alanine glyoxylate aminotransferase, uridylate kinase, acyl-CoA oxidase, dipeptidyl peptidase IV, MADS-box, serine/threonine protein kinase, ubiquitin conjugating enzyme, zinc finger C- × 8-C × 5-C × 3-H type, Hd3, acetyl CoA carboxylase, chlorophyll a/b binding protein, photolyase, protein phosphatase1 regulatory subunit SDS22 and two hypothetical proteins, co-mapping in this DT-QTL interval. Many of these candidate genes were found to have significant association with QTLs of grain yield, flowering time and leaf rolling under drought stress conditions. CONCLUSIONS: We have exploited available pearl millet EST sequences to generate a mapped resource of seventy five new gene-based markers for pearl millet and demonstrated its use in identifying candidate genes underlying a major DT-QTL in this species. The reported gene-based markers represent an important resource for identification of candidate genes for other mapped abiotic stress QTLs in pearl millet. They also provide a resource for initiating association studies using candidate genes and also for comparing the structure and function of distantly related plant genomes such as other Poaceae members

MINING SINGLE NUCLEOTIDE POLYMORPHISM FROM PUBLICLY AVAILABLE ESTS OF BREAD WHEAT (TRITICUM AESTIVUM L.)

Objective: The present study was undertaken to discover Single Nucleotide Polymorphisms (SNPs) in bread wheat with reference to leaf rust disease.Methods: Next Generation Sequencing platform sequencing by Oligonucleotide Ligation and Detection (SOLiD) was performed on four Serial Analysis of Gene Expression (SAGE) libraries of mock and leaf rust pathogen infected near-isogenic lines HD2329±Lr28. CLC Genomics Workbench was used for computational prediction of the SNPs. The predicted SNPs were filtered by Blast using wheat Expressed Sequence Tags (ESTs). The SNP-containing ESTs were annotated, and their expression was checked in response to inoculation of Puccinia triticina.Results: We have identified 191 SNPs from data obtained through the These EST-SNPs participated in various physiological and biochemical processes that influence important traits, such as cell rescue, defense and disease resistance.Conclusion: Very little knowledge exists on SNPs in hexaploid bread wheat (Triticum aestivum L.) because of the difficulty to discern the true polymorphic loci. This study has revealed fast and costs effective approach for SNP discovery which will be helpful in molecular breeding with important agronomic traits.Â

From RNA-seq to large-scale genotyping - genomics resources for rye (Secale cereale L.)

<p>Abstract</p> <p>Background</p> <p>The improvement of agricultural crops with regard to yield, resistance and environmental adaptation is a perpetual challenge for both breeding and research. Exploration of the genetic potential and implementation of genome-based breeding strategies for efficient rye (<it>Secale cereale </it>L.) cultivar improvement have been hampered by the lack of genome sequence information. To overcome this limitation we sequenced the transcriptomes of five winter rye inbred lines using Roche/454 GS FLX technology.</p> <p>Results</p> <p>More than 2.5 million reads were assembled into 115,400 contigs representing a comprehensive rye expressed sequence tag (EST) resource. From sequence comparisons 5,234 single nucleotide polymorphisms (SNPs) were identified to develop the Rye5K high-throughput SNP genotyping array. Performance of the Rye5K SNP array was investigated by genotyping 59 rye inbred lines including the five lines used for sequencing, and five barley, three wheat, and two triticale accessions. A balanced distribution of allele frequencies ranging from 0.1 to 0.9 was observed. Residual heterozygosity of the rye inbred lines varied from 4.0 to 20.4% with higher average heterozygosity in the pollen compared to the seed parent pool.</p> <p>Conclusions</p> <p>The established sequence and molecular marker resources will improve and promote genetic and genomic research as well as genome-based breeding in rye.</p

Future Directions in the Molecular Breeding of Forage and Turf

Key points Molecular breeding of forage and turf plants and their endosymbionts has entered the post-genomic era with a large amount of structural genomics information and genomic resources available for key forage and turf species and relevant model systems. A primary future challenge is the conversion of this information into useful functional knowledge for the development of molecular breeding technologies and products that address a range of high impact outcome scenarios in forage and turf. High-throughput approaches for spatial and temporal analysis, from genome to phenome, and the respective data integration in a systems biology context will be critical for the establishment of stringent gene-function correlations. Translational genomics will permit results obtained using model systems to have major impact on the understanding of the molecular basis of plant processes and direct application to the molecular breeding of forage and turf plants. These developments will be enhanced through applications of transgenesis and functionally-associated genetic markers in forage and turf molecular breeding building on genomic and post-genomic discoveries in these target species