Sripada M. Udupa

SuperSAGE: the drought stress-responsive transcriptome of chickpea roots

BackgroundDrought is the major constraint to increase yield in chickpea (Cicer arietinum). Improving drought tolerance is therefore of outmost importance for breeding. However, the complexity of the trait allowed only marginal progress. A solution to the current stagnation is expected from innovative molecular tools such as transcriptome analyses providing insight into stress-related gene activity, which combined with molecular markers and expression (e)QTL mapping, may accelerate knowledge-based breeding. SuperSAGE, an improved version of the serial analysis of gene expression (SAGE) technique, generating genome-wide, high-quality transcription profiles from any eukaryote, has been employed in the present study. The method produces 26 bp long fragments (26 bp tags) from defined positions in cDNAs, providing sufficient sequence information to unambiguously characterize the mRNAs. Further, SuperSAGE tags may be immediately used to produce microarrays and probes for real-time-PCR, thereby overcoming the lack of genomic tools in non-model organisms.ResultsWe applied SuperSAGE to the analysis of gene expression in chickpea roots in response to drought. To this end, we sequenced 80,238 26 bp tags representing 17,493 unique transcripts (UniTags) from drought-stressed and non-stressed control roots. A total of 7,532 (43%) UniTags were more than 2.7-fold differentially expressed, and 880 (5.0%) were regulated more than 8-fold upon stress. Their large size enabled the unambiguous annotation of 3,858 (22%) UniTags to genes or proteins in public data bases and thus to stress-response processes. We designed a microarray carrying 3,000 of these 26 bp tags. The chip data confirmed 79% of the tag-based results, whereas RT-PCR confirmed the SuperSAGE data in all cases.ConclusionThis study represents the most comprehensive analysis of the drought-response transcriptome of chickpea available to date. It demonstrates that – inter alias – signal transduction, transcription regulation, osmolyte accumulation, and ROS scavenging undergo strong transcriptional remodelling in chickpea roots already 6 h after drought stress. Certain transcript isoforms characterizing these processes are potential targets for breeding for drought tolerance. We demonstrate that these can be easily accessed by micro-arrays and RT-PCR assays readily produced downstream of SuperSAGE. Our study proves that SuperSAGE owns potential for molecular breeding also in non-model crops.

Allelic variation at (TAA) n microsatellite loci in a world collection of chickpea (Cicer arietinum L.) germplasm

Abstract A set of 12 randomly selected (TAA)n microsatellite loci of the cultivated chickpea (Cicer arietinum L.) were screened in a worldwide sample comprising 72 landraces, four improved cultivars and two wild species of the primary gene pool (C. reticulatum and C. echinosperum) to determine the level and pattern of polymorphism in these populations. A single fragment was amplified from all the accessions with each of 12 sequence-tagged microsatellite site markers, except for one locus where no fragment was obtained from either of the two wild species. There was a high degree of intraspecific polymorphism at these microsatellite loci, although isozymes, conventional RFLPs and RAPDs show very little or no polymorphism. Overall, the repeat number at a locus (excluding null alleles) ranged from 7 to 42. The average number of alleles per locus was 14.1 and the average genetic diversity was 0.86. Based on the estimates obtained, 11 out of the 12 frequency distributions of alleles at the loci tested can be considered to be non-normal. A significant positive correlation between the average number of repeats (size of the locus) and the amount of variation was observed, indicating that replication slippage may be the molecular mechanism involved in generation of variability at the loci. A comparison between the infinite allele and stepwise mutation models revealed that for 11 out of the 12 loci the number of alleles observed fell in between the values predicted by the two models. Phylogenetic analysis of microsatellite polymorphism in C. arietinum showed no relationship between accession and geographic origin, which is compatible with the recent expansion of this crop throughout the world.

Development of a composite collection for mining germplasm possessing allelic variation for beneficial traits in chickpea

Chickpea is one of the most important grain legume crops in the world. Large collections of genetic resources are maintained in the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) and International Center for Agricultural Research in the Dry Areas (ICARDA) genebanks. Association mapping using neutral markers has been suggested as a means to identify useful alleles in the vast reservoirs of genetic diversity existing in the germplasm collections that could be associated with the phenotypes among the population individuals. ICRISAT in collaboration with ICARDA developed a global composite collection of 3000 accessions that will be profiled using 50 polymorphic simple sequence repeat (SSR) markers. The data generated through this collaborative effort will be used to define the genetic structure of the global composite collection and to select a reference sample of 300 accessions representing the maximum diversity for the isolation of allelic variants of candidate gene associated with beneficial traits. It is then expected that molecular biologists and plant breeders will have opportunities to use diverse lines in functional and comparative genomics, in mapping and cloning gene(s), and in applied plant breeding to diversify the genetic base of the breeding populations which should lead to the development of broad-based elite breeding lines/cultivars with superior yield and enhanced adaptation to diverse environments.

Phenotypic and genetic diversity in Sinorhizobium meliloti and S. medicae from drought and salt affected regions of Morocco

BackgroundSinorhizobium meliloti and S. medicae are symbiotic nitrogen fixing bacteria in root nodules of forage legume alfalfa (Medicago sativa L.). In Morocco, alfalfa is usually grown in marginal soils of arid and semi-arid regions frequently affected by drought, extremes of temperature and soil pH, soil salinity and heavy metals, which affect biological nitrogen fixing ability of rhizobia and productivity of the host. This study examines phenotypic diversity for tolerance to the above stresses and genotypic diversity at Repetitive Extragenic Pallindromic DNA regions of Sinorhizobium nodulating alfalfa, sampled from marginal soils of arid and semi-arid regions of Morocco.ResultsRsa I digestion of PCR amplified 16S rDNA of the 157 sampled isolates, assigned 136 isolates as S. meliloti and the rest as S. medicae. Further phenotyping of these alfalfa rhizobia for tolerance to the environmental stresses revealed a large degree of variation: 55.41%, 82.16%, 57.96% and 3.18% of the total isolates were tolerant to NaCl (>513 mM), water stress (-1.5 MPa), high temperature (40°C) and low pH (3.5), respectively. Sixty-seven isolates of S. meliloti and thirteen isolates of S. medicae that were tolerant to salinity were also tolerant to water stress. Most of the isolates of the two species showed tolerance to heavy metals (Cd, Mn and Zn) and antibiotics (chloramphenicol, spectinomycin, streptomycin and tetracycline). The phenotypic clusters observed by the cluster analysis clearly showed adaptations of the S. meliloti and S. medicae strains to the multiple stresses. Genotyping with rep-PCR revealed higher genetic diversity within these phenotypic clusters and classified all the 157 isolates into 148 genotypes. No relationship between genotypic profiles and the phenotypes was observed. The Analysis of Molecular Variance revealed that largest proportion of significant (P < 0.01) genetic variation was distributed within regions (89%) than among regions (11%).ConclusionHigh degree of phenotypic and genotypic diversity is present in S. meliloti and S. medicae populations from marginal soils affected by salt and drought, in arid and semi-arid regions of Morocco. Some of the tolerant strains have a potential for exploitation in salt and drought affected areas for biological nitrogen fixation in alfalfa.

Asymmetric allele-specific expression in relation to developmental variation and drought stress in barley hybrids

In the present study, we analysed allele-specific expression (ASE) in the selfing species barley to assess the frequency of cis-acting regulatory variation and the effects of genetic background, developmental differences and drought stress on allelic expression levels. We measured ASE ratios in 30 genes putatively involved in stress responses in five hybrids and their reciprocals, namely Hordeum spontaneum 41-1/Alexis (HAl), Hordeum spontaneum 41-1/Arta (HAr), Sloop/WI3408 (SW), Tadmor/Sloop (TS) and Tadmor/WI3408 (TW). In order to detect cis-acting variation related to drought and developmental changes, the barley hybrids were grown under control and water-limited conditions, and leaf tissue was harvested at two developmental stages. The analysis demonstrated that more than half of the genes measured (63%) showed allelic differences in expression of up to 19-fold due to cis-regulatory variation in at least one cross by treatment/stage combination. Drought stress induced changes in allelic expression ratios, indicating differences between drought responsive cis-elements. In addition, ASE differences between developmental stages suggested the presence of cis-acting elements interacting with developmental cues. We were also able to demonstrate that the levels and frequency of allelic imbalance and hence differences in cis-regulatory elements are correlated with the genetic divergence between the parental lines, but may also arise as an adaptation to diverse habitats. Our findings suggest that cis-regulatory variation is a common phenomenon in barley, and may provide a molecular basis of transgression. Differential expression of near-isogenic members of the same gene family could potentially result in hybrid lines out performing their parents in terms of expression level, timing and response to developmental and environmental cues. Identification and targeted manipulation of cis-regulatory elements will assist in breeding improved crops with a better adaptation to changing environments.

Molecular Approaches and Breeding Strategies for Drought Tolerance in Barley

Barley genotypes, in particular landraces and wild species, represent an important source of variation for adaptive traits that may contribute to increase yield and yield stability under drought conditions, and that could be introgressed into improved varieties. Traits that have been investigated include physiological/biochemical and developmental/ morphological traits. Yield performance under drought is particularly a complex phenomenon, and plants exhibit a diverse range of genetically complex mechanisms for drought resistance. Quantitative trait loci (QTL) studies with and without H. spontaneum have shown that developmental genes, notably those involved in flowering time and plant stature show pleiotropic effects on abiotic stress tolerance and ultimately determine yield. Problems associated with the hybridization of H. spontaneum such as alleles with deleterious effects on field performance could be best addressed in the advanced backcross (AB-) QTL analysis. It was interesting to see that in AB-QTL populations like in balanced populations major QTL overshadowed minor QTL-alleles. Nevertheless, crosses with H. spontaneum, AB-QTL populations and association studies with H. spontaneum have also identified new alleles and genes that are related to abiotic stress tolerance. In order to identify genes that are related to drought tolerance microarrays analysis to monitor gene expression profiles for plants exposed to limited water environment is performed. Several studies with rapid dehydration treatment have shown that osmotic-stress-inducible genes could explain the response to drought stress in plants. Another development is the identification and use of nucleotide polymorphisms (SNP) in genes related to abiotic stress tolerance. An understanding of the combined function and expression of genes involved in various abiotic stresses, could help identify candidate genes underlying QTL of interest.

Genetic diversity reduction in improved durum wheat cultivars of Morocco as revealed by microsatellite markers

It has been argued that genetic diversity in crop varieties has been on the decline in recent times due to plant breeding. This can have serious consequences for both the genetic vulnerability of crops and their plasticity when responding to changes in production environments. It is, therefore, vital for plant breeding programs to maintain sufficient diversity in the cultivars deployed for multi-period cultivation. In this study, to understand the temporal genetic diversity in durum wheat, 21 improved durum wheat cultivars released in Morocco, since 1956 and five exotic cultivars currently used in crossing programs were analyzed using 13 microsatellite markers. The analysis revealed a total of 44 alleles and average genetic diversity of 0.485 with genetic distances ranging from 0.077 to 0.846 at 13 microsatellite loci in Moroccan durum wheat cultivars. All the durum cultivars of Morocco could be distinguished using the 13 microsatellite markers. The total number of alleles and unique alleles were highest in cultivars developed before 1990, decreasing in cultivars developed during the 1990s and 2000s, indicating that recent durum breeding efforts have reduced allelic richness in recent cultivars. Thus, deployment of exotic durum wheat lines in breeding programs could enhance genetic diversity in durum wheat cultivars.

Identification of Quantitative Trait Loci Controlling Root and Shoot Traits Associated with Drought Tolerance in a Lentil (Lens culinaris Medik.) Recombinant Inbred Line Population

Drought is one of the major abiotic stresses limiting lentil productivity in rainfed production systems. Specific rooting patterns can be associated with drought avoidance mechanisms that can be used in lentil breeding programs. In all, 252 co-dominant and dominant markers were used for Quantitative Trait Loci (QTL) analysis on 132 lentil recombinant inbred lines based on greenhouse experiments for root and shoot traits during two seasons under progressive drought-stressed conditions. Eighteen QTLs controlling a total of 14 root and shoot traits were identified. A QTL-hotspot genomic region related to a number of root and shoot characteristics associated with drought tolerance such as dry root biomass, root surface area, lateral root number, dry shoot biomass and shoot length was identified. Interestingly, a QTL (QRSratioIX-2.30) related to root-shoot ratio, an important trait for drought avoidance, explaining the highest phenotypic variance of 27.6 and 28.9% for the two consecutive seasons, respectively, was detected. This QTL was closed to the co-dominant SNP marker TP6337 and also flanked by the two SNP TP518 and TP1280. An important QTL (QLRNIII-98.64) related to lateral root number was found close to TP3371 and flanked by TP5093 and TP6072 SNP markers. Also, a QTL (QSRLIV-61.63) associated with specific root length was identified close to TP1873 and flanked by F7XEM6b SRAP marker and TP1035 SNP marker. These two QTLs were detected in both seasons. Our results could be used for marker-assisted selection in lentil breeding programs targeting root and shoot characteristics conferring drought avoidance as an efficient alternative to slow and labor-intensive conventional breeding methods.

A SSR kit to study genetic diversity in chickpea (Cicer arietinum L.)

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.

Marker technology for plant breeding

Numerous molecular markers, linked with traits of agronomic importance in the food legumes, pea, chickpea and lentil, have been identified. Microsatellite markers are being developed and mapped in a collaborative effort between the International Center for Agricultural Research in the Dry Areas (ICARDA) and the University of Frankfurt to overcome the relatively low amount of information that can be derived from the widely used dominant markers in chickpea. Besides mapping and identifying host plant resistance, efforts are being made to characterize the pathogen populations. Once host plant resistance has been identified and mapped, it will be feasible to deploy the relevant resistance genes when shifts occur in the pathogen population. The technology for using these markers in marker-assisted selection (MAS) has been greatly improved. The ability to use MAS to pyramid genes will make this technology an essential tool for legume breeders.