Candidate Genes For Tolerance Research Articles

Experimental Validation of Evolutionarily Conserved Genes Regulating Hypoxia Adaption in Drosophila and Humans

Hypoxia tolerance is regulated by polygenic mechanisms in metazoan organisms. The following study was performed to determine whether such polygenic mechanisms are shared evolutionarily between organisms like Drosophila and humans. Using a generalized linear mixed model we identified significant allele frequency differences between three independently evolved hypoxia‐tolerant populations and normoxic control populations of Drosophila. Around 50% of the ~3,800 SNP variants are clustered in 66 distinct genomic regions that contain genes that are differentially expressed when hypoxia‐tolerant and normoxic populations were compared. In addition, using genome‐wide SNP data from four high‐altitude human populations (i.e., Sherpas, Tibetans, Ethiopians, and Andeans), we found that several human orthologs of the genes under selection in flies are also likely under positive selection in all four high‐altitude human populations, suggesting that these conserved genes and pathways may regulate hypoxia tolerance in both flies and humans. Currently we have experimentally evaluated the role of these candidate genes in hypoxia tolerance in flies using RNAi‐mediated knockdown. We found that down‐regulation of several candidate genes’ expression, such as pyd, significantly enhanced survival in severe hypoxia in flies. Our results demonstrated that fundamental genetic mechanisms regulating hypoxia tolerance have indeed remained conserved throughout evolution. Such mechanisms may help us to develop novel therapeutic strategies to treat hypoxia‐related diseases in humans.Support or Funding InformationThis work was supported by the following grants from National Institute of Health: Training in Oxygen in Health and Disease Grant T32HL094282 to ARJ; 5P01HD32573 to GGH; and P50 GM081892 to KPW.

Transcriptome analysis in different rice cultivars provides novel insights into desiccation and salinity stress responses.

Drought and salinity are the major environmental factors that affect rice productivity. Comparative transcriptome analysis between tolerant and sensitive rice cultivars can provide insights into the regulatory mechanisms involved in these stress responses. In this study, the comparison of transcriptomes of a drought-tolerant [Nagina 22 (N22)] and a salinity-tolerant (Pokkali) rice cultivar with IR64 (susceptible cultivar) revealed variable transcriptional responses under control and stress conditions. A total of 801 and 507 transcripts were exclusively differentially expressed in N22 and Pokkali rice cultivars, respectively, under stress conditions. Gene ontology analysis suggested the enrichment of transcripts involved in response to abiotic stress and regulation of gene expression in stress-tolerant rice cultivars. A larger number of transcripts encoding for members of NAC and DBP transcription factor (TF) families in N22 and members of bHLH and C2H2 TF families in Pokkali exhibited differential regulation under desiccation and salinity stresses, respectively. Transcripts encoding for thioredoxin and involved in phenylpropanoid metabolism were up-regulated in N22, whereas transcripts involved in wax and terpenoid metabolism were up-regulated in Pokkali. Overall, common and cultivar-specific stress-responsive transcripts identified in this study can serve as a helpful resource to explore novel candidate genes for abiotic stress tolerance in rice.

Identification of Putative Candidate Genes for Water Stress Tolerance in Canola (Brassica napus).

Drought stress can directly inhibit seedling establishment in canola (Brassica napus), resulting in lower plant densities and reduced yields. To dissect this complex trait, 140 B. napus accessions were phenotyped under normal (0.0 MPa, S0) and water-stressed conditions simulated by polyethylene glycol (PEG) 6000 (−0.5 MPa, S5) in a hydroponic system. Phenotypic variation and heritability indicated that the root to shoot length ratio was a reliable indicator for water stress tolerance. Thereafter, 66 accessions (16 water stress tolerant, 34 moderate and 16 sensitive lines) were genotyped using 25,495 Brassica single nucleotide polymorphisms (SNPs). Genome-wide association studies (GWAS) identified 16 loci significantly associated with water stress response. Two B. napus accessions were used for RNA sequencing, with differentially-expressed genes under normal and water-stressed conditions examined. By combining differentially-expressed genes detected by RNA sequencing with significantly associated loci from GWAS, 79 candidate genes were identified, of which eight were putatively associated with drought tolerance based on gene ontology of Arabidopsis. Functional validation of these genes may confirm key drought-related genes for selection and breeding in B. napus. Our results provide insight into the genetic basis of water stress tolerance in canola.

Transcriptome Profiling of Wild Arachis from Water-Limited Environments Uncovers Drought Tolerance Candidate Genes.

Peanut (Arachis hypogaea L.) is an important legume cultivated mostly in drought-prone areas where its productivity can be limited by water scarcity. The development of more drought-tolerant varieties is, therefore, a priority for peanut breeding programs worldwide. In contrast to cultivated peanut, wild relatives have a broader genetic diversity and constitute a rich source of resistance/tolerance alleles to biotic and abiotic stresses. The present study takes advantage of this diversity to identify drought-responsive genes by analyzing the expression profile of two wild species, Arachis duranensis and Arachis magna (AA and BB genomes, respectively), in response to progressive water deficit in soil. Data analysis from leaves and roots of A. duranensis (454 sequencing) and A. magna (suppression subtractive hybridization (SSH)) stressed and control complementary DNA (cDNA) libraries revealed several differentially expressed genes in silico, and 44 of them were selected for further validation by quantitative RT-PCR (qRT-PCR). This allowed the identification of drought-responsive candidate genes, such as Expansin, Nitrilase, NAC, and bZIP transcription factors, displaying significant levels of differential expression during stress imposition in both species. This is the first report on identification of differentially expressed genes under drought stress and recovery in wild Arachis species. The generated transcriptome data, besides being a valuable resource for gene discovery, will allow the characterization of new alleles and development of molecular markers associated with drought responses in peanut. These together constitute important tools for the peanut breeding program and also contribute to a better comprehension of gene modulation in response to water deficit and rehydration.Electronic supplementary materialThe online version of this article (doi:10.1007/s11105-015-0882-x) contains supplementary material, which is available to authorized users.

Identification of Drought Tolerance Candidate Proteins and Genes in Reproductive Stage of Wheat

Reproductive development is the most water-stress-sensitive phase in the life of a cereal crop. We applied a proteomic approach to analysis the effects of drought stress on anther proteome and identification of candidate proteins and genes in drought tolerant, Dezful-10 and susceptible, Shiraz, wheat genotypes. Our results showed that short-term stresses at meiosis and mitosis stages declined plant seed setting and the meiosis stage is more sensitive to drought stress. Mass spectrometry analysis of responsive proteins revealed that several important protein in plant adaptation to drought stress are up-regulated only in tolerant plant including raftin, vacuolar invertase, putative PrMC3, Esterase/lipase, reversibly glycosylated polypeptide and subtilisin like serine protease. The expression of the selected proteins which were the representatives of the down-regulated proteins and of the up-regulated proteins in wheat genotypes were further assessed at the transcription level using real-time PCR.

Molecular evolution of drought tolerance and wood strength related candidate genes in loblolly pine (Pinus taeda L.)

AbstractLoblolly pine (Pinus taeda L.) is an intensely studied species that has become a model system for conifers. It is one of the most important commercial crops in the southeastern United States and grows across a vast territory. Due to exposure to this current diverse environment and the fluctuating climatic conditions of the past, it has likely accumulated substantial variation in adaptive trait and wood strength related genes. We merged a set of newly collected and previously published genomic DNA sequence data and analyzed them for departures from neutrality in 32 drought tolerance and wood strength related candidate genes using neutrality tests, such as Tajima’s D, HKA, MK and nonsynonymous to synonymous substitutions ratio (Z-test). Three other major Southern pines closely related to P. taeda (Pinus echinata Mill., P. elliottii Engelm., and P. palustris Mill.) were used as outgroups in interspecific tests. In three loci (4-coumarate: CoA ligase, putative cell-wall protein and trans-cinnamate 4-hydroxylase 2) neutrality was rejected by both intra- and interspecific tests, consistent with purifying selection. Neutrality was also rejected in several other loci (alpha-tubulin, arabinogalactan 4, arabinogalactan 6, cinnamate 4-hydroxylase 1, cinnamoyl CoA reductase, cinnamyl alcohol dehydrogenase, caffeoyl CoA O-methyltransferase 1, early response to drought 3, glycine hydroxymethyltransferase, ABI1 protein phosphatase 2C-like, putative wall-associated protein kinase, and unknown gene ug_2-498); however, these results are difficult to interpret because only one of the tests proved significant. This study contributes to the ongoing discussion about natural selection in putative adaptive genes in loblolly pine. However, unambiguous interpretation of the results often remains problematic.

The genome of the stress-tolerant wild tomato species Solanum pennellii.

Solanum pennellii is a wild tomato species endemic to Andean regions in South America, where it has evolved to thrive in arid habitats. Because of its extreme stress tolerance and unusual morphology, it is an important donor of germplasm for the cultivated tomato Solanum lycopersicum. Introgression lines (ILs) in which large genomic regions of S. lycopersicum are replaced with the corresponding segments from S. pennellii can show remarkably superior agronomic performance. Here we describe a high-quality genome assembly of the parents of the IL population. By anchoring the S. pennellii genome to the genetic map, we define candidate genes for stress tolerance and provide evidence that transposable elements had a role in the evolution of these traits. Our work paves a path toward further tomato improvement and for deciphering the mechanisms underlying the myriad other agronomic traits that can be improved with S. pennellii germplasm.

Inheritance of Rootstock Effects and Their Association with Salt Tolerance Candidate Genes in a Progeny Derived from ‘Volkamer’ Lemon

A seedling population from hybrids between ‘Volkamer’ lemon (Citrus volkameriana) and ‘Rubidoux’ trifoliate orange (Poncirus trifoliata) was grafted with ‘Hashimoto’ Satsuma mandarin (Citrus unshiu) to study the inheritance of rootstock effects on salt tolerance in terms of fruit yield. Trees were maintained in a screenhouse, and a salt treatment (25 mm NaCl) was applied to 32 genotypes from June to September every year for 5 years. Rootstocks were genotyped for five salt tolerance candidate genes. Significant effects of rootstock genotype (G) and treatment (E) were found for most traits. Salinity decreased yield and juice volume but improved soluble solids concentration (TSS) and rind thickness. Year effects were highly significant in most cases. G × E interactions were found for fruit weight, total fruit weight, juice volume (JV), leaf water content (LWC), and leaf [Na+]. Therefore, rootstocks that induce early fruit maturation under salinity (by increasing TSS and maintaining JV) can be selected to expand the harvesting calendar of mandarin cultivars. Salt tolerance candidate genes SOS1 and NHX1 were associated with fruit yield traits under normal conditions (1.4 dS·m−1), and SOS1 and CCC were associated with LWC under salinity conditions (4 dS·m−1). Only 5% progeny induced higher accumulated yield than ‘Volkamer’ lemon under salinity. Given the low heritability of rootstock effects on fruit yield under salinity conditions (0.18 at most), marker-assisted selection might be useful.

Characterization of the HMA7 gene and transcriptomic analysis of candidate genes for copper tolerance in two Silene vulgaris ecotypes

Silene vulgaris possesses ecotype-specific tolerance to high levels of copper in the soil. Although this was reported a few decades ago, little is known about this trait on a molecular level. The aim of this study was to analyze the transcription response to elevated copper concentrations in two S. vulgaris ecotypes originating from copper-contrasting soil types - copper-tolerant Lubietova and copper-sensitive Stranska skala. To reveal if plants are transcriptionally affected, we first analyzed the HMA7 gene, a known key player in copper metabolism. Based on BAC library screening, we identified a BAC clone containing a SvHMA7 sequence with all the structural properties specific for plant copper-transporting ATPases. The functionality of the gene was tested using heterologous complementation in yeast mutants. Analyses of SvHMA7 transcription patterns showed that both ecotypes studied up-regulated SvHMA7 transcription after the copper treatment. Our data are supported by analysis of appropriate reference genes based on RNA-Seq databases. To identify genes specifically involved in copper response in the studied ecotypes, we analyzed transcription profiles of genes coding Cu-transporting proteins and genes involved in the prevention of copper-induced oxidative stress in both ecotypes. Our data show that three genes (APx, POD and COPT5) differ in their transcription pattern between the ecotypes with constitutively increased transcription in Lubietova. Taken together, we have identified transcription differences between metallifferous and non-metalliferous ecotypes of S. vulgaris, and we have suggested candidate genes participating in metal tolerance in this species.

Identification of heat responsive genes in cotton

High summer temperature negatively affects cotton yield, and better understanding of genetic mechanisms of heat stress responses in cotton may facilitate development of new heat tolerant cultivars. We attempted to determine heat responsive genes in cotton using tolerant (Stoneville 453, BA 119) and susceptible (Nazilli 84S) cultivars. Twenty five expressed sequence tags (ESTs) were sequenced and studied for gene homology. Sixteen ESTs were significantly similar to known genes, whereas eight ESTs were similar to not annotated cDNA clones and 1 EST did not show homology to any known gene. Expression of some ESTs was analyzed by quantitative real-time PCR and IAA-ala hydrolase (IAR3), folylpolyglutamate synthase (FPGS3), and two not annotated ESTs (GhHS126 and GhHS128) were consistently up-regulated under both short- and long-term heat stress. Since cotton is considered relatively more heat tolerant than most of the other crops, it can be suggested that these genes and ESTs could play a significant role in heat tolerance. In addition, GhHS126 and GhHS128 might be parts of the new candidate genes for heat tolerance.

Mapping of STS markers developed from drought tolerance candidate genes and preliminary analysis of their association with yield-related traits in common wheat(Triticum aestivum)

Drought is a severe abiotic stress that affects wheat production worldwide. In order to identify candidate genes for tolerance to water stress in wheat, sequences of 11 genes that have function of drought tolerance in other plant species were used to identify the wheat ortholog genes via homology searching in the wheat EST database. Atotal of 11 primer pairs were identified and amplified PCR products in wheat. Of them, 10 STS markers were mapped on 11 chromosomes in a set of nulli-tetrasomic lines of ‘Chinese Spring’ wheat; six were mapped on chromosomes 1A, 1B, 4B, 7A, 2B and 5D, respectively, in a spring wheat mapping population (POP1). The marker XTaABH1 mapped on 7A in POP1 was the only one mapped but characterized in a winter wheat mapping population (POP2) for grain yield, kernel weight and diameter, and height in four-field trials applied different water stress or irrigation. The marker XTaABH1 was significantly associated with grain yield under rainfed condition, with kernel weight under terminal stress and non-irrigation conditions, with kernel diameter and height under non-irrigated condition. The STS primers, map information and marker-trait association produced in the currently study would be of interest to researchers working on drought tolerance.

Deciphering the genetic basis of microcystin tolerance.

BackgroundCyanobacteria constitute a serious threat to freshwater ecosystems by producing toxic secondary metabolites, e.g. microcystins. These microcystins have been shown to harm livestock, pets and humans and to affect ecosystem service and functioning. Cyanobacterial blooms are increasing worldwide in intensity and frequency due to eutrophication and global warming. However, Daphnia, the main grazer of planktonic algae and cyanobacteria, has been shown to be able to suppress bloom-forming cyanobacteria and to adapt to cyanobacteria that produce microcystins. Since Daphnia’s genome was published only recently, it is now possible to elucidate the underlying molecular mechanisms of microcystin tolerance of Daphnia.ResultsDaphnia magna was fed with either a cyanobacterial strain that produces microcystins or its genetically engineered microcystin knock-out mutant. Thus, it was possible to distinguish between effects due to the ingestion of cyanobacteria and effects caused specifically by microcystins. By using RNAseq the differentially expressed genes between the different treatments were analyzed and affected KOG-categories were calculated. Here we show that the expression of transporter genes in Daphnia was regulated as a specific response to microcystins. Subsequent qPCR and dietary supplementation with pure microcystin confirmed that the regulation of transporter gene expression was correlated with the tolerance of several Daphnia clones.ConclusionsHere, we were able to identify new candidate genes that specifically respond to microcystins by separating cyanobacterial effects from microcystin effects. The involvement of these candidate genes in tolerance to microcystins was validated by correlating the difference in transporter gene expression with clonal tolerance. Thus, the prevention of microcystin uptake most probably constitutes a key mechanism in the development of tolerance and adaptation of Daphnia. With the availability of clear candidate genes, future investigations examining the process of local adaptation of Daphnia populations to microcystins are now possible.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-776) contains supplementary material, which is available to authorized users.

SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativum L.).

BackgroundField pea (Pisum sativum L.) is a self-pollinating, diploid, cool-season food legume. Crop production is constrained by multiple biotic and abiotic stress factors, including salinity, that cause reduced growth and yield. Recent advances in genomics have permitted the development of low-cost high-throughput genotyping systems, allowing the construction of saturated genetic linkage maps for identification of quantitative trait loci (QTLs) associated with traits of interest. Genetic markers in close linkage with the relevant genomic regions may then be implemented in varietal improvement programs.ResultsIn this study, single nucleotide polymorphism (SNP) markers associated with expressed sequence tags (ESTs) were developed and used to generate comprehensive linkage maps for field pea. From a set of 36,188 variant nucleotide positions detected through in silico analysis, 768 were selected for genotyping of a recombinant inbred line (RIL) population. A total of 705 SNPs (91.7%) successfully detected segregating polymorphisms. In addition to SNPs, genomic and EST-derived simple sequence repeats (SSRs) were assigned to the genetic map in order to obtain an evenly distributed genome-wide coverage. Sequences associated with the mapped molecular markers were used for comparative genomic analysis with other legume species. Higher levels of conserved synteny were observed with the genomes of Medicago truncatula Gaertn. and chickpea (Cicer arietinum L.) than with soybean (Glycine max [L.] Merr.), Lotus japonicus L. and pigeon pea (Cajanus cajan [L.] Millsp.). Parents and RIL progeny were screened at the seedling growth stage for responses to salinity stress, imposed by addition of NaCl in the watering solution at a concentration of 18 dS m-1. Salinity-induced symptoms showed normal distribution, and the severity of the symptoms increased over time. QTLs for salinity tolerance were identified on linkage groups Ps III and VII, with flanking SNP markers suitable for selection of resistant cultivars. Comparison of sequences underpinning these SNP markers to the M. truncatula genome defined genomic regions containing candidate genes associated with saline stress tolerance.ConclusionThe SNP assays and associated genetic linkage maps developed in this study permitted identification of salinity tolerance QTLs and candidate genes. This constitutes an important set of tools for marker-assisted selection (MAS) programs aimed at performance enhancement of field pea cultivars.

Association Mapping and Nucleotide Sequence Variation in Five Drought Tolerance Candidate Genes in Spring Wheat

Functional markers are needed for key genes involved in drought tolerance to improve selection for crop yield under moisture stress conditions. The objectives of this study were to (i) characterize five drought tolerance candidate genes, namely dehydration responsive element binding 1A (DREB1A), enhanced response to abscisic acid (ERA1‐B and ERA1‐D), and fructan 1‐exohydrolase (1‐FEH‐A and 1‐FEH‐B), in wheat (Triticum aestivum L.) for nucleotide and haplotype diversity, Tajima's D value, and linkage disequilibrium (LD) and (ii) associate within‐gene single nucleotide polymorphisms (SNPs) with phenotypic traits in a spring wheat association mapping panel (n = 126). Field trials were grown under contrasting moisture regimes in Greeley, CO, and Melkassa, Ethiopia, in 2010 and 2011. Genome‐specific amplification and DNA sequence analysis of the genes identified SNPs and revealed differences in nucleotide and haplotype diversity, Tajima's D, and patterns of LD. DREB1A showed associations (false discovery rate adjusted probability value = 0.1) with normalized difference vegetation index, heading date, biomass, and spikelet number. Both ERA1‐A and ERA1‐B were associated with harvest index, flag leaf width, and leaf senescence. 1‐FEH‐A was associated with grain yield, and 1‐FEH‐B was associated with thousand kernel weight and test weight. If validated in relevant genetic backgrounds, the identified marker–trait associations may be applied to functional marker‐assisted selection.

Analysis of drought tolerance candidate genes in transgenic plants

Analysis of drought tolerance candidate genes in transgenic plants

Identification of quantitative trait loci and candidate genes for cadmium tolerance in Populus

Understanding genetic variation for the response of Populus to heavy metals like cadmium (Cd) is an important step in elucidating the underlying mechanisms of tolerance. In this study, a pseudo-backcross pedigree of Populus trichocarpa Torr. & Gray and Populus deltoides Bart. was characterized for growth and performance traits after Cd exposure. A total of 16 quantitative trait loci (QTL) at logarithm of odds (LOD) ratio ≥ 2.5 were detected for total dry weight, its components and root volume. Major QTL for Cd responses were mapped to two different linkage groups and the relative allelic effects were in opposing directions on the two chromosomes, suggesting differential mechanisms at these two loci. The phenotypic variance explained by Cd QTL ranged from 5.9 to 11.6% and averaged 8.2% across all QTL. A whole-genome microarray study led to the identification of nine Cd-responsive genes from these QTL. Promising candidates for Cd tolerance include an NHL repeat membrane-spanning protein, a metal transporter and a putative transcription factor. Additional candidates in the QTL intervals include a putative homolog of a glutamate cysteine ligase, and a glutathione-S-transferase. Functional characterization of these candidate genes should enhance our understanding of Cd metabolism and transport and phytoremediation capabilities of Populus.

Deciphering energy‐associated gene networks operating in the response of Arabidopsis plants to stress and nutritional cues

Plants need to continuously adjust their transcriptome in response to various stresses that lead to inhibition of photosynthesis and the deprivation of cellular energy. This adjustment is triggered in part by a coordinated re-programming of the energy-associated transcriptome to slow down photosynthesis and activate other energy-promoting gene networks. Therefore, understanding the stress-related transcriptional networks of genes belonging to energy-associated pathways is of major importance for engineering stress tolerance. In a bioinformatics approach developed by our group, termed 'gene coordination', we previously divided genes encoding for enzymes and transcription factors in Arabidopsis thaliana into three clusters, displaying altered coordinated transcriptional behaviors in response to multiple biotic and abiotic stresses (Plant Cell, 23, 2011, 1264). Enrichment analysis indicated further that genes controlling energy-associated metabolism operate as a compound network in response to stress. In the present paper, we describe in detail the network association of genes belonging to six central energy-associated pathways in each of these three clusters described in our previous paper. Our results expose extensive stress-associated intra- and inter-pathway interactions between genes from these pathways, indicating that genes encoding proteins involved in energy-associated metabolism are expressed in a highly coordinated manner. We also provide examples showing that this approach can be further utilized to elucidate candidate genes for stress tolerance and functions of isozymes.

Recent Updates on Salinity Stress in Rice: From Physiological to Molecular Responses

One-fifth of irrigated agriculture is negatively affected by high soil salinity. The expected population growth, over 9 billion by 2050, enhances the pressure for agricultural production in marginal saline lands. Rice (Oryza sativa L.), the staple food for more than half of the world's population, is the most salt-sensitive cereal. The need for salt-tolerant rice varieties able to cope with several other stress conditions obviously puts a lot of pressure on breeders who must better comprehend the physiology and genetic control of salt tolerance. In spite of several good reviews recently published, an integrated vision of current information on rice tolerance to salt stress has been lacking. Here we present the most recent data on the salinity effect on rice physiology and stress adaptation, including implications on growth regulation and reproductive development. We have included an inventory of salt tolerance donors available for breeding programs and a comprehensive survey of current work on QTL detection and cloning as well as marker-assisted selection to introgress favorable alleles into elite rice lines. A schematic view of the rice chromosomes on which salt tolerance QTLs and candidate genes are positioned is also included. Finally, we focus on the most promising candidate genes involved in salt stress response. There, we discuss the available knowledge on salt stress signaling and ion homeostasis, LEAs and other stress-induced proteins, genes with unknown function and transcription regulators as well as the present knowledge on the role of post-translational modifications on the modulation of the response to salinity in rice. We conclude by highlighting still missing clues that could help to design better salt tolerant varieties, and we evaluate the significance of the data presented for the future of rice breeding and sustainability of the culture in marginal saline soils.

Targeted association analysis identified japonica rice varieties achieving Na+/K+ homeostasis without the allelic make-up of the salt tolerant indica variety Nona Bokra

During the last decade, a large number of QTLs and candidate genes for rice tolerance to salinity have been reported. Using 124 SNP and 52 SSR markers, we targeted 14 QTLs and 65 candidate genes for association mapping within the European Rice Core collection (ERCC) comprising 180 japonica accessions. Significant differences in phenotypic response to salinity were observed. Nineteen distinct loci significantly associated with one or more phenotypic response traits were detected. Linkage disequilibrium between these loci was extremely low, indicating a random distribution of favourable alleles in the ERCC. Analysis of the function of these loci indicated that all major tolerance mechanisms were present in the ERCC although the useful level of expression of the different mechanisms was scattered among different accessions. Under moderate salinity stress some accessions achieved the same level of control of Na(+) concentration and Na(+)/K(+) equilibrium as the indica reference variety for salinity tolerance Nona Bokra, although without sharing the same alleles at several loci associated with Na(+) concentration. This suggests (a) differences between indica and japonica subspecies in the effect of QTLs and genes involved in salinity tolerance and (b) further potential for the improvement of tolerance to salinity above the tolerance level of Nona Bokra, provided the underlying mechanisms are complementary at the whole plant level. No accession carried all favourable alleles, or showed the best phenotypic responses for all traits measured. At least nine accessions were needed to assemble the favourable alleles and all the best phenotypic responses. An effective strategy for the accumulation of the favourable alleles would be marker-assisted population improvement.

DETERMINATION BY SSCP MARKERS OF THE ALLELIC DIVERSITY OF CANDIDATE GENES FOR TOLERANCE TO IRON CHLOROSIS IN CITRUS GERMPLASM

Iron chlorosis is one of the main abiotic constraints for Mediterranean citriculture and the development of marker assisted selection (MAS) for this trait would be a great aid for rootstock breeding. We performed SSCP (Single Stranded Conformation Polymorphism) analysis in order to discover allelic diversity of candidate genes for iron chlorosis tolerance in citrus. Two iron chlorosis tolerance candidate genes were selected from existing Citrus ESTs databases and Arabidopsis thaliana genome databases. Iron-Sulfur assembly protein (Fe_S=AT2G16710) and root iron transporter (IRT1=AT4G19690) candidate gene sequences were used to define primers in conserved regions. Six Citrus genotypes from the basic taxon of Citrus where used to identify polymorphic areas in the genes. Direct sequencing of amplified DNA fragments of candidate genes was performed and SNPs (single-nucleotide polymorphisms) and Indels where searched after sequence alignment. A total of 2215 bp DNA fragments were sequenced and 56 SNPs and 2 Indels were determined. New primers were defined, in conserved areas flanking polymorphic ones, for Single Strand Conformation Polymorphism (SSCP) analysis for further diversity and routine genotyping. Two pairs of primers were defined for each gene. SSCP-PCR analysis was performed with twenty-five Citrus genotypes. The neighbor joining method was used for cluster analysis. Poncirus trifoliate genotypes and their hybrids known to be sensitive to iron chlorosis clustered together and mandarins showed high diversity for both genes. Differences were found among sour orange genotypes known to have differential tolerance behavior to iron chlorosis. (Resume d'auteur)