Five Regions of the Pea Genome Co-Control Partial Resistance to D. pinodes, Tolerance to Frost, and Some Architectural or Phenological Traits.

Evidence for reciprocal links between plant responses to biotic or abiotic stresses and architectural and developmental traits has been raised using approaches based on epidemiology, physiology, or genetics. Winter pea has been selected for years for many agronomic traits contributing to yield, taking into account architectural or phenological traits such as height or flowering date. It remains nevertheless particularly susceptible to biotic and abiotic stresses, among which Didymella pinodes and frost are leading examples. The purpose of this study was to identify and resize QTL localizations that control partial resistance to D. pinodes, tolerance to frost, and architectural or phenological traits on pea dense genetic maps, considering how QTL colocalizations may impact future winter pea breeding. QTL analysis revealed five metaQTLs distributed over three linkage groups contributing to both D. pinodes disease severity and frost tolerance. At these loci, the haplotypes of alleles increasing both partial resistance to D. pinodes and frost tolerance also delayed the flowering date, increased the number of branches, and/or decreased the stipule length. These results question both the underlying mechanisms of the joint control of biotic stress resistance, abiotic stress tolerance, and plant architecture and phenology and the methods of marker-assisted selection optimizing stress control and productivity in winter pea breeding.

A QTL approach in faba bean highlights the conservation of genetic control of frost tolerance among legume species.

Frost is a major abiotic stress of winter type faba beans (Vica faba L.) and has adverse effects on crop yield. Climate change, far from reducing the incidence of frost events, is making these phenomena more and more common, severe, and prolonged. Despite the important interaction that the environment has in the tolerance of faba bean to frost, this trait seems to have good levels of heritability. Several QTLs for frost tolerance have already been reported, however, a more robust identification is needed to more precisely identify the genomic regions involved in faba bean tolerance to sub-zero temperatures. Several pea (Pisum sativum L.) and barrel medic (Medicago truncatula L.) frost tolerance QTLs appear to be conserved between these two species, furthering the hypothesis that the genetic control of frost tolerance in legume species might be more generally conserved. In this work, the QTL mapping in two faba bean recombinant inbred line (RIL) populations connected by a common winter-type parent has led to the identification of five genomic regions involved in the control of frost tolerance on linkage groups I, III, IV, and V. Among them, a major and robust QTL of great interest for marker-assisted selection was identified on the lower part of the long-arm of LGI. The synteny between the faba bean frost tolerance QTLs and those previously identified in other legume species such as barrel medic, pea or soybean highlighted at least partial conservation of the genetic control of frost tolerance among different faba bean genetic pools and legume species. Four novel RILs showing high and stable levels of tolerance and the ability to recover from freezing temperatures by accumulating frost tolerance QTLs are now available for breeding programs.

Integrated sRNA-seq and RNA-seq Analyses Reveal a microRNA Regulation Network Involved in Cold Response in Pisum sativum L.

(1) Background: Cold stress affects growth and development in plants and is a major environmental factor that decreases productivity. Over the past two decades, the advent of next generation sequencing (NGS) technologies has opened new opportunities to understand the molecular bases of stress resistance by enabling the detection of weakly expressed transcripts and the identification of regulatory RNAs of gene expression, including microRNAs (miRNAs). (2) Methods: In this study, we performed time series sRNA and mRNA sequencing experiments on two pea (Pisum sativum L., Ps) lines, Champagne frost-tolerant and Térèse frost-sensitive, during a low temperature treatment versus a control condition. (3) Results: An integrative analysis led to the identification of 136 miRNAs and a regulation network composed of 39 miRNA/mRNA target pairs with discordant expression patterns. (4) Conclusions: Our findings indicate that the cold response in pea involves 11 miRNA families as well as their target genes related to antioxidative and multi-stress defense mechanisms and cell wall biosynthesis.

Genome-wide association study identifies favorable SNP alleles and candidate genes for frost tolerance in pea.

BACKGROUND: Frost is a limiting abiotic stress for the winter pea crop (Pisum sativum L.) and identifying the genetic determinants of frost tolerance is a major issue to breed varieties for cold northern areas. Quantitative trait loci (QTLs) have previously been detected from bi-parental mapping populations, giving an overview of the genome regions governing this trait. The recent development of high-throughput genotyping tools for pea brings the opportunity to undertake genetic association studies in order to capture a higher allelic diversity within large collections of genetic resources as well as to refine the localization of the causal polymorphisms thanks to the high marker density. In this study, a genome-wide association study (GWAS) was performed using a set of 365 pea accessions. Phenotyping was carried out by scoring frost damages in the field and in controlled conditions. The association mapping collection was also genotyped using an Illumina Infinium® BeadChip, which allowed to collect data for 11,366 single nucleotide polymorphism (SNP) markers. RESULTS: GWAS identified 62 SNPs significantly associated with frost tolerance and distributed over six of the seven pea linkage groups (LGs). These results confirmed 3 QTLs that were already mapped in multiple environments on LG III, V and VI with bi-parental populations. They also allowed to identify one locus, on LG II, which has not been detected yet and two loci, on LGs I and VII, which have formerly been detected in only one environment. Fifty candidate genes corresponding to annotated significant SNPs, or SNPs in strong linkage disequilibrium with the formers, were found to underlie the frost damage (FD)-related loci detected by GWAS. Additionally, the analyses allowed to define favorable haplotypes of markers for the FD-related loci and their corresponding accessions within the association mapping collection. CONCLUSIONS: This study led to identify FD-related loci as well as corresponding favorable haplotypes of markers and representative pea accessions that might to be used in winter pea breeding programs. Among the candidate genes highlighted at the identified FD-related loci, the results also encourage further attention to the presence of C-repeat Binding Factors (CBF) as potential genetic determinants of the frost tolerance locus on LG VI.

Identification of Genes Differentially Expressed in Response to Cold in Pisum sativum Using RNA Sequencing Analyses.

Low temperature stress affects growth and development in pea (Pisum sativum L.) and decreases yield. In this study, RNA sequencing time series analyses performed on lines, Champagne frost-tolerant and Térèse frost-sensitive, during a low temperature treatment versus a control condition, led us to identify 4981 differentially expressed genes. Thanks to our experimental design and statistical analyses, we were able to classify these genes into three sets. The first one was composed of 2487 genes that could be related to the constitutive differences between the two lines and were not regulated during cold treatment. The second gathered 1403 genes that could be related to the chilling response. The third set contained 1091 genes, including genes that could be related to freezing tolerance. The identification of differentially expressed genes related to cold, oxidative stress, and dehydration responses, including some transcription factors and kinases, confirmed the soundness of our analyses. In addition, we identified about one hundred genes, whose expression has not yet been linked to cold stress. Overall, our findings showed that both lines have different characteristics for their cold response (chilling response and/or freezing tolerance), as more than 90% of differentially expressed genes were specific to each of them.

Genomic Tools in Pea Breeding Programs: Status and Perspectives.

Pea (Pisum sativum L.) is an annual cool-season legume and one of the oldest domesticated crops. Dry pea seeds contain 22-25% protein, complex starch and fiber constituents, and a rich array of vitamins, minerals, and phytochemicals which make them a valuable source for human consumption and livestock feed. Dry pea ranks third to common bean and chickpea as the most widely grown pulse in the world with more than 11 million tons produced in 2013. Pea breeding has achieved great success since the time of Mendel's experiments in the mid-1800s. However, several traits still require significant improvement for better yield stability in a larger growing area. Key breeding objectives in pea include improving biotic and abiotic stress resistance and enhancing yield components and seed quality. Taking advantage of the diversity present in the pea genepool, many mapping populations have been constructed in the last decades and efforts have been deployed to identify loci involved in the control of target traits and further introgress them into elite breeding materials. Pea now benefits from next-generation sequencing and high-throughput genotyping technologies that are paving the way for genome-wide association studies and genomic selection approaches. This review covers the significant development and deployment of genomic tools for pea breeding in recent years. Future prospects are discussed especially in light of current progress toward deciphering the pea genome.

Structural alteration of cell wall pectins accompanies pea development in response to cold.

Pea (Pisum sativum) cell wall metabolism in response to chilling was investigated in a frost-sensitive genotype 'Terese' and a frost-tolerant genotype 'Champagne'. Cell walls isolated from stipules of cold acclimated and non-acclimated plants showed that cold temperatures induce changes in polymers containing xylose, arabinose, galactose and galacturonic acid residues. In the tolerant cultivar Champagne, acclimation is accompanied by increases in homogalacturonan, xylogalacturonan and highly branched Rhamnogalacturonan I with branched and unbranched (1→5)-α-arabinans and (1→4)-ß-galactans. In contrast, the sensitive cultivar Terese accumulates substantial amounts of (1→4)-ß-xylans and glucuronoxylan, but not the pectins. Greater JIM7 labeling was observed in Champagne compared to Terese, indicating that cold acclimation also induces an increase in the degree of methylesterification of pectins. Significant decrease in polygalacturonase activities in both genotypes were observed at the end of cold acclimation. These data indicate a role for esterified pectins in cold tolerance. The possible functions for pectins and their associated arabinans and galactans in cold acclimation are discussed.

QTL analysis of frost damage in pea suggests different mechanisms involved in frost tolerance.

KEY MESSAGE: Avoidance mechanisms and intrinsic resistance are complementary strategies to improve winter frost tolerance and yield potential in field pea. The development of the winter pea crop represents a major challenge to expand plant protein production in temperate areas. Breeding winter cultivars requires the combination of freezing tolerance as well as high seed productivity and quality. In this context, we investigated the genetic determinism of winter frost tolerance and assessed its genetic relationship with yield and developmental traits. Using a newly identified source of frost resistance, we developed a population of recombinant inbred lines and evaluated it in six environments in Dijon and Clermont-Ferrand between 2005 and 2010. We developed a genetic map comprising 679 markers distributed over seven linkage groups and covering 947.1 cM. One hundred sixty-one quantitative trait loci (QTL) explaining 9-71 % of the phenotypic variation were detected across the six environments for all traits measured. Two clusters of QTL mapped on the linkage groups III and one cluster on LGVI reveal the genetic links between phenology, morphology, yield-related traits and frost tolerance in winter pea. QTL clusters on LGIII highlighted major developmental gene loci (Hr and Le) and the QTL cluster on LGVI explained up to 71 % of the winter frost damage variation. This suggests that a specific architecture and flowering ideotype defines frost tolerance in winter pea. However, two consistent frost tolerance QTL on LGV were independent of phenology and morphology traits, showing that different protective mechanisms are involved in frost tolerance. Finally, these results suggest that frost tolerance can be bred independently to seed productivity and quality.

Transcriptome sequencing for high throughput SNP development and genetic mapping in Pea.

BACKGROUND: Pea has a complex genome of 4.3 Gb for which only limited genomic resources are available to date. Although SNP markers are now highly valuable for research and modern breeding, only a few are described and used in pea for genetic diversity and linkage analysis. RESULTS: We developed a large resource by cDNA sequencing of 8 genotypes representative of modern breeding material using the Roche 454 technology, combining both long reads (400 bp) and high coverage (3.8 million reads, reaching a total of 1,369 megabases). Sequencing data were assembled and generated a 68 K unigene set, from which 41 K were annotated from their best blast hit against the model species Medicago truncatula. Annotated contigs showed an even distribution along M. truncatula pseudochromosomes, suggesting a good representation of the pea genome. 10 K pea contigs were found to be polymorphic among the genetic material surveyed, corresponding to 35 K SNPs.We validated a subset of 1538 SNPs through the GoldenGate assay, proving their ability to structure a diversity panel of breeding germplasm. Among them, 1340 were genetically mapped and used to build a new consensus map comprising a total of 2070 markers. Based on blast analysis, we could establish 1252 bridges between our pea consensus map and the pseudochromosomes of M. truncatula, which provides new insight on synteny between the two species. CONCLUSIONS: Our approach created significant new resources in pea, i.e. the most comprehensive genetic map to date tightly linked to the model species M. truncatula and a large SNP resource for both academic research and breeding.

A tandem array of CBF/DREB1 genes is located in a major freezing tolerance QTL region on Medicago truncatula chromosome 6.

BACKGROUND: Freezing provokes severe yield losses to different fall-sown annual legumes. Understanding the molecular bases of freezing tolerance is of great interest for breeding programs. Medicago truncatula Gaertn. is an annual temperate forage legume that has been chosen as a model species for agronomically and economically important legume crops. The present study aimed to identify positional candidate genes for a major freezing tolerance quantitative trait locus that was previously mapped to M. truncatula chromosome 6 (Mt-FTQTL6) using the LR3 population derived from a cross between the freezing-tolerant accession F83005-5 and the freezing-sensitive accession DZA045-5. RESULTS: The confidence interval of Mt-FTQTL6 was narrowed down to the region comprised between markers MTIC153 and NT6054 using recombinant F7 and F8 lines. A bacterial-artificial chromosome (BAC) clone contig map was constructed in an attempt to close the residual assembly gap existing therein. Twenty positional candidate genes including twelve C-repeat binding factor (CBF)/dehydration-responsive element binding factor 1 (DREB1) genes were identified from BAC-derived sequences and whole-genome shotgun sequences (WGS). CBF/DREB1 genes are organized in a tandem array within an approximately 296-Kb region. Eleven CBF/DREB1 genes were isolated and sequenced from F83005-5 and DZA045-5 which revealed high polymorphism among these accessions. Unique features characterizing CBF/DREB1 genes from M. truncatula, such as alternative splicing and large tandem duplication, are elucidated for the first time. CONCLUSIONS: Overall, twenty genes were identified as potential candidates to explain Mt-FTQTL6 effect. Their future functional characterization will uncover the gene(s) involved in freezing tolerance difference observed between F83005-5 and DZA045-5. Knowledge transfer for breeding improvement of crop legumes is expected. Furthermore, CBF/DREB1 related data will certainly have a large impact on research studies targeting this group of transcriptional activators in M. truncatula and other legume species.

Genetic variability and QTL mapping of freezing tolerance and related traits in Medicago truncatula.

Freezing is a major environmental limitation to crop productivity for a number of species including legumes. We investigated the genetic determinism of freezing tolerance in the model legume Medicago truncatula Gaertn (M. truncatula). After having observed a large variation for freezing tolerance among 15 M. truncatula accessions, the progeny of a F6 recombinant inbred line population, derived from a cross between two accessions, was acclimated to low above-freezing temperatures and assessed for: (a) number of leaves (NOL), leaf area (LA), chlorophyll content index (CCI), shoot and root dry weights (SDW and RDW) at the end of the acclimation period and (b) visual freezing damage (FD) during the freezing treatment and 2 weeks after regrowth and foliar electrolyte leakage (EL) 2 weeks after regrowth. Consistent QTL positions with additive effects for FD were found on LG1, LG4 and LG6, the latter being the most explanatory (R (2) ≈ 40 %). QTL for NOL, QTL for EL, NOL and RDW, and QTL for EL and CCI colocalized with FD QTL on LG1, LG4 and LG6, respectively. Favorable alleles for these additive effects were brought by the same parent suggesting that this accession contributes to superior freezing tolerance by affecting plants' capacity to maintain growth at low above-freezing temperatures. No epistatic effects were found between FD QTL, but for each of the studied traits, 3-6 epistatic effects were detected between loci not detected directly as QTL. These results open the way to the assessment of syntenic relationships between QTL for frost tolerance in M. truncatula and cultivated legume species.

Combining gene expression and genetic analyses to identify candidate genes involved in cold responses in pea.

Cold stress affects plant growth and development. In order to better understand the responses to cold (chilling or freezing tolerance), we used two contrasted pea lines. Following a chilling period, the Champagne line becomes tolerant to frost whereas the Terese line remains sensitive. Four suppression subtractive hybridisation libraries were obtained using mRNAs isolated from pea genotypes Champagne and Terese. Using quantitative polymerase chain reaction (qPCR) performed on 159 genes, 43 and 54 genes were identified as differentially expressed at the initial time point and during the time course study, respectively. Molecular markers were developed from the differentially expressed genes and were genotyped on a population of 164 RILs derived from a cross between Champagne and Terese. We identified 5 candidate genes colocalizing with 3 different frost damage quantitative trait loci (QTL) intervals and a protein quantity locus (PQL) rich region previously reported. This investigation revealed the role of constitutive differences between both genotypes in the cold responses, in particular with genes related to glycine degradation pathway that could confer to Champagne a better frost tolerance. We showed that freezing tolerance involves a decrease of expression of genes related to photosynthesis and the expression of a gene involved in the production of cysteine and methionine that could act as cryoprotectant molecules. Although it remains to be confirmed, this study could also reveal the involvement of the jasmonate pathway in the cold responses, since we observed that two genes related to this pathway were mapped in a frost damage QTL interval and in a PQL rich region interval, respectively.

Exploring chloroplastic changes related to chilling and freezing tolerance during cold acclimation of pea (Pisum sativum L.).

Pea (Pisum sativum L.) productivity is linked to its ability to cope with abiotic stresses such as low temperatures during fall and winter. In this study, we investigate the chloroplast-related changes occurring during pea cold acclimation, in order to further lead to genetic improvement of its field performance. Champagne and Térèse, two pea lines with different acclimation capabilities, were studied by physiological measurements, sub-cellular fractionation followed by relative protein quantification and two-dimensional DIGE. The chilling tolerance might be related to an increase in protein related to soluble sugar synthesis, antioxidant potential, regulation of mRNA transcription and translation through the chloroplast. Freezing tolerance, only observed in Champagne, seems to rely on a higher inherent photosynthetic potential at the beginning of the cold exposure, combined with an early ability to start metabolic processes aimed at maintaining the photosynthetic capacity, optimizing the stoichiometry of the photosystems and inducing dynamic changes in carbohydrate and protein synthesis and/or turnover.

Cell wall compositional modifications of Miscanthus ecotypes in response to cold acclimation.

Miscanthus, a potential energy crop grass, can be damaged by late frost when shoots emerge too early in the spring and during the first winter after planting. The effects of cold acclimation on cell wall composition were investigated in a frost-sensitive clone of Miscanthus x giganteus compared to frost-tolerant clone, Miscanthus sinensis August Feder, and an intermediate frost-tolerant clone, M. sinensis Goliath. Cellulose and lignin contents were higher in M. x giganteus than in the M. sinensis genotypes. In ambient temperature controls, each clone displayed different glucuronoarabinoxylan (GAX) contents and degree of arabinose substitution on the xylan backbone. During cold acclimation, an increase in (1→3),(1→4)-ß-D-glucan content was observed in all genotypes. Uronic acid level increased in the frost sensitive genotype but decreased in the frost tolerant genotypes in response to cold. In all clones, major changes in cell wall composition were observed with modifications in phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) activities in both non- and cold-acclimated experiments. A large increase in CAD activity under cold stress was displayed in each clone, but it was largest in the frost-tolerant clone, M. sinensis August Feder. The marked increase in PAL activity observed in the frost-tolerant clones under cold acclimation, suggests a reorientation of the products towards the phenylpropanoid pathway or aromatic synthesis. How changes in cell wall physical properties can impact frost tolerance is discussed.

A conserved molecular basis for photoperiod adaptation in two temperate legumes.

Legumes were among the first plant species to be domesticated, and accompanied cereals in expansion of agriculture from the Fertile Crescent into diverse environments across the Mediterranean basin, Europe, Central Asia, and the Indian subcontinent. Although several recent studies have outlined the molecular basis for domestication and eco-geographic adaptation in the two main cereals from this region, wheat and barley, similar questions remain largely unexplored in their legume counterparts. Here we identify two major loci controlling differences in photoperiod response between wild and domesticated pea, and show that one of these, high response to photoperiod (HR), is an ortholog of early flowering 3 (ELF3), a gene involved in circadian clock function. We found that a significant proportion of flowering time variation in global pea germplasm is controlled by HR, with a single, widespread functional variant conferring altered circadian rhythms and the reduced photoperiod response associated with the spring habit. We also present evidence that ELF3 has a similar role in lentil, another major legume crop, with a distinct functional variant contributing to reduced photoperiod response in cultivars widely deployed in short-season environments. Our results identify the factor likely to have permitted the successful prehistoric expansion of legume cultivation to Northern Europe, and define a conserved genetic basis for major adaptive changes in flowering phenology and growth habit in an important crop group.

A proteomic approach to decipher chilling response from cold acclimation in pea (Pisum sativum L.).

Two pea lines (Pisum sativum L.) with contrasted behaviours towards chilling and subsequent frost were studied by a proteomic approach to better understand cold acclimation. Following a chilling period, the Champagne line becomes tolerant to frost whereas Terese remains sensitive. Variance analysis allowed to select 260 statistically variable spots with 68 identified proteins (35 in leaves, 18 in stems, and 15 in roots). These proteins were shared out in proteins related to chilling response or cold acclimation. The better adaptation of Champagne to chilling might be related to a higher content in proteins involved in photosynthesis and in defence mechanisms. Moreover Champagne might prevent freezing damage particularly thanks to a higher constitutive expression of housekeeping proteins related to Terese. After three days of subsequent frost, proteomes of previously chilled plants also showed significant differences compared to unchilled plants. Out of 112 statistically variable spots (44 in leaves, 38 in stems, and 30 in roots), 32 proteins were identified. These proteins were related to frost response or frost resistance. It seems that Champagne could resist to frost with the reorientation of the energy metabolism.

Highly-multiplexed SNP genotyping for genetic mapping and germplasm diversity studies in pea.

BACKGROUND: Single Nucleotide Polymorphisms (SNPs) can be used as genetic markers for applications such as genetic diversity studies or genetic mapping. New technologies now allow genotyping hundreds to thousands of SNPs in a single reaction.In order to evaluate the potential of these technologies in pea, we selected a custom 384-SNP set using SNPs discovered in Pisum through the resequencing of gene fragments in different genotypes and by compiling genomic sequence data present in databases. We then designed an Illumina GoldenGate assay to genotype both a Pisum germplasm collection and a genetic mapping population with the SNP set. RESULTS: We obtained clear allelic data for more than 92% of the SNPs (356 out of 384). Interestingly, the technique was successful for all the genotypes present in the germplasm collection, including those from species or subspecies different from the P. sativum ssp sativum used to generate sequences. By genotyping the mapping population with the SNP set, we obtained a genetic map and map positions for 37 new gene markers. CONCLUSION: Our results show that the Illumina GoldenGate assay can be used successfully for high-throughput SNP genotyping of diverse germplasm in pea. This genotyping approach will simplify genotyping procedures for association mapping or diversity studies purposes and open new perspectives in legume genomics.

Association of sugar content QTL and PQL with physiological traits relevant to frost damage resistance in pea under field and controlled conditions.

To increase yield in pea (Pisum sativum L.), autumn sowing would be preferable. Hence, frost tolerance of pea became a major trait of interest for breeders. In order to better understand the cold acclimation in pea, Champagne a frost tolerant line and Terese, a frost sensitive line, and their recombinant inbred lines (RIL) were studied. RIL frost tolerance was evaluated by a frost damage scale under field as well as controlled conditions. A quantitative trait loci (QTL) approach was used to identify chromosomal regions linked to frost tolerance. The detected QTL explained from 6.5 to 46.5% of the phenotypic variance. Amongst them, those located on linkage groups 5 and 6 were consistent with over all experiments, in field as well as in controlled environments. In order to improve the understanding of the frost tolerance mechanisms, several cold acclimation key characters such as concentration of sugars, electrolyte leakage, osmotic pressure, and activity of RuBisCO were assessed. Some of these physiological QTL colocalised with QTL for frost damage, in particular two raffinose QTL on LG5 and LG6 and one RuBisCO activity QTL on LG6, explaining 8.8 to 27.0% of the phenotypic variance. In addition, protein quantitative loci were mapped; some of them colocalised with frost damage and physiological QTL on LG5 and LG6, explaining 16.0-43.6% of the phenotypic variance. Raffinose metabolism and RuBisCO activity and its effect on photosynthesis might play a major role in cold acclimation of pea.