The giant diploid faba genome unlocks variation in a global protein crop.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.

afila, the origin and nature of a major innovation in the history of pea breeding.

The afila (af) mutation causes the replacement of leaflets by a branched mass of tendrils in the compound leaves of pea - Pisum sativum L. This mutation was first described in 1953, and several reports of spontaneous af mutations and induced mutants with a similar phenotype exist. Despite widespread introgression into breeding material, the nature of af and the origin of the alleles used remain unknown. Here, we combine comparative genomics with reverse genetic approaches to elucidate the genetic determinants of af. We also investigate haplotype diversity using a set of AfAf and afaf cultivars and breeding lines and molecular markers linked to seven consecutive genes. Our results show that deletion of two tandemly arranged genes encoding Q-type Cys(2)His(2) zinc finger transcription factors, PsPALM1a and PsPALM1b, is responsible for the af phenotype in pea. Eight haplotypes were identified in the af-harbouring genomic region on chromosome 2. These haplotypes differ in the size of the deletion, covering more or less genes. Diversity at the af locus is valuable for crop improvement and sheds light on the history of pea breeding for improved standing ability. The results will be used to understand the function of PsPALM1a/b and to transfer the knowledge for innovation in related crops.

MtEFD and MtEFD2: Two transcription factors with distinct neofunctionalization in symbiotic nodule development.

Rhizobium-legume nitrogen-fixing symbiosis involves the formation of a specific organ, the root nodule, which provides bacteria with the proper cellular environment for atmospheric nitrogen fixation. Coordinated differentiation of plant and bacterial cells is an essential step of nodule development, for which few transcriptional regulators have been characterized. Medicago truncatula ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION (MtEFD) encodes an APETALA2/ETHYLENE RESPONSIVE FACTOR (ERF) transcription factor, the mutation of which leads to both hypernodulation and severe defects in nodule development. MtEFD positively controls a negative regulator of cytokinin signaling, the RESPONSE REGULATOR 4 (MtRR4) gene. Here we showed that that the Mtefd-1 mutation affects both plant and bacterial endoreduplication in nodules, as well as the expression of hundreds of genes in young and mature nodules, upstream of known regulators of symbiotic differentiation. MtRR4 expressed with the MtEFD promoter complemented Mtefd-1 hypernodulation but not the nodule differentiation phenotype. Unexpectedly, a nonlegume homolog of MtEFD, AtERF003 in Arabidopsis (Arabidopsis thaliana), could efficiently complement both phenotypes of Mtefd-1, in contrast to the MtEFD paralog MtEFD2 expressed in the root and nodule meristematic zone. A domain swap experiment showed that MtEFD2 differs from MtEFD by its C-terminal fraction outside the DNA binding domain. Furthermore, clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) mutagenesis of MtEFD2 led to a reduction in the number of nodules formed in Mtefd-1, with downregulation of a set of genes, including notably NUCLEAR FACTOR-YA1 (MtNF-YA1) and MtNF-YB16, which are essential for nodule meristem establishment. We, therefore, conclude that nitrogen-fixing symbiosis recruited two proteins originally expressed in roots, MtEFD and MtEFD2, with distinct functions and neofunctionalization processes for each of them.

Integration of the SMXL/D53 strigolactone signalling repressors in the model of shoot branching regulation in Pisum sativum.

DWARF53 (D53) in rice (Oryza sativa) and its homologs in Arabidopsis (Arabidopsis thaliana), SUPPRESSOR OF MAX2-LIKE 6 (SMXL6), SMXL7 and SMXL8, are well established negative regulators of strigolactone (SL) signalling in shoot branching regulation. Little is known of pea (Pisum sativum) homologs and whether D53 and related SMXLs are specific to SL signalling pathways. Here, we identify two allelic pea mutants, dormant3 (dor3), and demonstrate through gene mapping and sequencing that DOR3 corresponds to a homolog of D53 and SMXL6/SMXL7, designated PsSMXL7. Phenotype analysis, gene expression, protein and hormone quantification assays were performed to determine the role of PsSMXL7 in regulation of bud outgrowth and the role of PsSMXL7 and D53 in integrating SL and cytokinin (CK) responses. Like D53 and related SMXLs, we show that PsSMXL7 can be degraded by SL and induces feedback upregulation of PsSMXL7 transcript. Here we reveal a system conserved in pea and rice, whereby CK also upregulates PsSMXL7/D53 transcripts, providing a clear mechanism for SL and CK cross-talk in the regulation of branching. To further deepen our understanding of the branching network in pea, we provide evidence that SL acts via PsSMXL7 to modulate auxin content via PsAFB5, which itself regulates expression of SL biosynthesis genes. We therefore show that PsSMXL7 is key to a triple hormone network involving an auxin-SL feedback mechanism and SL-CK cross-talk.

A major-effect genetic locus, ApRVII, controlling resistance against both adapted and non-adapted aphid biotypes in pea.

KEY MESSAGE: A genome-wide association study for pea resistance against a pea-adapted biotype and a non-adapted biotype of the aphid, Acyrthosiphon pisum, identified a genomic region conferring resistance to both biotypes. In a context of reduced insecticide use, the development of cultivars resistant to insect pests is crucial for an integrated pest management. Pea (Pisum sativum) is a crop of major importance among cultivated legumes, for the supply of dietary proteins and nitrogen in low-input cropping systems. However, yields of the pea crop have become unstable due to plant parasites. The pea aphid (Acyrthosiphon pisum) is an insect pest species forming a complex of biotypes, each one adapted to feed on one or a few related legume species. This study aimed to identify resistance to A. pisum and the underlying genetic determinism by examining a collection of 240 pea genotypes. The collection was screened against a pea-adapted biotype and a non-adapted biotype of A. pisum to characterize their resistant phenotype. Partial resistance was observed in some pea genotypes exposed to the pea-adapted biotype. Many pea genotypes were completely resistant to non-adapted biotype, but some exhibited partial susceptibility. A genome-wide association study, using pea exome-capture sequencing data, enabled the identification of the major-effect quantitative trait locus ApRVII on the chromosome 7. ApRVII includes linkage disequilibrium blocks significantly associated with resistance to one or both of the two aphid biotypes studied. Finally, we identified candidate genes underlying ApRVII that are potentially involved in plant-aphid interactions and marker haplotypes linked with aphid resistance. This study sets the ground for the functional characterization of molecular pathways involved in pea defence to the aphids but also is a step forward for breeding aphid-resistant cultivars.

Nitric Oxide Metabolic Pathway in Drought-Stressed Nodules of Faba Bean (Vicia faba L.).

Drought is an environmental stress that strongly impacts plants. It affects all stages of growth and induces profound disturbances that influence all cellular functions. Legumes can establish a symbiosis with Rhizobium-type bacteria, whose function is to fix atmospheric nitrogen in organs called nodules and to meet plant nitrogen needs. Symbiotic nitrogen fixation (SNF) is particularly sensitive to drought. We raised the hypothesis that, in drought-stressed nodules, SNF inhibition is partly correlated to hypoxia resulting from nodule structure compaction and an increased O2 diffusion barrier, and that the nodule energy regeneration involves phytoglobin-nitric oxide (Pgb-NO) respiration. To test this hypothesis, we subjected faba bean (Vicia faba L.) plants nodulated with a Rhizobium laguerreae strain to either drought or osmotic stress. We monitored the N2-fixation activity, the energy state (ATP/ADP ratio), the expression of hypoxia marker genes (alcohol dehydrogenase and alanine aminotransferase), and the functioning of the Pgb-NO respiration in the nodules. The collected data confirmed our hypothesis and showed that (1) drought-stressed nodules were subject to more intense hypoxia than control nodules and (2) NO production increased and contributed via Pgb-NO respiration to the maintenance of the energy state of drought-stressed nodules.

ß-Amyrin Synthase1 Controls the Accumulation of the Major Saponins Present in Pea (Pisum sativum).

The use of pulses as ingredients for the production of food products rich in plant proteins is increasing. However, protein fractions prepared from pea or other pulses contain significant amounts of saponins, glycosylated triterpenes that can impart an undesirable bitter taste when used as an ingredient in foodstuffs. In this article, we describe the identification and characterization of a gene involved in saponin biosynthesis during pea seed development, by screening mutants obtained from two Pisum sativum TILLING (Targeting Induced Local Lesions IN Genomes) populations in two different genetic backgrounds. The mutations studied are located in a gene designated PsBAS1 (ß-amyrin synthase1), which is highly expressed in maturing pea seeds and which encodes a protein previously shown to correspond to an active ß-amyrin synthase. The first allele is a nonsense mutation, while the second mutation is located in a splice site and gives rise to a mis-spliced transcript encoding a truncated, nonfunctional protein. The homozygous mutant seeds accumulated virtually no saponin without affecting the seed nutritional or physiological quality. Interestingly, BAS1 appears to control saponin accumulation in all other tissues of the plant examined. These lines represent a first step in the development of pea varieties lacking bitterness off-flavors in their seeds. Our work also shows that TILLING populations in different genetic backgrounds represent valuable genetic resources for both crop improvement and functional genomics.

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.

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop.

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner.

Identification of LATE BLOOMER2 as a CYCLING DOF FACTOR Homolog Reveals Conserved and Divergent Features of the Flowering Response to Photoperiod in Pea.

The molecular pathways responsible for the flowering response to photoperiod have been extensively studied in Arabidopsis thaliana and cereals but remain poorly understood in other major plant groups. Here, we describe a dominant mutant at the LATE BLOOMER2 (LATE2) locus in pea (Pisum sativum) that is late-flowering with a reduced response to photoperiod. LATE2 acts downstream of light signaling and the circadian clock to control expression of the main photoperiod-regulated FT gene, FTb2, implying that it plays a primary role in photoperiod measurement. Mapping identified the CYCLING DOF FACTOR gene CDFc1 as a strong candidate for LATE2, and the late2-1D mutant was found to carry a missense mutation in CDFc1 that impairs its capacity to bind to the blue-light photoreceptor FKF1 in yeast two-hybrid assays and delays flowering in Arabidopsis when overexpressed. Arabidopsis CDF genes are important negative regulators of CONSTANS (CO) transcription, but we found no effect of LATE2 on the transcription of pea CO-LIKE genes, nor on genes in any other families previously implicated in the activation of FT in Arabidopsis. Our results reveal an important component of the pea photoperiod response pathway and support the view that regulation of FTb2 expression by photoperiod occurs via a CO-independent mechanism.

EARLY FLOWERING3 Redundancy Fine-Tunes Photoperiod Sensitivity.

Three pea (Pisum sativum) loci controlling photoperiod sensitivity, HIGH RESPONSE (HR), DIE NEUTRALIS (DNE), and STERILE NODES (SN), have recently been shown to correspond to orthologs of Arabidopsis (Arabidopsis thaliana) circadian clock genes EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHMO, respectively. A fourth pea locus, PHOTOPERIOD (PPD), also contributes to the photoperiod response in a similar manner to SN and DNE, and recessive ppd mutants on a spring-flowering hr mutant background show early, photoperiod-insensitive flowering. However, the molecular identity of PPD has so far remained elusive. Here, we show that the PPD locus also has a role in maintenance of diurnal and circadian gene expression rhythms and identify PPD as an ELF3 co-ortholog, termed ELF3b Genetic interactions between pea ELF3 genes suggest that loss of PPD function does not affect flowering time in the presence of functional HR, whereas PPD can compensate only partially for the lack of HR These results provide an illustration of how gene duplication and divergence can generate potential for the emergence of more subtle variations in phenotype that may be adaptively significant.

Evidence that auxin is required for normal seed size and starch synthesis in pea.

In recent years the biosynthesis of auxin has been clarified with the aid of mutations in auxin biosynthesis genes. However, we know little about the effects of these mutations on the seed-filling stage of seed development. Here we investigate a key auxin biosynthesis mutation of the garden pea, which results in auxin deficiency in developing seeds. We exploit the large seed size of this model species, which facilitates the measurement of compounds in individual seeds. The mutation results in small seeds with reduced starch content and a wrinkled phenotype at the dry stage. The phenotypic effects of the mutation were fully reversed by introduction of the wild-type gene as a transgene, and partially reversed by auxin application. The results indicate that auxin is required for normal seed size and starch accumulation in pea, an important grain legume crop.

Full-length de novo assembly of RNA-seq data in pea (Pisum sativum L.) provides a gene expression atlas and gives insights into root nodulation in this species.

Next-generation sequencing technologies allow an almost exhaustive survey of the transcriptome, even in species with no available genome sequence. To produce a Unigene set representing most of the expressed genes of pea, 20 cDNA libraries produced from various plant tissues harvested at various developmental stages from plants grown under contrasting nitrogen conditions were sequenced. Around one billion reads and 100 Gb of sequence were de novo assembled. Following several steps of redundancy reduction, 46 099 contigs with N50 length of 1667 nt were identified. These constitute the 'Caméor' Unigene set. The high depth of sequencing allowed identification of rare transcripts and detected expression for approximately 80% of contigs in each library. The Unigene set is now available online (http://bios.dijon.inra.fr/FATAL/cgi/pscam.cgi), allowing (i) searches for pea orthologs of candidate genes based on gene sequences from other species, or based on annotation, (ii) determination of transcript expression patterns using various metrics, (iii) identification of uncharacterized genes with interesting patterns of expression, and (iv) comparison of gene ontology pathways between tissues. This resource has allowed identification of the pea orthologs of major nodulation genes characterized in recent years in model species, as a major step towards deciphering unresolved pea nodulation phenotypes. In addition to a remarkable conservation of the early transcriptome nodulation apparatus between pea and Medicago truncatula, some specific features were highlighted. The resource provides a reference for the pea exome, and will facilitate transcriptome and proteome approaches as well as SNP discovery in pea.

Development of two major resources for pea genomics: the GenoPea 13.2K SNP Array and a high-density, high-resolution consensus genetic map.

Single nucleotide polymorphism (SNP) arrays represent important genotyping tools for innovative strategies in both basic research and applied breeding. Pea is an important food, feed and sustainable crop with a large (about 4.45 Gbp) but not yet available genome sequence. In the present study, 12 pea recombinant inbred line populations were genotyped using the newly developed GenoPea 13.2K SNP Array. Individual and consensus genetic maps were built providing insights into the structure and organization of the pea genome. Largely collinear genetic maps of 3918-8503 SNPs were obtained from all mapping populations, and only two of these exhibited putative chromosomal rearrangement signatures. Similar distortion patterns in different populations were noted. A total of 12 802 transcript-derived SNP markers placed on a 15 079-marker high-density, high-resolution consensus map allowed the identification of ohnologue-rich regions within the pea genome and the localization of local duplicates. Dense syntenic networks with sequenced legume genomes were further established, paving the way for the identification of the molecular bases of important agronomic traits segregating in the mapping populations. The information gained on the structure and organization of the genome from this research will undoubtedly contribute to the understanding of the evolution of the pea genome and to its assembly. The GenoPea 13.2K SNP Array and individual and consensus genetic maps are valuable genomic tools for plant scientists to strengthen pea as a model for genetics and physiology and enhance breeding.

Genome-wide association mapping of partial resistance to Aphanomyces euteiches in pea.

BACKGROUND: Genome-wide association (GWA) mapping has recently emerged as a valuable approach for refining the genetic basis of polygenic resistance to plant diseases, which are increasingly used in integrated strategies for durable crop protection. Aphanomyces euteiches is a soil-borne pathogen of pea and other legumes worldwide, which causes yield-damaging root rot. Linkage mapping studies reported quantitative trait loci (QTL) controlling resistance to A. euteiches in pea. However the confidence intervals (CIs) of these QTL remained large and were often linked to undesirable alleles, which limited their application in breeding. The aim of this study was to use a GWA approach to validate and refine CIs of the previously reported Aphanomyces resistance QTL, as well as identify new resistance loci. METHODS: A pea-Aphanomyces collection of 175 pea lines, enriched in germplasm derived from previously studied resistant sources, was evaluated for resistance to A. euteiches in field infested nurseries in nine environments and with two strains in climatic chambers. The collection was genotyped using 13,204 SNPs from the recently developed GenoPea Infinium® BeadChip. RESULTS: GWA analysis detected a total of 52 QTL of small size-intervals associated with resistance to A. euteiches, using the recently developed Multi-Locus Mixed Model. The analysis validated six of the seven previously reported main Aphanomyces resistance QTL and detected novel resistance loci. It also provided marker haplotypes at 14 consistent QTL regions associated with increased resistance and highlighted accumulation of favourable haplotypes in the most resistant lines. Previous linkages between resistance alleles and undesired late-flowering alleles for dry pea breeding were mostly confirmed, but the linkage between loci controlling resistance and coloured flowers was broken due to the high resolution of the analysis. A high proportion of the putative candidate genes underlying resistance loci encoded stress-related proteins and others suggested that the QTL are involved in diverse functions. CONCLUSION: This study provides valuable markers, marker haplotypes and germplasm lines to increase levels of partial resistance to A. euteiches in pea breeding.

Genetic diversity and trait genomic prediction in a pea diversity panel.

BACKGROUND: Pea (Pisum sativum L.), a major pulse crop grown for its protein-rich seeds, is an important component of agroecological cropping systems in diverse regions of the world. New breeding challenges imposed by global climate change and new regulations urge pea breeders to undertake more efficient methods of selection and better take advantage of the large genetic diversity present in the Pisum sativum genepool. Diversity studies conducted so far in pea used Simple Sequence Repeat (SSR) and Retrotransposon Based Insertion Polymorphism (RBIP) markers. Recently, SNP marker panels have been developed that will be useful for genetic diversity assessment and marker-assisted selection. RESULTS: A collection of diverse pea accessions, including landraces and cultivars of garden, field or fodder peas as well as wild peas was characterised at the molecular level using newly developed SNP markers, as well as SSR markers and RBIP markers. The three types of markers were used to describe the structure of the collection and revealed different pictures of the genetic diversity among the collection. SSR showed the fastest rate of evolution and RBIP the slowest rate of evolution, pointing to their contrasted mode of evolution. SNP markers were then used to predict phenotypes -the date of flowering (BegFlo), the number of seeds per plant (Nseed) and thousand seed weight (TSW)- that were recorded for the collection. Different statistical methods were tested including the LASSO (Least Absolute Shrinkage ans Selection Operator), PLS (Partial Least Squares), SPLS (Sparse Partial Least Squares), Bayes A, Bayes B and GBLUP (Genomic Best Linear Unbiased Prediction) methods and the structure of the collection was taken into account in the prediction. Despite a limited number of 331 markers used for prediction, TSW was reliably predicted. CONCLUSION: The development of marker assisted selection has not reached its full potential in pea until now. This paper shows that the high-throughput SNP arrays that are being developed will most probably allow for a more efficient selection in this species.

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.

Unexpectedly low nitrogen acquisition and absence of root architecture adaptation to nitrate supply in a Medicago truncatula highly branched root mutant.

To complement N2 fixation through symbiosis, legumes can efficiently acquire soil mineral N through adapted root architecture. However, root architecture adaptation to mineral N availability has been little studied in legumes. Therefore, this study investigated the effect of nitrate availability on root architecture in Medicago truncatula and assessed the N-uptake potential of a new highly branched root mutant, TR185. The effects of varying nitrate supply on both root architecture and N uptake were characterized in the mutant and in the wild type. Surprisingly, the root architecture of the mutant was not modified by variation in nitrate supply. Moreover, despite its highly branched root architecture, TR185 had a permanently N-starved phenotype. A transcriptome analysis was performed to identify genes differentially expressed between the two genotypes. This analysis revealed differential responses related to the nitrate acquisition pathway and confirmed that N starvation occurred in TR185. Changes in amino acid content and expression of genes involved in the phenylpropanoid pathway were associated with differences in root architecture between the mutant and the wild type.

Gene-based SNP discovery and genetic mapping in pea.

KEY MESSAGE: Gene-based SNPs were identified and mapped in pea using five recombinant inbred line populations segregating for traits of agronomic importance. Pea (Pisum sativum L.) is one of the world's oldest domesticated crops and has been a model system in plant biology and genetics since the work of Gregor Mendel. Pea is the second most widely grown pulse crop in the world following common bean. The importance of pea as a food crop is growing due to its combination of moderate protein concentration, slowly digestible starch, high dietary fiber concentration, and its richness in micronutrients; however, pea has lagged behind other major crops in harnessing recent advances in molecular biology, genomics and bioinformatics, partly due to its large genome size with a large proportion of repetitive sequence, and to the relatively limited investment in research in this crop globally. The objective of this research was the development of a genome-wide transcriptome-based pea single-nucleotide polymorphism (SNP) marker platform using next-generation sequencing technology. A total of 1,536 polymorphic SNP loci selected from over 20,000 non-redundant SNPs identified using deep transcriptome sequencing of eight diverse Pisum accessions were used for genotyping in five RIL populations using an Illumina GoldenGate assay. The first high-density pea SNP map defining all seven linkage groups was generated by integrating with previously published anchor markers. Syntenic relationships of this map with the model legume Medicago truncatula and lentil (Lens culinaris Medik.) maps were established. The genic SNP map establishes a foundation for future molecular breeding efforts by enabling both the identification and tracking of introgression of genomic regions harbouring QTLs related to agronomic and seed quality traits.

Identification of QTLs associated with seed protein concentration in two diverse recombinant inbred line populations of pea.

Improving the seed protein concentration (SPC) of pea (Pisum sativum L.) has turned into an important breeding objective because of the consumer demand for plant-based protein and demand from protein fractionation industries. To support the marker-assisted selection (MAS) of SPC towards accelerated breeding of improved cultivars, we have explored two diverse recombinant inbred line (RIL) populations to identify the quantitative trait loci (QTLs) associated with SPC. The two RIL populations, MP 1918 × P0540-91 (PR-30) and Ballet × Cameor (PR-31), were derived from crosses between moderate SPC × high SPC accessions. A total of 166 and 159 RILs of PR-30 and PR-31, respectively, were genotyped using an Axiom® 90K SNP array and 13.2K SNP arrays, respectively. The RILs were phenotyped in replicated trials in two and three locations of Saskatchewan, Canada in 2020 and 2021, respectively, for agronomic assessment and SPC. Using composite interval mapping, we identified three QTLs associated with SPC in PR-30 and five QTLs in PR-31, with the LOD value ranging from 3.0 to 11.0. A majority of these QTLs were unique to these populations compared to the previously known QTLs for SPC. The QTL SPC-Ps-5.1 overlapped with the earlier reported SPC associated QTL PC-QTL-3. Three QTLs, SPC-Ps-4.2, SPC-Ps-5.1, and SPC-Ps-7.2 with LOD scores of 7.2, 7.9, and 11.3, and which explained 14.5%, 11.6%, and 11.3% of the phenotypic variance, respectively, can be used for marker-assisted breeding to increase SPC in peas. Eight QTLs associated with the grain yield were identified with LOD scores ranging from 3.1 to 8.2. Two sets of QTLs, SPC-Ps-2.1 and GY-Ps-2.1, and SPC-Ps-5.1 and GY-Ps-5.3, shared the QTL/peak regions. Each set of QTLs contributed to either SPC or grain yield depending on which parent the QTL region is derived from, thus confirming that breeding for SPC should take into consideration the effects on grain yield.