Julien Frouin

Genome-wide association mapping of root traits in a japonica rice panel.

Rice is a crop prone to drought stress in upland and rainfed lowland ecosystems. A deep root system is recognized as the best drought avoidance mechanism. Genome-wide association mapping offers higher resolution for locating quantitative trait loci (QTLs) than QTL mapping in biparental populations. We performed an association mapping study for root traits using a panel of 167 japonica accessions, mostly of tropical origin. The panel was genotyped at an average density of one marker per 22.5 kb using genotyping by sequencing technology. The linkage disequilibrium in the panel was high (r2>0.6, on average, for 20 kb mean distances between markers). The plants were grown in transparent 50 cm × 20 cm × 2 cm Plexiglas nailboard sandwiches filled with 1.5 mm glass beads through which a nutrient solution was circulated. Root system architecture and biomass traits were measured in 30-day-old plants. The panel showed a moderate to high diversity in the various traits, particularly for deep (below 30 cm depth) root mass and the number of deep roots. Association analyses were conducted using a mixed model involving both population structure and kinship to control for false positives. Nineteen associations were significant at P<1e-05, and 78 were significant at P<1e-04. The greatest numbers of significant associations were detected for deep root mass and the number of deep roots, whereas no significant associations were found for total root biomass or deep root proportion. Because several QTLs for different traits were co-localized, 51 unique loci were detected; several co-localized with meta-QTLs for root traits, but none co-localized with rice genes known to be involved in root growth. Several likely candidate genes were found in close proximity to these loci. Additional work is necessary to assess whether these markers are relevant in other backgrounds and whether the genes identified are robust candidates.

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.

Specific patterns of genetic diversity among aromatic rice varieties in Myanmar

BackgroundAfter observing peculiar rice varieties in Myanmar, in terms of classification in varietal groups and of grain quality, we focused on Myanmar varieties and analyzed variations at 19 microsatellite loci as well as sequences of the aroma gene BADH2.ResultsMicrosatellites were able to retrieve the well-established classification into Indica (isozyme group 1), Japonica (group 6, comprising temperate and tropical forms) and specific groups from the Himalayan foothills including some Aus varieties (group 2) and some aromatic varieties (group 5). They revealed a new cluster of accessions close to, but distinct from, non-Myanmar varieties in group 5. With reference to earlier terminology, we propose to distinguish a group “5A” including group 5 varieties from the Indian subcontinent (South and West Asia) and a group “5B” including most group 5 varieties from Myanmar. In Myanmar varieties, aroma was distributed in group 1 (Indica) and in group 5B. New BADH2 variants were found. Some accessions carried a 43 bp deletion in the 3’ UTR that was not completely associated with aroma. Other accessions, all of group 5B, displayed a particular BADH2 allele with a 3 bp insertion and 100% association with aroma.ConclusionWith the new group and the new alleles found in Myanmar varieties, our study shows that the Himalayan foothills contain series of non-Indica and non-Japonica varietal types with novel variations for useful traits.

Genome-wide association analysis for heat tolerance at flowering detected a large set of genes involved in adaptation to thermal and other stresses

Fertilization sensitivity to heat in rice is a major issue within climate change scenarios in the tropics. A panel of 167 indica landraces and improved varieties was phenotyped for spikelet sterility (SPKST) under 38°C during anthesis and for several secondary traits potentially affecting panicle micro-climate and thus the fertilization process. The panel was genotyped with an average density of one marker per 29 kb using genotyping by sequencing. Genome-wide association analyses (GWAS) were conducted using three methods based on single marker regression, haplotype regression and simultaneous fitting of all markers, respectively. Fourteen loci significantly associated with SPKST under at least two GWAS methods were detected. A large number of associations was also detected for the secondary traits. Analysis of co-localization of SPKST associated loci with QTLs detected in progenies of bi-parental crosses reported in the literature allowed to narrow -down the position of eight of those QTLs, including the most documented one, qHTSF4.1. Gene families underlying loci associated with SPKST corresponded to functions ranging from sensing abiotic stresses and regulating plant response, such as wall-associated kinases and heat shock proteins, to cell division and gametophyte development. Analysis of diversity at the vicinity of loci associated with SPKST within the rice three thousand genomes, revealed widespread distribution of the favourable alleles across O. sativa genetic groups. However, few accessions assembled the favourable alleles at all loci. Effective donors included the heat tolerant variety N22 and some Indian and Taiwanese varieties. These results provide a basis for breeding for heat tolerance during anthesis and for functional validation of major loci governing this trait.

Positional cloning of the rice male sterility gene ms-IR36, widely used in the inter-crossing phase of recurrent selection schemes

The monogenetic recessive male-sterile gene ms-IR36 is widely used to facilitate the inter-crossing phase of recurrent selection in rice (Oryza sativa), but its segregation within the progeny disturbs other breeding phases. Marker-assisted early identification of msms and Msms seedlings would help overcome this drawback. Using successively bulked segregant analysis and large F2 populations, we mapped the ms-IR36 gene to a 33-kb region on the short arm of chromosome 2 that includes 10 candidate genes. Sequencing of these candidates together with checking rice genome annotations and expression databases allowed the target to be narrowed down to one candidate gene already isolated and characterized as the tapetum degeneration retardation (TDR) gene and reported to be involved in tapetal programmed cell death. Comparison of the sequence of the TDR gene between male-sterile (MS) and male-fertile (MF) IR36 plants detected one non-synonymous nucleotide substitution affecting the active domain of the encoded protein. Perfect co-segregation was observed between polymorphism at this nucleotide (SNP) and the MS/MF phenotype of 946 F2 plants. Spatial modelling of the active domain of the candidate protein reinforced the candidate status of the only SNP identified. Histological characterization of anther development in MS IR36 revealed defects identical to the ones observed in mutants used for the isolation and characterization of the TDR gene: delayed/non-degradation of tapetum tissue and collapse of the haploid microspores. We concluded that ms-IR36 corresponded to the TDR gene with a different mutation from the earlier one described in the same gene. No significant linkage drag was associated with ms-IR36. A SNP-based marker that enables simple early identification of MS plants and MF plants with the Msms genotype was designed.

Tolerance to mild salinity stress in japonica rice: A genome-wide association mapping study highlights calcium signaling and metabolism genes

Salinity tolerance is an important quality for European rice grown in river deltas. We evaluated the salinity tolerance of a panel of 235 temperate japonica rice accessions genotyped with 30,000 SNP markers. The panel was exposed to mild salt stress (50 mM NaCl; conductivity of 6 dS m-1) at the seedling stage. Eight different root and shoot growth parameters were measured for both the control and stressed treatments. The Na+ and K+ mass fractions of the stressed plants were measured using atomic absorption spectroscopy. The salt treatment affected plant growth, particularly the shoot parameters. The panel showed a wide range of Na+/K+ ratio and the temperate accessions were distributed over an increasing axis, from the most resistant to the most susceptible checks. We conducted a genome-wide association study on indices of stress response and ion mass fractions in the leaves using a classical mixed model controlling structure and kinship. A total of 27 QTLs validated by sub-sampling were identified. For indices of stress responses, we also used another model that focused on marker × treatment interactions and detected 50 QTLs, three of which were also identified using the classical method. We compared the positions of the significant QTLs to those of approximately 300 genes that play a role in rice salt tolerance. The positions of several QTLs were close to those of genes involved in calcium signaling and metabolism, while other QTLs were close to those of kinases. These results reveal the salinity tolerance of accessions with a temperate japonica background. Although the detected QTLs must be confirmed by other approaches, the number of associations linked to candidate genes involved in calcium-mediated ion homeostasis highlights pathways to explore in priority to understand the salinity tolerance of temperate rice.

Genomic prediction offers the most effective marker assisted breeding approach for ability to prevent arsenic accumulation in rice grains

The high concentration of arsenic in the paddy fields and, consequently, in the rice grains is a critical issue in many rice-growing areas. Breeding arsenic tolerant rice varieties that prevent As uptake and its accumulation in the grains is a major mitigation options. However, the genetic control of the trait is complex, involving large number of gene of limited individual effect, and raises the question of the most efficient breeding method. Using data from three years of experiment in a naturally arsenic-reach field, we analysed the performances of the two major breeding methods: conventional, quantitative trait loci based, selection targeting loci involved in arsenic tolerance, and the emerging, genomic selection, predicting genetic values without prior hypotheses on causal relationships between markers and target traits. We showed that once calibrated in a reference population the accuracy of genomic prediction of arsenic content in the grains of the breeding population was rather high, ensuring genetic gains per time unite close to phenotypic selection. Conversely, selection targeting quantitative loci proved to be less robust as, though in agreement with the literature on the genetic bases of arsenic tolerance, few target loci identified in the reference population could be validated in the breeding population.

Additional file 3: Table S3. of Association mapping of resistance to rice blast in upland field conditions

Blast disease scores measured on 18 differential lines in Andranomanelatra in 2011 and 2015. (XLSX 9 kb)