A cluster of Ankyrin and Ankyrin-TPR repeat genes is associated with panicle branching diversity in rice.

The number of grains per panicle is an important yield-related trait in cereals which depends in part on panicle branching complexity. One component of this complexity is the number of secondary branches per panicle. Previously, a GWAS site associated with secondary branch and spikelet numbers per panicle in rice was identified. Here we combined gene capture, bi-parental genetic population analysis, expression profiling and transgenic approaches in order to investigate the functional significance of a cluster of 6 ANK and ANK-TPR genes within the QTL. Four of the ANK and ANK-TPR genes present a differential expression associated with panicle secondary branch number in contrasted accessions. These differential expression patterns correlate in the different alleles of these genes with specific deletions of potential cis-regulatory sequences in their promoters. Two of these genes were confirmed through functional analysis as playing a role in the control of panicle architecture. Our findings indicate that secondary branching diversity in the rice panicle is governed in part by differentially expressed genes within this cluster encoding ANK and ANK-TPR domain proteins that may act as positive or negative regulators of panicle meristem's identity transition from indeterminate to determinate state.

Genome-wide association mapping for root traits in a panel of rice accessions from Vietnam.

BACKGROUND: Despite recent sequencing efforts, local genetic resources remain underexploited, even though they carry alleles that can bring agronomic benefits. Taking advantage of the recent genotyping with 22,000 single-nucleotide polymorphism markers of a core collection of 180 Vietnamese rice varieties originating from provinces from North to South Vietnam and from different agrosystems characterized by contrasted water regimes, we have performed a genome-wide association study for different root parameters. Roots contribute to water stress avoidance and are a still underexploited target for breeding purpose due to the difficulty to observe them. RESULTS: The panel of 180 rice varieties was phenotyped under greenhouse conditions for several root traits in an experimental design with 3 replicates. The phenotyping system consisted of long plastic bags that were filled with sand and supplemented with fertilizer. Root length, root mass in different layers, root thickness, and the number of crown roots, as well as several derived root parameters and shoot traits, were recorded. The results were submitted to association mapping using a mixed model involving structure and kinship to enable the identification of significant associations. The analyses were conducted successively on the whole panel and on its indica (115 accessions) and japonica (64 accessions) subcomponents. The two associations with the highest significance were for root thickness on chromosome 2 and for crown root number on chromosome 11. No common associations were detected between the indica and japonica subpanels, probably because of the polymorphism repartition between the subspecies. Based on orthology with Arabidopsis, the possible candidate genes underlying the quantitative trait loci are reviewed. CONCLUSIONS: Some of the major quantitative trait loci we detected through this genome-wide association study contain promising candidate genes encoding regulatory elements of known key regulators of root formation and development.

Characterization of a panel of Vietnamese rice varieties using DArT and SNP markers for association mapping purposes.

BACKGROUND: The development of genome-wide association studies (GWAS) in crops has made it possible to mine interesting alleles hidden in gene bank resources. However, only a small fraction of the rice genetic diversity of any given country has been exploited in the studies with worldwide sampling conducted to date. This study presents the development of a panel of rice varieties from Vietnam for GWAS purposes. RESULTS: The panel, initially composed of 270 accessions, was characterized for simple agronomic traits (maturity class, grain shape and endosperm type) commonly used to classify rice varieties. We first genotyped the panel using Diversity Array Technology (DArT) markers. We analyzed the panel structure, identified two subpanels corresponding to the indica and japonica sub-species and selected 182 non-redundant accessions. However, the number of usable DArT markers (241 for an initial library of 6444 clones) was too small for GWAS purposes. Therefore, we characterized the panel of 182 accessions with 25,971 markers using genotyping by sequencing. The same indica and japonica subpanels were identified. The indica subpanel was further divided into six populations (I1 to I6) using a model-based approach. The japonica subpanel, which was more highly differentiated, was divided into 4 populations (J1 to J4), including a temperate type (J2). Passport data and phenotypic traits were used to characterize these populations. Some populations were exclusively composed of glutinous types (I3 and J2). Some of the upland rice varieties appeared to belong to indica populations, which is uncommon in this region of the world. Linkage disequilibrium decayed faster in the indica subpanel (r2 below 0.2 at 101 kb) than in the japonica subpanel (r2 below 0.2 at 425 kb), likely because of the strongest differentiation of the japonica subpanel. A matrix adapted for GWAS was built by eliminating the markers with a minor allele frequency below 5% and imputing the missing data. This matrix contained 21,814 markers. A GWAS was conducted on time to flowering to prove the utility of this panel. CONCLUSIONS: This publicly available panel constitutes an important resource giving access to original allelic diversity. It will be used for GWAS on root and panicle traits.

Genome-wide Association Study of a Panel of Vietnamese Rice Landraces Reveals New QTLs for Tolerance to Water Deficit During the Vegetative Phase.

BACKGROUND: Drought tolerance is a major challenge in breeding rice for unfavorable environments. In this study, we used a panel of 180 Vietnamese rice landraces genotyped with 21,623 single-nucleotide polymorphism markers to perform a genome-wide association study (GWAS) for different drought response and recovery traits during the vegetative stage. These landraces originate from different geographical locations and are adapted to different agrosystems characterized by contrasted water regimes. Vietnamese landraces are often underrepresented in international panels used for GWAS, but they can contain original genetic determinants related to drought resistance. RESULTS: The panel of 180 rice varieties was phenotyped under greenhouse conditions for several drought-related traits in an experimental design with 3 replicates. Plants were grown in pots for 4 weeks and drought-stressed by stopping irrigation for an additional 4 weeks. Drought sensitivity scores and leaf relative water content were measured throughout the drought stress. The recovery capacity was measured 2 weeks after plant rewatering. Several QTLs associated with these drought tolerance traits were identified by GWAS using a mixed model with control of structure and kinship. The number of detected QTLs consisted of 14 for leaf relative water content, 9 for slope of relative water content, 12 for drought sensitivity score, 3 for recovery ability and 1 for relative crop growth rate. This set of 39 QTLs actually corresponded to a total of 17 different QTLs because 9 were simultaneously associated with two or more traits, which indicates that these common loci may have pleiotropic effects on drought-related traits. No QTL was found in association with the same traits in both the indica and japonica subpanels. The possible candidate genes underlying the quantitative trait loci are reviewed. CONCLUSIONS: Some of the identified QTLs contain promising candidate genes with a function related to drought tolerance by osmotic stress adjustment.

Customization of Artificial MicroRNA Design.

RNAi approaches, including microRNA (miRNA) regulatory pathway, offer great tools for functional characterization of unknown genes. Moreover, the applications of artificial microRNA (amiRNA) in the field of plant transgenesis have also been advanced to engineer pathogen-resistant or trait-improved transgenic plants. Until now, despite the high potency of amiRNA approach, no commercial plant cultivar expressing amiRNAs with improved traits has been released yet. Beside the issues of biosafety policies, the specificity and efficacy of amiRNAs are of major concerns. Sufficient cares should be taken for the specificity and efficacy of amiRNAs due to their potential off-target effects and other issues relating to in vivo expression of pre-amiRNAs. For these reasons, the proper design of amiRNAs with the lowest off-target possibility is very important for successful applications of the approach in plant. Therefore, there are many studies with the aim to improve the amiRNA design and amiRNA expressing backbones for obtaining better specificity and efficacy. However, the requirement for an efficient reference for the design is still needed. In the present chapter, we attempt to summarize and discuss all the major concerns relating to amiRNA design with the hope to provide a significant guideline for this approach.