Unravelling marker trait associations linking nutritional value with pigmentation in rice seed.

While considerable breeding effort has focused on increasing the yields of staple crops such as rice and the levels of micronutrients such as iron and zinc, breeding to address the problems of the double-burden of malnutrition has received less attention. Pigmented rice has higher nutritional value and greater health benefits compared to white rice. However, the genetic associations underlying pericarp coloration and accumulation of nutritionally valuable compounds is still poorly understood. Here we report the targeted genetic analysis of 364 rice accessions, assessing the genetic relationship between pericarp coloration (measured using multi-spectral imaging) and a range of phenolic compounds with potential nutritional and health-promoting characteristics. A genome-wide association study resulted in the identification of over 280 single nucleotide polymorphisms (SNPs) associated with the traits of interest. Many of the SNPs were associated with more than one trait, colocalization occurring between nutritional traits, and nutritional and color-related traits. Targeted association analysis identified 67 SNPs, located within 52 candidate genes and associated with 24 traits. Six haplotypes identified within the genes Rc/bHLH17 and OsIPT5 indicated that these genes have an important role in the regulation of a wide range of phenolic compounds, and not only those directly conferring pericarp color. These identified genetic linkages between nutritionally valuable phenolic compounds and pericarp color present not only a valuable resource for the enhancement of the nutritional value of rice but an easy method of selection of suitable genotypes.

Unraveling the genetics underlying micronutrient signatures of diversity panel present in brown rice through genome-ionome linkages.

Rice (Oryza sativa) is an important staple crop to address the Hidden Hunger problem not only in Asia but also in Africa where rice is fast becoming an important source of calories. The brown rice (whole grain with bran) is known to be more nutritious due to elevated mineral composition. The genetics underlying brown rice ionome (sum total of such mineral composition) remains largely unexplored. Hence, we conducted a comprehensive study to dissect the genetic architecture of the brown rice ionome. We used genome-wide association studies, gene set analysis, and targeted association analysis for 12 micronutrients in the brown rice grains. A diverse panel of 300 resequenced indica accessions, with more than 1.02 million single nucleotide polymorphisms, was used. We identified 109 candidate genes with 5-20% phenotypic variation explained for the 12 micronutrients and identified epistatic interactions with multiple micronutrients. Pooling all candidate genes per micronutrient exhibited phenotypic variation explained values ranging from 11% to almost 40%. The key donor lines with larger concentrations for most of the micronutrients possessed superior alleles, which were absent in the breeding lines. Through gene regulatory networks we identified enriched functional pathways for central regulators that were detected as key candidate genes through genome-wide association studies. This study provided important insights on the ionome variations in rice, on the genetic basis of the genome-ionome relationships and on the molecular mechanisms underlying micronutrient signatures.

What happens at night? Physiological mechanisms related to maintaining grain yield under high night temperature in rice.

High night temperature (HNT) causes substantial yield loss in rice (Oryza sativa L.). In this study, the physiological processes related to flag leaf dark respiration (Rn) and grain filling under HNT were explored in a multi-parent advanced generation intercross population developed for heat tolerance (MAGICheat ) along with selected high temperature tolerant breeding lines developed with heat-tolerant parents. Within a subset of lines, flag leaf Rn under HNT treatment was related to lower spikelet number per panicle and thus reduced yield. HNT enhanced the nighttime reduction of non-structural carbohydrates (NSC) in stem tissue, but not in leaves, and stem nighttime NSC reduction was negatively correlated with yield. Between heading and harvest, the major difference in NSC concentration was found for starch, but not for soluble sugar. HNT weakened the relationship between NSC remobilization and harvest index at both the phenotypic and genetic level. By using genome-wide association studies, an invertase inhibitor, MADS box transcription factors and a UDP-glycosyltransferase that were identified as candidate genes orchestrating stem NSC remobilization in the control treatment were lost under HNT. With the identification of physiological and genetic components related to rice HNT response, this study offers promising prebreeding materials and trait targets to sustain yield stability under climate change.

OsTPR boosts the superior grains through increase in upper secondary rachis branches without incurring a grain quality penalty.

To address the future food security in Asia, we need to improve the genetic gain of grain yield while ensuring the consumer acceptance. This study aimed to identify novel genes influencing the number of upper secondary rachis branches (USRB) to elevate superior grains without compromising grain quality by studying the genetic variance of 310 diverse O. sativa var. indica panel using single- and multi-locus genome-wide association studies (GWAS), gene set analyses and gene regulatory network analysis. GWAS of USRB identified 230 significant (q-value < 0.05) SNPs from chromosomes 1 and 2. GWAS targets narrowed down using gene set analyses identified large effect association on an important locus LOC_Os02g50790/LOC_Os02g50799 encoding a nuclear-pore anchor protein (OsTPR). The superior haplotype derived from non-synonymous SNPs identified in OsTPR was specifically associated with increase in USRB with superior grains being low chalk. Through haplotype mining, we further demonstrated the synergy of offering added yield advantage due to superior allele of OsTPR in elite materials with low glycaemic index (GI) property. We further validated the importance of OsTPR using recombinant inbred lines (RILs) population by introgressing a superior allele of OsTPR into elite materials resulted in raise in productivity in high amylose background. This confirmed a critical role for OsTPR in influencing yield while maintaining grain and nutritional quality.

The genetics underlying metabolic signatures in a brown rice diversity panel and their vital role in human nutrition.

Brown rice (Oryza sativa) possesses various nutritionally dense bioactive phytochemicals exhibiting a wide range of antioxidant, anti-cancer, and anti-diabetic properties known to promote various human health benefits. However, despite the wide claims made about the importance of brown rice for human nutrition the underlying metabolic diversity has not been systematically explored. Non-targeted metabolite profiling of developing and mature seeds of a diverse genetic panel of 320 rice cultivars allowed quantification of 117 metabolites. The metabolite genome-wide association study (mGWAS) detected genetic variants influencing diverse metabolic targets in developing and mature seeds. We further interlinked genetic variants on chromosome 7 (6.06-6.43 Mb region) with complex epistatic genetic interactions impacting multi-dimensional nutritional targets, including complex carbohydrate starch quality, the glycemic index, antioxidant catechin, and rice grain color. Through this nutrigenomics approach rare gene bank accessions possessing genetic variants in bHLH and IPT5 genes were identified through haplotype enrichment. These variants were associated with a low glycemic index, higher catechin levels, elevated total flavonoid contents, and heightened antioxidant activity in the whole grain with elevated anti-cancer properties being confirmed in cancer cell lines. This multi-disciplinary nutrigenomics approach thus allowed us to discover the genetic basis of human health-conferring diversity in the metabolome of brown rice.

Genome-wide association coupled gene to gene interaction studies unveil novel epistatic targets among major effect loci impacting rice grain chalkiness.

Rice varieties whose quality is graded as excellent have a lower percent grain chalkiness (PGC) of two per cent and below with higher whole grain yields upon milling, leading to higher economic returns for farmers. We have conducted a genome-wide association study (GWAS) using a combined population panel of indica and japonica rice varieties, and identified a total of 746 single nucleotide polymorphisms (SNPs) that were strongly associated with the chalk phenotype, covered 78 Quantitative Trait Loci (QTL) regions. Among them, 21 were high-value QTLs, as they explained at least 10 % of the phenotypic variance for PGC. A combined epistasis and GWAS was applied to dissect the genetics of the complex chalkiness trait, and its regulatory cascades were validated using gene regulatory networks. Promising novel epistatic interactions were found between the loci of chromosomes 6 (PGC6.1) and 7 (PGC7.8) that contributed to lower PGC. Based on haplotype mining only a few modern rice varieties confounded with a lower chalkiness, and they possess several PGC QTLs. The importance of PGC6.1 was validated through multi-parent advanced generation intercrosses and several low-chalk lines possessing superior haplotypes were identified. The results of this investigation have deciphered the underlying genetic networks that can reduce PGC to 2%, and will thus support future breeding programs to improve the grain quality of elite genetic material with high-yielding potentials.

Balancing the double-edged sword effect of increased resistant starch content and its impact on rice texture: its genetics and molecular physiological mechanisms.

Resistant starch (RS) is the portion of starch that escapes gastrointestinal digestion and acts as a substrate for fermentation of probiotic bacteria in the gut. Aside from enhancing gut health, RS contributes to a lower glycemic index. A genome-wide association study coupled with targeted gene association studies was conducted utilizing a diverse panel of 281 resequenced Indica rice lines comprising of ~2.2 million single nucleotide polymorphisms. Low-to-intermediate RS phenotypic variations were identified in the rice diversity panel, resulting in novel associations of RS to several genes associated with amylopectin biosynthesis and degradation. Selected rice lines encoding superior alleles of SSIIa with medium RS and inferior alleles with low RS groups were subjected to detailed transcriptomic, metabolomic, non-starch dietary fibre (DF), starch structural and textural attributes. The gene regulatory networks highlighted the importance of a protein phosphatase alongside multiple genes of starch metabolism. Metabolomics analyses resulted in the identification of several metabolite hubs (carboxylic acid, sugars and polyamines) in the medium RS group. Among DF, mannose and galactose from the water-insoluble fraction were found to be highly associated with low and medium RS lines, respectively. Starch structural analyses revealed that a moderate increase in RS is also linked to an elevation of amylose 1 and amylose 2 fractions. Although rice lines with medium RS content negatively affected textural and viscosity properties in comparison to low RS, the textural property of medium RS lines was in the same acceptable range as IR64, a rice mega variety popular in Asia.

Dissecting the genome-wide genetic variants of milling and appearance quality traits in rice.

Higher head rice yield (HRY), which represents the proportion of intact grains that survive milling, and lower grain chalkiness (opacity) are key quality traits. We investigated the genetic basis of HRY and chalkiness in 320 diverse resequenced accessions of indica rice with integrated single- and multi-locus genome-wide association studies using 2.26 million single-nucleotide polymorphisms. We identified novel haplotypes that underly higher HRY on chromosomes 3, 6, 8, and 11, and that lower grain chalkiness in a fine-mapped region on chromosome 5. Whole-genome sequencing of 92 IRRI breeding lines was performed to identify the genetic variants of HRY and chalkiness. Rare and novel haplotypes were found for lowering chalkiness, but missing alleles hindered progress towards enhancing HRY in breeding material. The novel haplotypes that we identified have potential use in breeding programs aimed at improving these important traits in the rice crop.

Integrating a genome-wide association study with a large-scale transcriptome analysis to predict genetic regions influencing the glycaemic index and texture in rice.

Reliably generating rice varieties with low glycaemic index (GI) is an important nutritional intervention given the high rates of Type II diabetes incidences in Asia where rice is staple diet. We integrated a genome-wide association study (GWAS) with a transcriptome-wide association study (TWAS) to determine the genetic basis of the GI in rice. GWAS utilized 305 re-sequenced diverse indica panel comprising ~2.4 million single nucleotide polymorphisms (SNPs) enriched in genic regions. A novel association signal was detected at a synonymous SNP in exon 2 of LOC_Os05g03600 for intermediate-to-high GI phenotypic variation. Another major hotspot region was predicted for contributing intermediate-to-high GI variation, involves 26 genes on chromosome 6 (GI6.1). These set of genes included GBSSI, two hydrolase genes, genes involved in signalling and chromatin modification. The TWAS and methylome sequencing data revealed cis-acting functionally relevant genetic variants with differential methylation patterns in the hot spot GI6.1 region, narrowing the target to 13 genes. Conversely, the promoter region of GBSSI and its alternative splicing allele (G allele of Wxa ) explained the intermediate-to-high GI variation. A SNP (C˃T) at exon-10 was also highlighted in the preceding analyses to influence final viscosity (FV), which is independent of amylose content/GI. The low GI line with GC haplotype confirmed soft texture, while other two low GI lines with GT haplotype were characterized as hard and cohesive. The low GI lines were further confirmed through clinical in vivo studies. Gene regulatory network analysis highlighted the role of the non-starch polysaccharide pathway in lowering GI.

Deciphering the Genetic Architecture of Cooked Rice Texture.

The textural attributes of cooked rice determine palatability and consumer acceptance. Henceforth, understanding the underlying genetic basis is pivotal for the genetic improvement of preferred textural attributes in breeding programs. We characterized diverse set of 236 Indica accessions from 37 countries for textural attributes, which includes adhesiveness (ADH), hardness (HRD), springiness (SPR), and cohesiveness (COH) as well as amylose content (AC). A set of 147,692 high quality SNPs resulting from genotyping data of 700K high Density Rice Array (HDRA) derived from the Indica diversity panels of 218 lines were retained for marker-trait associations of textural attributes using single-locus (SL) genome wide association studies (GWAS) which resulted in identifying hotspot on chromosome 6 for AC and ADH attributes. Four independent multi-locus approaches (ML-GWAS) including FASTmrEMMA, pLARmEB, mrMLM, and ISIS_EM-BLASSO were implemented to dissect additional loci of major/minor effects influencing the rice texture and to overcome limitations of SL-based GWAS approach. In total 224 significant quantitative trait nucleotide (QTNs) were identified using ML-GWAS, of which 97 were validated with at least two out of the four multi-locus methods. The GWAS results were in accordance with the very significant negative correlation (r = -0.83) observed between AC and ADH, and the significant correlation exhibited by AC (r < 0.4) with HRD, SPR, and COH. The novel haplotypes and putative candidate genes influencing textural properties beyond AC will be a useful resource for deployment into the marker assisted program to capture consumer preferences influencing rice texture and palatability.

Whole genome sequencing-based association study to unravel genetic architecture of cooked grain width and length traits in rice.

In this study, we used 2.9 million single nucleotide polymorphisms (SNP) and 393,429 indels derived from whole genome sequences of 591 rice landraces to determine the genetic basis of cooked and raw grain length, width and shape using genome-wide association study (GWAS). We identified a unique fine-mapped genetic region GWi7.1 significantly associated with cooked and raw grain width. Additionally, GWi7.2 that harbors GL7/GW7 a cloned gene for grain dimension was found. Novel regions in chromosomes 10 and 11 were also found to be associated with cooked grain shape and raw grain width, respectively. The indel-based GWAS identified fine-mapped genetic regions GL3.1 and GWi5.1 that matched synteny breakpoints between indica and japonica. GL3.1 was positioned a few kilobases away from GS3, a cloned gene for cooked and raw grain lengths in indica. GWi5.1 found to be significantly associated with cooked and raw grain width. It anchors upstream of cloned gene GW5, which varied between indica and japonica accessions. GWi11.1 is present inside the 3'-UTR of a functional gene in indica that corresponds to a syntenic break in chromosome 11 of japonica. Our results identified novel allelic structural variants and haplotypes confirmed using single locus and multilocus SNP and indel-based GWAS.

Systems Genetics Identifies a Novel Regulatory Domain of Amylose Synthesis.

A deeper understanding of the regulation of starch biosynthesis in rice (Oryza sativa) endosperm is crucial in tailoring digestibility without sacrificing grain quality. In this study, significant association peaks on chromosomes 6 and 7 were identified through a genomewide association study (GWAS) of debranched starch structure from grains of a 320 indica rice diversity panel using genotyping data from the high-density rice array. A systems genetics approach that interrelates starch structure data from GWAS to functional pathways from a gene regulatory network identified known genes with high correlation to the proportion of amylose and amylopectin. An SNP in the promoter region of Granule Bound Starch Synthase I was identified along with seven other SNPs to form haplotypes that discriminate samples into different phenotypic ranges of amylose. A GWAS peak on chromosome 7 between LOC_Os07g11020 and LOC_Os07g11520 indexed by a nonsynonymous SNP mutation on exon 5 of a bHLH transcription factor was found to elevate the proportion of amylose at the expense of reduced short-chain amylopectin. Linking starch structure with starch digestibility by determining the kinetics of cooked grain amylolysis of selected haplotypes revealed strong association of starch structure with estimated digestibility kinetics. Combining all results from grain quality genomics, systems genetics, and digestibility phenotyping, we propose target haplotypes for fine-tuning starch structure in rice through marker-assisted breeding that can be used to alter the digestibility of rice grain, thus offering rice consumers a new diet-based intervention to mitigate the impact of nutrition-related noncommunicable diseases.

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest.

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars.

An advanced draft genome assembly of a desi type chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is an important pulse legume crop. We previously reported a draft genome assembly of the desi chickpea cultivar ICC 4958. Here we report an advanced version of the ICC 4958 genome assembly (version 2.0) generated using additional sequence data and an improved genetic map. This resulted in 2.7-fold increase in the length of the pseudomolecules and substantial reduction of sequence gaps. The genome assembly covered more than 94% of the estimated gene space and predicted the presence of 30,257 protein-coding genes including 2230 and 133 genes encoding potential transcription factors (TF) and resistance gene homologs, respectively. Gene expression analysis identified several TF and chickpea-specific genes with tissue-specific expression and displayed functional diversification of the paralogous genes. Pairwise comparison of pseudomolecules in the desi (ICC 4958) and the earlier reported kabuli (CDC Frontier) chickpea assemblies showed an extensive local collinearity with incongruity in the placement of large sequence blocks along the linkage groups, apparently due to use of different genetic maps. Single nucleotide polymorphism (SNP)-based mining of intra-specific polymorphism identified more than four thousand SNPs differentiating a desi group and a kabuli group of chickpea genotypes.

Designing climate-resilient rice with ideal grain quality suited for high-temperature stress.

To ensure rice food security, the target outputs of future rice breeding programmes should focus on developing climate-resilient rice varieties with emphasis on increased head rice yield coupled with superior grain quality. This challenge is made greater by a world that is increasingly becoming warmer. Such environmental changes dramatically impact head rice and milling yield as well as increasing chalkiness because of impairment in starch accumulation and other storage biosynthetic pathways in the grain. This review highlights the knowledge gained through gene discovery via quantitative trait locus (QTL) cloning and structural-functional genomic strategies to reduce chalk, increase head rice yield, and develop stable lines with optimum grain quality in challenging environments. The newly discovered genes and the knowledge gained on the influence of specific alleles related to stability of grain quality attributes provide a robust platform for marker-assisted selection in breeding to design heat-tolerant rice varieties with superior grain quality. Using the chalkiness trait in rice as a case study, we demonstrate here that the emerging field of systems genetics can help fast-track the identification of novel alleles and gene targets that can be pyramided for the development of environmentally robust rice varieties that possess improved grain quality.

The chickpea genomic web resource: visualization and analysis of the desi-type Cicer arietinum nuclear genome for comparative exploration of legumes.

BACKGROUND: Availability of the draft nuclear genome sequences of small-seeded desi-type legume crop Cicer arietinum has provided an opportunity for investigating unique chickpea genomic features and evaluation of their biological significance. The increasing number of legume genome sequences also presents a challenge for developing reliable and information-driven bioinformatics applications suitable for comparative exploration of this important class of crop plants. RESULTS: The Chickpea Genomic Web Resource (CGWR) is an implementation of a suite of web-based applications dedicated to chickpea genome visualization and comparative analysis, based on next generation sequencing and assembly of Cicer arietinum desi-type genotype ICC4958. CGWR has been designed and configured for mapping, scanning and browsing the significant chickpea genomic features in view of the important existing and potential roles played by the various legume genome projects in mutant mapping and cloning. It also enables comparative informatics of ICC4958 DNA sequence analysis with other wild and cultivated genotypes of chickpea, various other leguminous species as well as several non-leguminous model plants, to enable investigations into evolutionary processes that shape legume genomes. CONCLUSIONS: CGWR is an online database offering a comprehensive visual and functional genomic analysis of the chickpea genome, along with customized maps and gene-clustering options. It is also the only plant based web resource supporting display and analysis of nucleosome positioning patterns in the genome. The usefulness of CGWR has been demonstrated with discoveries of biological significance made using this server. The CGWR is compatible with all available operating systems and browsers, and is available freely under the open source license at http://www.nipgr.res.in/CGWR/home.php.

Tomato genomic resources database: an integrated repository of useful tomato genomic information for basic and applied research.

Tomato Genomic Resources Database (TGRD) allows interactive browsing of tomato genes, micro RNAs, simple sequence repeats (SSRs), important quantitative trait loci and Tomato-EXPEN 2000 genetic map altogether or separately along twelve chromosomes of tomato in a single window. The database is created using sequence of the cultivar Heinz 1706. High quality single nucleotide polymorphic (SNP) sites between the genes of Heinz 1706 and the wild tomato S. pimpinellifolium LA1589 are also included. Genes are classified into different families. 5'-upstream sequences (5'-US) of all the genes and their tissue-specific expression profiles are provided. Sequences of the microRNA loci and their putative target genes are catalogued. Genes and 5'-US show presence of SSRs and SNPs. SSRs located in the genomic, genic and 5'-US can be analysed separately for the presence of any particular motif. Primer sequences for all the SSRs and flanking sequences for all the genic SNPs have been provided. TGRD is a user-friendly web-accessible relational database and uses CMAP viewer for graphical scanning of all the features. Integration and graphical presentation of important genomic information will facilitate better and easier use of tomato genome. TGRD can be accessed as an open source repository at http://59.163.192.91/tomato2/.

FmMDb: a versatile database of foxtail millet markers for millets and bioenergy grasses research.

The prominent attributes of foxtail millet (Setaria italica L.) including its small genome size, short life cycle, inbreeding nature, and phylogenetic proximity to various biofuel crops have made this crop an excellent model system to investigate various aspects of architectural, evolutionary and physiological significances in Panicoid bioenergy grasses. After release of its whole genome sequence, large-scale genomic resources in terms of molecular markers were generated for the improvement of both foxtail millet and its related species. Hence it is now essential to congregate, curate and make available these genomic resources for the benefit of researchers and breeders working towards crop improvement. In view of this, we have constructed the Foxtail millet Marker Database (FmMDb; http://www.nipgr.res.in/foxtail.html), a comprehensive online database for information retrieval, visualization and management of large-scale marker datasets with unrestricted public access. FmMDb is the first database which provides complete marker information to the plant science community attempting to produce elite cultivars of millet and bioenergy grass species, thus addressing global food insecurity.