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.

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.

Novel Imaging Techniques to Analyze Panicle Architecture.

Panicle architecture is known to directly influence grain yield in rice, and thus is an important trait for rice varietal improvement. However, spike branching consequences trigger variation in number of superior and inferior grains and thus affect grain quality. The genetics behind the length of both primary and secondary branches were studied resulting in the identification of cloned genes. Extending this knowledge to include other physiological parameters of panicle architecture is not yet well studied, and it requires high-throughput imaging techniques that are accurate. In this chapter we put the spotlight on Panicle Trait Phenotyping Tool (P-TRAP), a freely available platform independent software to analyze the panicle architecture of rice, as one of such methods that can be used to generate a comprehensive and reproducible panicle architecture data and identify superior breeding lines. P-TRAP measures 15 panicle structure and nine spikelet traits. These quantitative traits can be used in genome-wide association studies to understand their genetic basis.

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.

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.

Investigating glycemic potential of rice by unraveling compositional variations in mature grain and starch mobilization patterns during seed germination.

Rice lines with slower starch digestibility provide opportunities in mitigating the global rise in type II diabetes and related non-communicable diseases. However, screening for low glycemic index (GI) in rice breeding programs is not possible due to time and cost constraints. This study evaluated the feasibility of using in vitro cooked grain amylolysis, starch mobilization patterns during seed germination, and variation in starch structure and composition in the mature seed to differentiate patterns of starch digestibility. Mobilization patterns of total starch, resistant starch, amylose and amylopectin chains, and free sugars during seed germination revealed that the process is analogous to digestion in the human gastrointestinal tract. The combination of these biochemical markers can be used as an alternative measure to predict GI. Additionally, transcriptome analysis of stored mRNA transcripts in high and low GI lines detected differences in starch metabolism and confirmed the importance of seed storage pathways in influencing digestibility. Pathway analyses supported by metabolomics data revealed that resistant starch, cell wall non-starch polysaccharides and flavonoids potentially contribute to slower digestibility. These new insights can guide precision breeding programs to produce low GI rice with acceptable cooking quality to help mitigate the burden of diet-associated lifestyle diseases.

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.

Prospects of breeding high-quality rice using post-genomic tools.

KEY MESSAGE: The holistic understanding derived from integrating grain quality and sensory research outcomes in breeding high-quality rice in the light of post-genomics resources has been synthesized. Acceptance of new rice genotypes by producers and consumers hinges not only on their potential for higher yield but recent emphasis has also been on premium-value genotypes that have the ability to satisfy consumer preferences for grain quality. This review article provides insights into how to link grain quality attributes and sensory perception to support breeding superior rice varieties. Recent advances in quality profiling and omics technologies have provided efficient approaches to identify the key genes and biochemical markers involved in rice quality traits. Emphasis has been given to the upcoming area of holistic understanding of grain quality and attributes derived from sensory evaluation to leverage integrative gene discovery strategies that enable breeding programs to efficiently tap the huge genetic diversity in rice for novel genes that enhance rice food quality.

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.