Why not XY? Male monoecious sexual phenotypes challenge the female monoecious paradigm in Cannabis sativa L.

Monoecy in Cannabis sativa L. has long been considered an industrially important trait due to the increased uniformity it offers and was thought to be exclusively associated with XX females. The isolation and characterisation of a monoecious individual with XY chromosomes sourced from non-proprietary germplasm is reported for the first time. The chromosomal make up of this trait was confirmed through inflorescence structure, growth habit, PCR analysis and sexual phenotypes of progeny from a series of targeted crosses. The identification of an XY monoecious phenotype widens our understanding of monoecy in Cannabis and has important implications for breeding, particularly for producing F1-hybrid seed.

Accumulation patterns of anthocyanin and γ-oryzanol during black rice grain development.

Pigmented rice, especially black rice, is gaining popularity as it is rich in antioxidants such as anthocyanins and γ-oryzanol. At present, knowledge about temporal control of biosynthesis and accumulation of antioxidants during grain development is limited. To address this, the accumulation patterns of anthocyanins and γ-oryzanol were assessed in two distinct black rice genotypes over the course of grain development, and the expression of known regulatory genes for anthocyanin biosynthesis was examined. The results indicated that total γ-oryzanol content increased continuously throughout grain development, while total anthocyanins peaked at dough stage (15 to 21 days after flowering) followed by a decline until grain maturity in both genotypes. However, the rate of decrease in anthocyanin content differed between genotypes, and a more prominent decline in cyanidin 3-O-glucoside (C3G) relative to peonidin 3-O-glucoside (P3G) was observed for both. Anthocyanin content was closely linked with the expression of key regulatory genes in the MBW (MYB-bHLH-WD40) complex. This improved knowledge of the genotype-specific biosynthesis (anthocyanins only) and accumulation patterns of anthocyanins and γ-oryzanol can inform subsequent research efforts to increase concentrations of these key antioxidants in black rice grains.

From dawn 'til dusk: daytime progression regulates primary and secondary metabolism in Cannabis glandular trichomes.

Cannabis sativa L. glandular trichomes (GTs) synthesise large amounts of secondary metabolites, predominantly cannabinoids and terpenoids. The associated demand for carbon and energy makes GTs strong sink tissues with indications that their secondary metabolism is coupled to the availability of photoassimilates. Many metabolites show diurnal patterns of flux, but it is unknown whether cannabinoids and terpenoids are regulated by time of day. We quantified cannabinoids, terpenoids and the GT proteome over a 12-hour light period in flowers of Hindu Kush, a high-tetrahydrocannabinol (THC) cultivar. Major cannabinoids changed significantly over the course of day, resulting in an increase in total measured cannabinoids. Major terpenoids also changed, with sesquiterpenes generally decreasing with day progression. While monoterpenes generally did not decrease, the second most abundant, α-pinene, increased. The GT proteome changed the most within the first six hours of the day and analysis of differentially abundant proteins indicated upregulation of primary metabolism. Surprisingly, key cannabinoid biosynthetic enzymes decreased with daytime progression despite increases in cannabinoid content, which indicate that daytime increases of photoassimilates are the main driver of cannabinoid regulation. This first reporting of variability of cannabinoid and terpenoid biosynthesis over the course of the day has implications for Cannabis research and production.

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.

Moving Away from 12:12; the Effect of Different Photoperiods on Biomass Yield and Cannabinoids in Medicinal Cannabis.

The standard practice to initiate flowering in medicinal cannabis involves reducing the photoperiod from a long-day period to an equal duration cycle of 12 h light (12L)/12 h dark (12D). This method reflects the short-day flowering dependence of many cannabis varieties but may not be optimal for all. We sought to identify the effect of nine different flowering photoperiod treatments on the biomass yield and cannabinoid concentration of three medicinal cannabis varieties. The first, "Cannatonic", was a high cannabidiol (CBD)-accumulating line, whereas the other two, "Northern Lights" and "Hindu Kush", were high Δ9-tetrahydrocannabinol (THC) accumulators. The nine treatments tested, following 18 days under 18 h light/6 h dark following cloning and propagation included a standard 12L:12D period, a shortened period of 10L:14D, and a lengthened period of 14L:10D. The other six treatments started in one of the aforementioned and then 28 days later (mid-way through flowering) were switched to one of the other treatments, thus causing either an increase of 2 or 4 h, or a decrease of 2 or 4 h. Measured parameters included the timing of reproductive development; the dry weight flower yield; and the % dry weight of the main target cannabinoids, CBD and THC, from which the total g cannabinoid per plant was calculated. Flower biomass yields were highest for all lines when treatments started with 14L:10D; however, in the two THC lines, a static 14L:10D photoperiod caused a significant decline in THC concentration. Conversely, in Cannatonic, all treatments starting with 14L:10D led to a significant increase in the CBD concentration, which led to a 50-100% increase in total CBD yield. The results show that the assumption that a 12L:12D photoperiod is optimal for all lines is incorrect as, in some lines, yields can be greatly increased by a lengthened light period during flowering.

Using a global diversity panel of Cannabis sativa L. to develop a near InfraRed-based chemometric application for cannabinoid quantification.

C. sativa has gained renewed interest as a cash crop for food, fibre and medicinal markets. Irrespective of the final product, rigorous quantitative testing for cannabinoids, the regulated biologically active constituents of C. sativa, is a legal prerequisite across the supply chains. Currently, the medicinal cannabis and industrial hemp industries depend on costly chromatographic analysis for cannabinoid quantification, limiting production, research and development. Combined with chemometrics, Near-InfraRed spectroscopy (NIRS) has potential as a rapid, accurate and economical alternative method for cannabinoid analysis. Using chromatographic data on 12 therapeutically relevant cannabinoids together with spectral output from a diffuse reflectance NIRS device, predictive chemometric models were built for major and minor cannabinoids using dried, homogenised C. sativa inflorescences from a diverse panel of 84 accessions. Coefficients of determination (r2) of the validation models for 10 of the 12 cannabinoids ranged from 0.8 to 0.95, with models for major cannabinoids showing best performance. NIRS was able to discriminate between neutral and acidic forms of cannabinoids as well as between C3-alkyl and C5-alkyl cannabinoids. The results show that NIRS, when used in conjunction with chemometrics, is a promising method to quantify cannabinoids in raw materials with good predictive results.

Autoimmune activation and hypersensitization of the AT1 and ETA receptors contributes to vascular injury in scleroderma renal crisis.

OBJECTIVES: Scleroderma renal crisis (SRC) is a rare vascular complication of systemic sclerosis with substantial risks for end-stage renal disease and premature death. Activating autoantibodies (Abs) targeting the angiotensin II type 1 (AT1R) and the endothelin-1 type A receptor (ETAR) have been identified as predictors for SRC. Here, we sought to determine their pathogenic significance for acute renal vascular injury potentially triggering kidney failure and malignant hypertension. METHODS: IgG from patients with SRC was studied for AT1R and ETAR dependent biologic effects on isolated rat renal interlobar arteries and vascular cells including contraction, signalling and mechanisms of receptor activation. RESULTS: In myography experiments, patient IgG exerted vasoconstriction sensitive to inhibition of AT1R and ETAR. This relied on MEK-ERK signalling indicating functional relevance of anti-AT1R and anti-ETAR Abs. The contractile response to angiotensin II and endothelin-1 was amplified by patient IgG containing anti-AT1R and anti-ETAR Abs with substantial crosstalk between both receptors implicating autoimmune receptor hypersensitization. Co-immunoprecipitation experiments indicated heterodimerization between both receptor types which may enable the observed functional interrelation by direct structural interactions. CONCLUSION: We provide experimental evidence that agonistic Abs may contribute to SRC. This effect is presumably related to direct receptor stimulation and additional allosteric effects, at least in heterodimeric receptor constellations. Novel therapies targeted at autoimmune hyperactivation of AT1R and ETAR might improve outcomes in severe cases of SRC.

Genome-Wide Association Reveals Trait Loci for Seed Glucosinolate Accumulation in Indian Mustard (Brassica juncea L.).

Glucosinolates (GSLs) are sulphur- and nitrogen-containing secondary metabolites implicated in the fitness of Brassicaceae and appreciated for their pungency and health-conferring properties. In Indian mustard (Brassica juncea L.), GSL content and composition are seed-quality-determining traits affecting its economic value. Depending on the end use, i.e., condiment or oil, different GSL levels constitute breeding targets. The genetic control of GSL accumulation in Indian mustard, however, is poorly understood, and current knowledge of GSL biosynthesis and regulation is largely based on Arabidopsis thaliana. A genome-wide association study was carried out to dissect the genetic architecture of total GSL content and the content of two major GSLs, sinigrin and gluconapin, in a diverse panel of 158 Indian mustard lines, which broadly grouped into a South Asia cluster and outside-South-Asia cluster. Using 14,125 single-nucleotide polymorphisms (SNPs) as genotyping input, seven distinct significant associations were discovered for total GSL content, eight associations for sinigrin content and 19 for gluconapin. Close homologues of known GSL structural and regulatory genes were identified as candidate genes in proximity to peak SNPs. Our results provide a comprehensive map of the genetic control of GLS biosynthesis in Indian mustard, including priority targets for further investigation and molecular marker development.

Characterization of the Cannabis sativa glandular trichome proteome.

Cannabis sativa has been cultivated since antiquity as a source of fibre, food and medicine. The recent resurgence of C. sativa as a cash crop is mainly driven by the medicinal and therapeutic properties of its resin, which contains compounds that interact with the human endocannabinoid system. Compared to other medicinal crops of similar value, however, little is known about the biology of C. sativa. Glandular trichomes are small hair-like projections made up of stalk and head tissue and are responsible for the production of the resin in C. sativa. Trichome productivity, as determined by C. sativa resin yield and composition, is only beginning to be understood at the molecular level. In this study the proteomes of glandular trichome stalks and heads, were investigated and compared to the proteome of the whole flower tissue, to help further elucidate C. sativa glandular trichome biochemistry. The data suggested that the floral tissue acts as a major source of carbon and energy to the glandular trichome head sink tissue, supplying sugars which drive secondary metabolite biosynthesis. The trichome stalk seems to play only a limited role in secondary metabolism and acts as both source and sink.

Enriched-GWAS and Transcriptome Analysis to Refine and Characterize a Major QTL for Anaerobic Germination Tolerance in Rice.

Tolerance of anaerobic germination (AG) is a key trait in the development of direct seeded rice. Through rapid and sustained coleoptile elongation, AG tolerance enables robust seedling establishment under flooded conditions. Previous attempts to fine map and characterize AG2 (qAG7.1), a major centromere-spanning AG tolerance QTL, derived from the indica variety Ma-Zhan Red, have failed. Here, a novel approach of "enriched haplotype" genome-wide association study based on the Ma-Zhan Red haplotype in the AG2 region was successfully used to narrow down AG2 from more than 7 Mb to less than 0.7 Mb. The AG2 peak region contained 27 genes, including the Rc gene, responsible for red pericarp development in pigmented rice. Through comparative variant and transcriptome analysis between AG tolerant donors and susceptible accessions several candidate genes potentially controlling AG2 were identified, among them several regulatory genes. Genome-wide comparative transcriptome analysis suggested differential regulation of sugar metabolism, particularly trehalose metabolism, as well as differential regulation of cell wall modification and chloroplast development to be implicated in AG tolerance mechanisms.

Genetic Mapping by Sequencing More Precisely Detects Loci Responsible for Anaerobic Germination Tolerance in Rice.

Direct seeded rice (DSR) is a mainstay for planting rice in the Americas, and it is rapidly becoming more popular in Asia. It is essential to develop rice varieties that are suitable for this type of production system. ASD1, a landrace from India, possesses several traits desirable for direct-seeded fields, including tolerance to anaerobic germination (AG). To map the genetic basis of its tolerance, we examined a population of 200 F2:3 families derived from a cross between IR64 and ASD1 using the restriction site-associated DNA sequencing (RAD-seq) technology. This genotyping platform enabled the identification of 1921 single nucleotide polymorphism (SNP) markers to construct a high-resolution genetic linkage map with an average interval of 0.9 cM. Two significant quantitative trait loci (QTLs) were detected on chromosomes 7 and 9, qAG7 and qAG9, with LOD scores of 7.1 and 15.0 and R2 values of 15.1 and 29.4, respectively. Here, we obtained more precise locations of the QTLs than traditional simple sequence repeat and low-density SNP genotyping methods and may help further dissect the genetic factors of these QTLs.

An extreme-phenotype genome-wide association study identifies candidate cannabinoid pathway genes in Cannabis.

Cannabis produces a class of isoprenylated resorcinyl polyketides known as cannabinoids, a subset of which are medically important and exclusive to this plant. The cannabinoid alkyl group is a critical structural feature that governs therapeutic activity. Genetic enhancement of the alkyl side-chain could lead to the development of novel chemical phenotypes (chemotypes) for pharmaceutical end-use. However, the genetic determinants underlying in planta variation of cannabinoid alkyl side-chain length remain uncharacterised. Using a diversity panel derived from the Ecofibre Cannabis germplasm collection, an extreme-phenotype genome-wide association study (XP-GWAS) was used to enrich for alkyl cannabinoid polymorphic regions. Resequencing of chemotypically extreme pools revealed a known cannabinoid synthesis pathway locus as well as a series of chemotype-associated genomic regions. One of these regions contained a candidate gene encoding a ß-keto acyl carrier protein (ACP) reductase (BKR) putatively associated with polyketide fatty acid starter unit synthesis and alkyl side-chain length. Association analysis revealed twenty-two polymorphic variants spanning the length of this gene, including two nonsynonymous substitutions. The success of this first reported application of XP-GWAS for an obligate outcrossing and highly heterozygote plant genus suggests that this approach may have generic application for other plant species.

Comparative Transcriptomics and Co-Expression Networks Reveal Tissue- and Genotype-Specific Responses of qDTYs to Reproductive-Stage Drought Stress in Rice (Oryza sativa L.).

Rice (Oryza sativa L.) is more sensitive to drought stress than other cereals. To dissect molecular mechanisms underlying drought-tolerant yield in rice, we applied differential expression and co-expression network approaches to transcriptomes from flag-leaf and emerging panicle tissues of a drought-tolerant yield introgression line, DTY-IL, and the recurrent parent Swarna, under moderate reproductive-stage drought stress. Protein turnover and efficient reactive oxygen species scavenging were found to be the driving factors in both tissues. In the flag-leaf, the responses further included maintenance of photosynthesis and cell wall reorganization, while in the panicle biosynthesis of secondary metabolites was found to play additional roles. Hub genes of importance in differential drought responses included an expansin in the flag-leaf and two peroxidases in the panicle. Overlaying differential expression data with allelic variation in DTY-IL quantitative trait loci allowed for the prioritization of candidate genes. They included a differentially regulated auxin-responsive protein, with DTY-IL-specific amino acid changes in conserved domains, as well as a protein kinase with a DTY-IL-specific frameshift in the C-terminal region. The approach highlights how the integration of differential expression and allelic variation can aid in the discovery of mechanism and putative causal contribution underlying quantitative trait loci for drought-tolerant yield.

An improved 7K SNP array, the C7AIR, provides a wealth of validated SNP markers for rice breeding and genetics studies.

Single nucleotide polymorphisms (SNPs) are highly abundant, amendable to high-throughput genotyping, and useful for a number of breeding and genetics applications in crops. SNP frequencies vary depending on the species and populations under study, and therefore target SNPs need to be carefully selected to be informative for each application. While multiple SNP genotyping systems are available for rice (Oryza sativa L. and its relatives), they vary in their informativeness, cost, marker density, speed, flexibility, and data quality. In this study, we report the development and performance of the Cornell-IR LD Rice Array (C7AIR), a second-generation SNP array containing 7,098 markers that improves upon the previously released C6AIR. The C7AIR is designed to detect genome-wide polymorphisms within and between subpopulations of O. sativa, as well as O. glaberrima, O. rufipogon and O. nivara. The C7AIR combines top-performing SNPs from several previous rice arrays, including 4,007 SNPs from the C6AIR, 2,056 SNPs from the High Density Rice Array (HDRA), 910 SNPs from the 384-SNP GoldenGate sets, 189 SNPs from the 44K array selected to add information content for elite U.S. tropical japonica rice varieties, and 8 trait-specific SNPs. To demonstrate its utility, we carried out a genome-wide association analysis for plant height, employing the C7AIR across a diversity panel of 189 rice accessions and identified 20 QTLs contributing to plant height. The C7AIR SNP chip has so far been used for genotyping >10,000 rice samples. It successfully differentiates the five subpopulations of Oryza sativa, identifies introgressions from wild and exotic relatives, and is useful for quantitative trait loci (QTL) and association mapping in diverse materials. Moreover, data from the C7AIR provides valuable information that can be used to select informative and reliable SNP markers for conversion to lower-cost genotyping platforms for genomic selection and other downstream applications in breeding.

The Genetic Basis and Nutritional Benefits of Pigmented Rice Grain.

Improving the nutritional quality of rice grains through modulation of bioactive compounds and micronutrients represents an efficient means of addressing nutritional security in societies which depend heavily on rice as a staple food. White rice makes a major contribution to the calorific intake of Asian and African populations, but its nutritional quality is poor compared to that of pigmented (black, purple, red orange, or brown) variants. The compounds responsible for these color variations are the flavonoids anthocyanin and proanthocyanidin, which are known to have nutritional value. The rapid progress made in the technologies underlying genome sequencing, the analysis of gene expression and the acquisition of global 'omics data, genetics of grain pigmentation has created novel opportunities for applying molecular breeding to improve the nutritional value and productivity of pigmented rice. This review provides an update on the nutritional value and health benefits of pigmented rice grain, taking advantage of both indigenous and modern knowledge, while also describing the current approaches taken to deciphering the genetic basis of pigmentation.

Methodology: ssb-MASS: a single seed-based sampling strategy for marker-assisted selection in rice.

BACKGROUND: Integrated breeding approaches such as combining marker-assisted selection and rapid line fixation through single-seed-descent, can effectively increase the frequency of desirable alleles in a breeding program and increase the rate of genetic gain for quantitative traits by shortening the breeding cycle. However, with most genotyping being outsourced to 3rd party service providers' nowadays, sampling has become the bottleneck for many breeding programs. While seed-chipping as prevailed as an automatable seed sampling protocol in many species, the symmetry of rice seeds makes this solution as laborious and costly as sampling leaf tissue. The aim of this study is to develop, validate and deploy a single seed sampling strategy for marker-assisted selection of fixed lines in rice that is more efficient, cost-effective and convenient compared to leaf-based sampling protocols without compromising the accuracy of the marker-assisted selection results. RESULTS: Evaluations replicated across accessions and markers showed that a single rice seed is sufficient to generate enough DNA (7-8 ng/µL) to run at least ten PCR trait-markers suitable for marker-assisted selection strategies in rice. The DNA quantity and quality extracted from single seeds from fixed lines (F6) with different physical and/or chemical properties were not significantly different. Nor were there significant differences between single seeds collected 15 days after panicle initiation compared to those harvested at maturity. A large-scale comparison between single seed and leaf-based methodologies showed not only high levels of genotypic concordance between both protocols (~ 99%) but also higher SNP call rates in single seed (99.24% vs. 97.5% in leaf). A cost-benefit analysis showed that this single seed sampling strategy decreased the cost of sampling fourfold. An advantage of this approach is that desirable genotypes can be selected before investing in planting activities reducing the cost associated with field operations. CONCLUSION: This study reports the development of a cost-effective and simple single seed genotyping strategy that facilitates the adoption and deployment of marker-assisted selection strategies in rice. This will allow breeders to increase the frequency of favorable alleles and combine rapid generation advancement techniques much more cost-effectively accelerating the process and efficiency of parental selection and varietal development.

Complex Patterns of Cannabinoid Alkyl Side-Chain Inheritance in Cannabis.

The cannabinoid alkyl side-chain represents an important pharmacophore, where genetic targeting of alkyl homologs has the potential to provide enhanced forms of Cannabis for biopharmaceutical manufacture. Delta(9)-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) synthase genes govern dicyclic (CBDA) and tricyclic (THCA) cannabinoid composition. However, the inheritance of alkyl side-chain length has not been resolved, and few studies have investigated the contributions and interactions between cannabinoid synthesis pathway loci. To examine the inheritance of chemical phenotype (chemotype), THCAS and CBDAS genotypes were scored and alkyl cannabinoid segregation analysed in 210 F2 progeny derived from a cross between two Cannabis chemotypes divergent for alkyl and cyclic cannabinoids. Inheritance patterns of F2 progeny were non-Gaussian and deviated from Mendelian expectations. However, discrete alkyl cannabinoid segregation patterns consistent with digenic as well as epistatic modes of inheritance were observed among F2 THCAS and CBDAS genotypes. These results suggest linkage between cannabinoid pathway loci and highlight the need for further detailed characterisation of cannabinoid inheritance to facilitate metabolic engineering of chemically elite germplasm.

1k-RiCA (1K-Rice Custom Amplicon) a novel genotyping amplicon-based SNP assay for genetics and breeding applications in rice.

BACKGROUND: While a multitude of genotyping platforms have been developed for rice, the majority of them have not been optimized for breeding where cost, turnaround time, throughput and ease of use, relative to density and informativeness are critical parameters of their utility. With that in mind we report the development of the 1K-Rice Custom Amplicon, or 1k-RiCA, a robust custom sequencing-based amplicon panel of ~ 1000-SNPs that are uniformly distributed across the rice genome, designed to be highly informative within indica rice breeding pools, and tailored for genomic prediction in elite indica rice breeding programs. RESULTS: Empirical validation tests performed on the 1k-RiCA showed average marker call rates of 95% with marker repeatability and concordance rates of 99%. These technical properties were not affected when two common DNA extraction protocols were used. The average distance between SNPs in the 1k-RiCA was 1.5 cM, similar to the theoretical distance which would be expected between 1,000 uniformly distributed markers across the rice genome. The average minor allele frequencies on a panel of indica lines was 0.36 and polymorphic SNPs estimated on pairwise comparisons between indica by indica accessions and indica by japonica accessions were on average 430 and 450 respectively. The specific design parameters of the 1k-RiCA allow for a detailed view of genetic relationships and unambiguous molecular IDs within indica accessions and good cost vs. marker-density balance for genomic prediction applications in elite indica germplasm. Predictive abilities of Genomic Selection models for flowering time, grain yield, and plant height were on average 0.71, 0.36, and 0.65 respectively based on cross-validation analysis. Furthermore the inclusion of important trait markers associated with 11 different genes and QTL adds value to parental selection in crossing schemes and marker-assisted selection in forward breeding applications. CONCLUSIONS: This study validated the marker quality and robustness of the 1k-RiCA genotypic platform for genotyping populations derived from indica rice subpopulation for genetic and breeding purposes including MAS and genomic selection. The 1k-RiCA has proven to be an alternative cost-effective genotyping system for breeding applications.

Variation in seed longevity among diverse Indica rice varieties.

BACKGROUND AND AIMS: Understanding variation in seed longevity, especially within closely related germplasm, will lead to better understanding of the molecular basis of this trait, which is particularly important for seed genebanks, but is also relevant to anyone handling seeds. We therefore set out to determine the relative seed longevity of diverse Indica rice accessions through storage experiments. Since antioxidants are purported to play a role in seed storability, the antioxidant activity and phenolic content of caryopses were determined. METHODS: Seeds of 299 Indica rice accessions harvested at 31, 38 and 45 d after heading (DAH) between March and May 2015 and differing in harvest moisture content (MC) were subsequently stored at 10.9 % MC and 45 °C. Samples were taken at regular intervals and sown for germination. Germination data were subjected to probit analysis and the resulting parameters that describe the loss of viability during storage were used for genome-wide association (GWA) analysis. KEY RESULTS: The seed longevity parameters, Ki [initial viability in normal equivalent deviates (NED)], -σ-1 (σ is the time for viability to fall by 1 NED in experimental storage) and p50 [time for viability to fall to 50 % (0 NED)], varied considerably across the 299 Indica accessions. Seed longevity tended to increase as harvest MC decreased and to decrease as harvest MC increased. Eight major loci associated with seed longevity parameters were identified through GWA analysis. The favourable haplotypes on chromosomes 1, 3, 4, 9 and 11 enhanced p50 by ratios of 0.22-1.86. CONCLUSIONS: This is the first study to describe the extent of variation in σ within a species' variety group. A priori candidate genes selected based on rice genome annotation and gene network ontology databases suggested that the mechanisms conferring high seed longevity might be related to DNA repair and transcription, sugar metabolism, reactive oxygen species scavenging and embryonic/root development.