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.

PENTOSAN POLYSULFATE SODIUM (ELMIRON) MACULOPATHY: A Genetic Perspective.

PURPOSE: To assess genetic associations for pentosan polysufate sodium maculopathy. METHODS: Genetic testing for inherited retinal dystrophy genes using exome testing and for 14 age-related macular degeneration-associated single nucleotide polymorphisms (SNPs) using panel testing were performed. In addition, full-field electroretinograms (ffERG) were obtained to identify any cone-rod dystrophy. RESULTS: Eleven of 15 patients were women, with a mean age of 69 (range 46-85). Inherited retinal dystrophy exome testing in five patients revealed six pathogenic variants, but failed to confirm inherited retinal dystrophy in any patient genetically. FfERG performed in 12 patients demonstrated only nonspecific a- and b-wave abnormalities in 11 cases and was normal in one case. For age-related macular degeneration single nucleotide polymorphisms, CFH rs3766405 ( P = 0.003) and CETP ( P = 0.027) were found to be statistically significantly associated with pentosan polysulfate maculopathy phenotype compared with the control population. CONCLUSION: Pentosan polysulfate maculopathy is not associated with Mendelian inherited retinal dystrophy genes. However, several age-related macular degeneration risk alleles were identified to be associated with maculopathy compared with their frequency in the normal population. This suggests a role for genes in disease pathology, particularly the alternative complement pathway. These findings would benefit from further investigation to understand the risk of developing maculopathy in taking pentosan polysulfate.

Development of a new AgriSeq 4K mid-density SNP genotyping panel and its utility in pearl millet breeding.

Pearl millet is a crucial nutrient-rich staple food in Asia and Africa and adapted to the climate of semi-arid topics. Since the genomic resources in pearl millet are very limited, we have developed a brand-new mid-density 4K SNP panel and demonstrated its utility in genetic studies. A set of 4K SNPs were mined from 925 whole-genome sequences through a comprehensive in-silico pipeline. Three hundred and seventy-three genetically diverse pearl millet inbreds were genotyped using the newly-developed 4K SNPs through the AgriSeq Targeted Genotyping by Sequencing technology. The 4K SNPs were uniformly distributed across the pearl millet genome and showed considerable polymorphism information content (0.23), genetic diversity (0.29), expected heterozygosity (0.29), and observed heterozygosity (0.03). The SNP panel successfully differentiated the accessions into two major groups, namely B and R lines, through genetic diversity, PCA, and structure models as per their pedigree. The linkage disequilibrium (LD) analysis showed Chr3 had higher LD regions while Chr1 and Chr2 had more low LD regions. The genetic divergence between the B- and R-line populations was 13%, and within the sub-population variability was 87%. In this experiment, we have mined 4K SNPs and optimized the genotyping protocol through AgriSeq technology for routine use, which is cost-effective, fast, and highly reproducible. The newly developed 4K mid-density SNP panel will be useful in genomics and molecular breeding experiments such as assessing the genetic diversity, trait mapping, backcross breeding, and genomic selection in pearl millet.

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.

Flood resilience loci SUBMERGENCE 1 and ANAEROBIC GERMINATION 1 interact in seedlings established underwater.

Crops with resilience to multiple climatic stresses are essential for increased yield stability. Here, we evaluate the interaction between two loci associated with flooding survival in rice (Oryza sativa L.). ANAEROBIC GERMINATION 1 (AG1), encoding trehalose 6-phosphate phosphatase 7 (TPP7), promotes mobilization of endosperm reserves to enhance the elongation of a hollow coleoptile in seeds that are seeded directly into shallow paddies. SUBMERGENCE 1 (SUB1), encoding the ethylene-responsive transcription factor SUB1A-1, confers tolerance to complete submergence by dampening carbohydrate catabolism, to enhance recovery upon desubmergence. Interactions between AG1/TPP7 and SUB1/SUB1A-1 were investigated under three flooding scenarios using four near-isogenic lines by surveying growth and survival. Pyramiding of the two loci does not negatively affect anaerobic germination or vegetative-stage submergence tolerance. However, the pyramided AG1 SUB1 genotype displays reduced survival when seeds are planted underwater and maintained under submergence for 16 d. To better understand the roles of TPP7 and SUB1A-1 and their interaction, temporal changes in carbohydrates and shoot transcriptomes were monitored in the four genotypes varying at the two loci at four developmental timeponts, from day 2 after seeding through day 14 of complete submergence. TPP7 enhances early coleoptile elongation, whereas SUB1A-1 promotes precocious photoautotrophy and then restricts underwater elongation. By contrast, pyramiding of the AG1 and SUB1 slows elongation growth, the transition to photoautotrophy, and survival. mRNA-sequencing highlights time-dependent and genotype-specific regulation of mRNAs associated with DNA repair, cell cycle, chromatin modification, plastid biogenesis, carbohydrate catabolism and transport, elongation growth, and other processes. These results suggest that interactions between AG1/TPP7 and SUB1/SUB1A-1 could impact seedling establishment if paddy depth is not effectively managed after direct seeding.

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.

QTL mapping for tolerance to anaerobic germination in rice from IR64 and the aus landrace Kharsu 80A.

Direct seeding of rice often results in poor crop establishment due to unlevelled fields, unpredicted heavy rains after sowing, and weed and pest invasion. Thus, it is important to develop varieties able to tolerate flooding during germination, also known as anaerobic germination (AG), to address these constraints. A study was conducted to identify QTLs associated with AG tolerance from an IR64/Kharsu 80A F2:3 mapping population using 190 lines phenotyped for seedling survival under the stress. Genotyping was performed using a genomewide 384-plex Indica/Indica SNP set. Four QTLs derived from Kharsu 80A providing increased tolerance to anaerobic germination were identified: three on chromosome 7 (qAG7.1, qAG7.2 and qAG7.3) and one on chromosome 3 (qAG3), with LOD values ranging from 5.7 to 7.7, and phenotypic variance explained (R2) from 8.1% to 12.6%. The QTLs identified in this study can be further investigated to better understand the genetic bases of AG tolerance in rice, and used for marker-assisted selection to develop more robust direct-seeded rice varieties.

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.

Exploring the genetic diversity within traditional Philippine pigmented Rice.

BACKGROUND: The wild ancestors of domesticated rice had red seed, white rice being the result of a mutation in the rice domestication gene Rc. Many pigmented rice landraces are still grown by ethnic communities for their nutritional and cultural value. This study assesses the genetic diversity in a collection of pigmented rice accessions from the Philippines. RESULTS: We undertook an analysis of the genetic and colour variation in a collection of 696 pigmented rice accessions held at PhilRice in the Philippines. The collection was reduced to 589 genotypes after removal of accessions with limited passport data or with low SNP marker call rates. Removal of duplicate genotypes resulted in a final, core collection of 307 accessions, representing all administrative districts of the Philippines, and composed predominately of japonica and indica sub-species. No genetic structure was observed in the core collection based on geographic origin. A pairwise comparison of accessions by region indicating that both local and long-distance exchange of rice accessions had occurred. The majority of the genetic variation was within regions (82.38%), rather than between regions (10.23%), with the remaining variation being within rice accession variance (7.39%). The most genetically diverse rice accessions originated from the Cordillera Administrative Region (CAR) in the far north of the Philippines, and in the regions of Davao and Caraga in the southeast. A comparison with pigmented rice accessions from the neighbouring countries Taiwan, Laos, China and India revealed a close relationship between accessions from Taiwan, supporting the hypothesis of southward diffusion of Austronesians from Taiwan to the Philippine. The 14-bp deletion within the gene Rc, known to result in loss of red pigmentation, was found in 30 accessions that still had coloured pericarps. Multi-spectral phenotyping was used to measure seed geometric and colour-appearance traits in 197 accessions from the core collection. The purple and variable purple rice accessions had the lowest values for the seed colour parameters - lightness (L*), intensity, saturation, a* (green - red; redness) and b* (blue - yellow; yellowness). CONCLUSION: These pigmented rice accessions represent a diverse genetic resource of value for further study and nutritional improvement of commercial rice varieties.

QTL mapping and confirmation for tolerance of anaerobic conditions during germination derived from the rice landrace Ma-Zhan Red.

Wide adoption of direct-seeded rice practices has been hindered by poorly leveled fields, heavy rainfall and poor drainage, which cause accumulation of water in the fields shortly after sowing, leading to poor crop establishment. This is due to the inability of most rice varieties to germinate and reach the water surface under complete submergence. Hence, tolerance of anaerobic conditions during germination is an essential trait for direct-seeded rice cultivation in both rainfed and irrigated ecosystems. A QTL study was conducted to unravel the genetic basis of tolerance of anaerobic conditions during germination using a population derived from a cross between IR42, a susceptible variety, and Ma-Zhan Red, a tolerant landrace from China. Phenotypic data was collected based on the survival rates of the seedlings at 21 days after sowing of dry seeds under 10 cm of water. QTL analysis of the mapping population consisting of 175 F2:3 families genotyped with 118 SSR markers identified six significant QTLs on chromosomes 2, 5, 6, and 7, and in all cases the tolerant alleles were contributed by Ma-Zhan Red. The largest QTL on chromosome 7, having a LOD score of 14.5 and an R (2) of 31.7 %, was confirmed using a BC2F3 population. The QTLs detected in this study provide promising targets for further genetic characterization and for use in marker-assisted selection to rapidly develop varieties with improved tolerance to anaerobic condition during germination. Ultimately, this trait can be combined with other abiotic stress tolerance QTLs to provide resilient varieties for direct-seeded systems.