Marker-Assisted Introgression and Stacking of Major QTLs Controlling Grain Number (Gn1a) and Number of Primary Branching (WFP) to NERICA Cultivars.

The era of the green revolution has significantly improved rice yield productivity. However, with the growing population and decreasing arable land, rice scientists must find new ways to improve rice productivity. Although hundreds of rice yield-related QTLs were already mapped and some of them were cloned, only a few were utilized for actual systematic introgression breeding programs. In this study, the major yield QTLs Grain Number 1a (Gn1a) and Wealthy Farmer's Panicle (WFP) were introgressed and stacked in selected NERICA cultivars by marker-assisted backcross breeding (MABB). The DNA markers RM3360, RM3452, and RM5493 were used for foreground selection. At BC3F4 and BC3F5 generation, a combination of marker-assisted selection and phenotypic evaluation were carried out to select lines with target alleles and traits. Further, genotyping-by-sequencing (GBS) was conducted to validate the introgression and determine the recurrent parent genome recovery (RPGR) of the selected lines. The Gn1a and/or WFP introgression lines showed significantly higher numbers of spikelets per panicle and primary branching compared to the recurrent parents. In addition, lines with Gn1a and/or WFP alleles were comparatively similar to the recurrent parents (RP) in most yield-related traits. This study demonstrates the success of utilizing yield QTLs and marker-assisted selection to develop and improve rice cultivars.

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.

Genetic Analysis of Agronomic Traits and Grain Iron and Zinc Concentrations in a Doubled Haploid Population of Rice (Oryza sativa L.).

The development of micronutrient dense rice varieties with good agronomic traits is one of the sustainable and cost-effective approaches for reducing malnutrition. Identification of QTLs for high grain Fe and Zn, yield and yield components helps in precise and faster development of high Fe and Zn rice. We carried out a three-season evaluation using IR05F102 x IR69428 derived doubled-haploid population at IRRI. Inclusive composite interval mapping was carried out using SNP markers and Best Linear Unbiased Estimates of the phenotypic traits. A total of 23 QTLs were identified for eight agronomic traits and grain Fe and Zn concentration that explained 7.2 to 22.0% PV. A QTL by environment interaction analysis confirmed the stability of nine QTLs, including two QTLs for Zn on chromosomes 5 and 12. One epistatic interaction for plant height was significant with 28.4% PVE. Moreover, five QTLs were identified for Fe and Zn that harbor several candidate genes, e.g. OsZIP6 on QTL qZn5.1. A number of QTLs were associated with a combination of greater yield and increased grain Zn levels. These results are useful for development of new rice varieties with good agronomic traits and high grain Zn using MAS, and identification of genetic resources with the novel QTLs for grain Zn.

Trait-based mapping to identify the genetic factors underlying anaerobic germination of rice: Phenotyping, GXE, and QTL mapping.

BACKGROUND: Anaerobic germination is one of the most important traits for rice under direct-seeded conditions. The trait reduces risk of crop failure due to waterlogged conditions after seeding and allows water to be used as a means of weed control. The identification of QTLs and causal genes for anaerobic germination will facilitate breeding for improved direct-seeded rice varieties. In this study, we explored a BC1F2:3 population developed from a cross between BJ1, an indica landrace, and NSIC Rc222, a high-yielding recurrent parent. The population was phenotyped under different screening methods (anaerobic screenhouse, anaerobic tray, and aerobic screenhouse) to establish the relationship among the methods and to identify the most suitable screening method, followed by bulk segregant analysis (BSA) to identify large-effect QTLs. RESULTS: The study showed high heritability for survival (SUR) under all three phenotyping conditions. Although high correlation was observed within screening environments between survival at 14 and 21 days after seeding, the correlation across environments was low. Germination under aerobic and anaerobic conditions showed very low correlation, indicating the independence of their genetic control. The results were further confirmed through AMMI analysis. Four significant markers with an effect on anaerobic germination were identified through BSA. CIM analysis revealed qAG1-2, qAG6-2, qAG7-4, and qAG10-1 having significant effects on the trait. qAG6-2 and qAG10-1 were consistent across screening conditions and seedling age while qAG1-2 and qAG7-4 were specific to screening methods. All QTLs showed an effect when survival across all screening methods was analyzed. Together, the QTLs explained 39 to 55% of the phenotypic variation for survival under anaerobic conditions. No QTL effects were observed under aerobic conditions. CONCLUSIONS: The study helped us understand the effect of phenotyping method on anaerobic germination, which will lead to better phenotyping for this trait in future studies. The QTLs identified through this study will allow the improvement of breeding lines for the trait through marker-assisted selection or through forward breeding approaches such as genomic selection. The high frequency of the BJ1 allele of these QTLs will enhance the robustness of germination under anaerobic conditions in inbred and hybrid rice varieties.

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.

Exploring genetic architecture of grain yield and quality traits in a 16-way indica by japonica rice MAGIC global population.

Identification of Quantitative Trait Loci (QTL) has been a challenge for complex traits due to the use of populations with narrow genetic base. Most of QTL mapping studies were carried out from crosses made within the subspecies, either indica × indica or japonica × japonica. In this study we report advantages of using Multi-parent Advanced Generation Inter-Crosses global population, derived from a combination of eight indica and eight japonica elite parents, in QTL discovery for yield and grain quality traits. Genome-wide association study and interval mapping identified 38 and 34 QTLs whereas Bayesian networking detected 60 QTLs with 22 marker-marker associations, 32 trait-trait associations and 65 marker-trait associations. Notably, nine known QTLs/genes qPH1/OsGA20ox2, qDF3/OsMADS50, PL, QDg1, qGW-5b, grb7-2, qGL3/GS3, Amy6/Wx gene and OsNAS3 were consistently identified by all approaches for nine traits whereas qDF3/OsMADS50 was co-located for both yield and days-to-flowering traits on chromosome 3. Moreover, we identified a number of candidate QTLs in either one or two analyses but further validations will be needed. The results indicate that this new population has enabled identifications of significant QTLs and interactions for 16 traits through multiple approaches. Pyramided recombinant inbred lines provide a valuable source for integration into future breeding programs.

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.

Deciphering Genetics Underlying Stable Anaerobic Germination in Rice: Phenotyping, QTL Identification, and Interaction Analysis.

Anaerobic germination (AG) is an important trait for direct-seeded rice (DSR) to be successful. Rice usually has low germination under anaerobic conditions, which leads to a poor crop stand in DSR when rain occurs after seeding. The ability of rice to germinate under water reduces the risk of poor crop stand. Further, this allows the use of water as a method of weed control. The identification of the genetic factors leading to high anaerobic germination is required to develop improved DSR varieties. In the present study, two BC1F2:3 mapping families involving a common parent with anaerobic germination potential, Kalarata, an indica landrace, and two recurrent parents, NSIC Rc222 and NSIC Rc238, were used. Phenotyping was done under two environmental conditions and genotyping was carried out through the KASP SNP genotyping platform. A total of 185 and 189 individuals genotyped with 170 and 179 polymorphic SNPs were used for QTL analysis for the two populations, Kalarata/NSIC Rc238 and Kalarata/NSIC Rc222, respectively. A total of five QTLs on chromosomes 3, 5, 6, 7, and 8 for survival (SUR) and four QTLs on chromosomes 1, 3 (two locations), and 7 for the trait seedling height (SH) across the populations and over the screening conditions were identified. Except for the QTLs on chromosomes 5 and 8, the parent with AG potential, Kalarata, contributed all the other QTLs. Among the five QTLs for SUR, the second-largest QTL (qSUR6-1) was novel for AG potential in rice, showing a stable expression in terms of genetic background and screening conditions explaining 11.96% to 16.01% of the phenotypic variation. The QTL for SH (qSH1-1) was also novel. Considering different genetic backgrounds and different screening conditions, the QTLs identified for the trait SUR explained phenotypic variation in the range of 57.60% to 73.09% while that for the trait SH ranged from 13.53% to 34.30%.

Genome-Wide Association Mapping in a Rice MAGIC Plus Population Detects QTLs and Genes Useful for Biofortification.

The development of rice genotypes with micronutrient-dense grains and disease resistance is one of the major priorities in rice improvement programs. We conducted Genome-wide association studies (GWAS) using a Multi-parent Advanced Generation Inter-Cross (MAGIC) Plus population to identify QTLs and SNP markers that could potentially be integrated in biofortification and disease resistance breeding. We evaluated 144 MAGIC Plus lines for agronomic and biofortification traits over two locations for two seasons, while disease resistance was screened for one season in the screen house. X-ray fluorescence technology was used to measure grain Fe and Zn concentrations. Genotyping was carried out by genotype by sequencing and a total of 14,242 SNP markers were used in the association analysis. We used Mixed linear model (MLM) with kinship and detected 57 significant genomic regions with a -log10 (P-value) ≥ 3.0. The PH 1.1 and Zn 7.1 were consistently identified in all the four environments, ten QTLs qDF 3.1, qDF 6.2 qDF 9.1 qPH 5.1 qGL 3.1, qGW 3.1, qGW 11.1, and qZn 6.2 were detected in two environments, while two major loci qBLB 11.1 and qBLB 5.1 were identified for Bacterial Leaf Blight (BLB) resistance. The associated SNP markers were found to co-locate with known major genes and QTLs such as OsMADS50 for days to flowering, osGA20ox2 for plant height, and GS3 for grain length. Similarly, Xa4 and xa5 genes were identified for BLB resistance and Pi5(t), Pi28(t), and Pi30(t) genes were identified for Blast resistance. A number of metal homeostasis genes OsMTP6, OsNAS3, OsMT2D, OsVIT1, and OsNRAMP7 were co-located with QTLs for Fe and Zn. The marker-trait relationships from Bayesian network analysis showed consistency with the results of GWAS. A number of promising candidate genes reported in our study can be further validated. We identified several QTLs/genes pyramided lines with high grain Zn and acceptable yield potential, which are a good resource for further evaluation to release as varieties as well as for use in breeding programs.

A trehalose-6-phosphate phosphatase enhances anaerobic germination tolerance in rice.

Global socioeconomic developments create strong incentives for farmers to shift from transplanted to direct-seeded rice (DSR) as a means of intensification and economization(1). Rice production must increase to ensure food security(2) and the bulk of this increase will have to be achieved through intensification of cultivation, because expansion of cultivated areas is reaching sustainable limits(3). Anaerobic germination tolerance, which enables uniform germination and seedling establishment under submergence(4), is a key trait for the development of tropical DSR varieties(5,6). Here, we identify a trehalose-6-phosphate phosphatase gene, OsTPP7, as the genetic determinant in qAG-9-2, a major quantitative trait locus (QTL) for anaerobic germination tolerance(7). OsTPP7 is involved in trehalose-6-phosphate (T6P) metabolism, central to an energy sensor that determines anabolism or catabolism depending on local sucrose availability(8,9). OsTPP7 activity may increase sink strength in proliferating heterotrophic tissues by indicating low sugar availability through increased T6P turnover, thus enhancing starch mobilization to drive growth kinetics of the germinating embryo and elongating coleoptile, which consequently enhances anaerobic germination tolerance.

qEMF3, a novel QTL for the early-morning flowering trait from wild rice, Oryza officinalis, to mitigate heat stress damage at flowering in rice, O. sativa.

A decline in rice (Oryza sativa L.) production caused by heat stress is one of the biggest concerns resulting from future climate change. Rice spikelets are most susceptible to heat stress at flowering. The early-morning flowering (EMF) trait mitigates heat-induced spikelet sterility at the flowering stage by escaping heat stress during the daytime. We attempted to develop near-isogenic lines (NILs) for EMF in the indica-type genetic background by exploiting the EMF locus from wild rice, O. officinalis (CC genome). A stable quantitative trait locus (QTL) for flower opening time (FOT) was detected on chromosome 3. A QTL was designated as qEMF3 and it shifted FOT by 1.5-2.0 h earlier for cv. Nanjing 11 in temperate Japan and cv. IR64 in the Philippine tropics. NILs for EMF mitigated heat-induced spikelet sterility under elevated temperature conditions completing flower opening before reaching 35°C, a general threshold value leading to spikelet sterility. Quantification of FOT of cultivars popular in the tropics and subtropics did not reveal the EMF trait in any of the cultivars tested, suggesting that qEMF3 has the potential to advance FOT of currently popular cultivars to escape heat stress at flowering under future hotter climates. This is the first report to examine rice with the EMF trait through marker-assisted breeding using wild rice as a genetic resource.

Mapping quantitative trait loci associated with yield and yield components under reproductive stage salinity stress in rice (Oryza sativa L.).

Salinity tolerance in rice is critical at reproductive stage because it ultimately determines grain yield. An F2 mapping population derived from a Sadri/FL478 cross was exposed to saline field conditions (6-8 dS m(-1)) after the active tillering stage to identify reproductive stage specific QTLs for salinity tolerance. Genetic linkage map was constructed using 123 microsatellite markers on 232 F2 progenies. Totally 35 QTLs for 11 traits under salinity stress were detected with LOD > 3, out of which 28 QTLs that explained from 5.9 to 30.0% phenotypic variation were found to be significant based on permutation test. Three major QTL clusters were found on chromosomes 2 (RM423-RM174), 4 (RM551-RM518) and 6 (RM20224-RM528) for multiple traits under salinity stress. Both parental lines contributed additively for QTLs identified for the yield components. A majority of the QTLs detected in our study are reported for the first time for reproductive stage salinity stress. Fine-mapping of selected putative QTLs will be the next step to facilitate marker-assisted backcrossing and to detect useful genes for salinity tolerance at the reproductive stage in rice.