Proof of concept and early development stage of market-oriented high iron and zinc rice expressing dicot ferritin and rice nicotianamine synthase genes.

Micronutrient deficiencies such as iron (Fe), zinc (Zn), and vitamin A, constitute a severe global public health phenomenon. Over half of preschool children and two-thirds of nonpregnant women of reproductive age worldwide have micronutrient deficiencies. Biofortification is a cost-effective strategy that comprises a meaningful and sustainable means of addressing this issue by delivering micronutrients through staple foods to populations with limited access to diverse diets and other nutritional interventions. Here, we report on the proof-of-concept and early development stage of a collection of biofortified rice events with a high density of Fe and Zn in polished grains that have been pursued further to advance development for product release. In total, eight constructs were developed specifically expressing dicot ferritins and the rice nicotianamine synthase 2 (OsNAS2) gene under different combinations of promoters. A large-scale transformation of these constructs to Bangladesh and Philippines commercial indica cultivars and subsequent molecular screening and confined field evaluations resulted in the identification of a pool of ten events with Fe and Zn concentrations in polished grains of up to 11 µg g-1 and up to 37 µg g-1, respectively. The latter has the potential to reduce the prevalence of inadequate Zn intake for women of childbearing age in Bangladesh and in the Philippines by 30% and 50%, respectively, compared to the current prevalence. To our knowledge, this is the first potential biotechnology public-sector product that adopts the product cycle phase-gated approach, routinely applied in the private sector.

CRISPR-mediated promoter editing of a cis-regulatory element of OsNAS2 increases Zn uptake/translocation and plant yield in rice.

Developing nutritious rice with a higher yield is one approach to alleviating the problem of micronutrient deficiency in developing countries, especially human malnutrition involving zinc and iron (Fe) deficiency, and achieving better adoption. The transport of micronutrients such as Fe and Zn is mainly regulated via the nicotianamine synthase (OsNAS) gene family, whereas yield is a complex trait that involves multiple loci. Genome editing via CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, focusing on the OsNAS2 promoter, particularly the deletion of the cis-regulatory element ARR1AT at position -933, was conducted for an enhanced accumulation of Zn in the grain and per plant. The results showed that our promoter editing increased Zn concentration per plant. Evidence also showed that an improved spikelet number per main panicle led to increased grain per plant. The traits were inherited in "transgene-free" and homozygous plant progenies. Further investigation needs to be conducted to validate trait performance under field conditions and elucidate the cause of the spikelet increase.

Development and Field Evaluation of Near-Isogenic Lines of GR2-EBRRI dhan29 Golden Rice.

Vitamin A deficiency remains a common public health problem among the rice-dependent poor people in the developing countries of Asia. Conventional milled rice does not contain provitamin A (ß-carotene) in is edible part (endosperm) and is also deficient in essential minerals, such as iron and zinc. Transgenic Golden Rice event GR2E, which produces ß-carotene in its endosperm, was used as a parent to introgress the transgene locus conferring ß-carotene biosynthesis into a widely grown rice variety, BRRI dhan29, which covers around 26.1% of the irrigated rice area (4.901 Mha) of Bangladesh in the dry season. The current study reports the introgression process and field performance of GR2E BRRI dhan29 Golden Rice. The background recovery of GR2E BRRI dhan29 lines at BC5F2 generation was more than 98% with a 6K SNP-chip set. The transgenic GR2E BRRI dhan29 yielded 6.2 t/ha to 7.7 t/ha with an average of 7.0 ± 0.38 t/ha, while the non-transgenic BRRI dhan29 yielded 7.0 t/ha under confined field conditions in Bangladesh. Moreover, no significant difference between GR2-E BRRI dhan29 Golden Rice and non-transgenic BRRI dhan29 in any measured trait was observed in the multi-location trials conducted at five locations across the country. Furthermore, the appearance of cooked and uncooked rice was similar to that of BRRI dhan29 except for the yellow color indicating the presence of carotenoids. Total carotenoid content in the selected introgression lines ranged from 8.5 to 12.5 µg/g with an average of 10.6 ± 1.16 µg/g. This amount is sufficient to deliver approximately 66 and 80% of the recommended daily intake of vitamin A for children and women, respectively, assuming complete substitution of white rice in the diet with Golden Rice. However, the lead selected line(s) need further evaluation at open field conditions before deciding for commercial cultivation. A large-scale feeding trial among the malnourished community with this newly developed GR2-E BRRI dhan29 Golden Rice is also required to validate its efficacy in alleviating vitamin A deficiency.

Development and characterization of GR2E Golden rice introgression lines.

Golden Rice with ß-carotene in the grain helps to address the problem of vitamin A deficiency. Prior to commercialize Golden Rice, several performance and regulatory checkpoints must be achieved. We report results of marker assisted backcross breeding of the GR2E trait into three popular rice varieties followed by a series of confined field tests of event GR2E introgression lines to assess their agronomic performance and carotenoid expression. Results from confined tests in the Philippines and Bangladesh have shown that GR2E introgression lines matched the performance of the recurrent parents for agronomic and yield performance, and the key components of grain quality. Moreover, no differences were observed in terms of pest and disease reaction. The best performing lines identified in each genetic background had significant amounts of carotenoids in the milled grains. These lines can supply 30-50% of the estimated average requirements of vitamin A.

Genotype × environment interactions for grain iron and zinc content in rice.

BACKGROUND: Nutrient deficiency in humans, especially in children and lactating women, is a major concern. Increasing the micronutrient concentration in staple crops like rice is one way to overcome this. The micronutrient content in rice, especially the iron (Fe) and zinc (Zn) content, is highly variable. The identification of rice genotypes in which there are naturally high Fe and Zn concentrations across environments is an important target towards the production of biofortified rice. RESULTS: Phenotypic correlations between grain Fe and Zn content were positive and significant in all environments but a significant negative association was observed between grain yield and grain Fe and Zn. Promising breeding lines with higher Zn or Fe content, or both, were: IR 82475-110-2-2-1-2 (Zn: 20.24-37.33 mg kg-1 ; Fe: 7.47-14.65 mg kg-1 ); IR 83294-66-2-2-3-2 (Zn: 22-37-41.97 mg kg-1 ; Fe: 9.43-17.16); IR 83668-35-2-2-2 (Zn: 27.15-42.73 mg kg-1 ; Fe: 6.01-14.71); IR 68144-2B-2-2-3-1-166 (Zn: 23.53-40.30 mg kg-1 ; Fe: 10.53-17.80 mg kg-1 ) and RP Bio 5478-185M7 (Zn: 22.60-40.07 mg kg-1 ; Fe: 7.64-14.73 mg kg-1 ). Among these, IR82475-110-2-2-1-2 (Zn: 20.24-37.33 mg kg-1 ; Fe: 7.47-14.65 mg kg-1 ) is also high yielding with 3.75 t ha-1 . Kelhrie Cha (Zn: 17.76-36.45 mg kg-1 ; Fe: 7.17-14.77 mg kg-1 ), Dzuluorhe (Zn: 17.48-39.68 mg kg-1 ; Fe: 7.89-19.90 mg kg-1 ), Nedu (Zn: 18.97-43.55 mg kg-1 Fe: 8.01-19.51 mg kg-1 ), Kuhusoi-Ri-Sareku (Zn: 17.37-44.14 mg kg-1 ; Fe: 8.99-14.30 mg kg-1 ) and Mima (Zn: 17.10-45.64 mg kg-1 ; Fe: 9.97-17.40 mg kg-1 ) were traditional donor genotypes that possessed both high grain Fe and high Zn content. CONCLUSION: Significant genotype × location (G × L) effects were observed in all traits except Fe. Genetic variance was significant and was considerably larger than the variance of G × L for grain Zn and Fe content traits, except grain yield. The G × L × year variance component was significant in all cases. © 2020 Society of Chemical Industry.

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.

Molecular characterization and safety assessment of biofortified provitamin A rice.

Part of the studies involved in safety assessment of genetically engineered crops includes characterizing the organization, integrity, and stability of the inserted DNA and evaluating the potential allergenicity and toxicity of newly-expressed proteins. Molecular characterization of the introduced DNA in provitamin A biofortified rice event GR2E confirmed insertion of a single copy of the transfer-DNA in the genome and its inheritance as a single locus. Nucleotide sequencing of the inserted DNA confirmed it was introduced without modifications. The phytoene synthase, and carotene desaturase proteins did not display sequence similarity with allergens or toxins. Both proteins were rapidly digested in simulated gastric fluid and their enzymatic activity was inhibited upon heat treatment. Acute oral toxicity testing of the protein in mice demonstrated lack of adverse effects. These evidences substantiated the lack of any identifiable hazards for both proteins and in combination with other existing comparative analyses provided assurance that food derived from this rice is safe. This conclusion is in line with those of the regulatory agencies of US Food and Drug Administration, Health Canada and Food Standard Australia and New Zealand.

Compositional Analysis of Genetically Engineered GR2E "Golden Rice" in Comparison to That of Conventional Rice.

Compositional analyses were performed on samples of rice grain, straw, and derived bran obtained from golden rice event GR2E and near-isogenic control PSBRc82 rice grown at four locations in the Philippines during 2015 and 2016. Grain samples were analyzed for key nutritional components, including proximates, fiber, polysaccharides, fatty acids, amino acids, minerals, vitamins, and antinutrients. Samples of straw and bran were analyzed for proximates and minerals. The only biologically meaningful difference between GR2E and control rice was in levels of ß-carotene and other provitamin A carotenoids in the grain. Except for ß-carotene and related carotenoids, the compositional parameters of GR2E rice were within the range of natural variability of those components in conventional rice varieties with a history of safe consumption. Mean provitamin A concentrations in milled rice of GR2E can contribute up to 89-113% and 57-99% of the estimated average requirement for vitamin A for preschool children in Bangladesh and the Philippines, respectively.

Zinc and Iron Nutrition Status in the Philippines Population and Local Soils.

The Philippines is one of the major rice-producing and rice-consuming countries of Asia. A large portion of its population depends on rice for their daily caloric intake and nutritional needs. The lack of dietary diversity among poor communities has led to nutritional consequences, particularly micronutrient deficiencies. Iron-deficiency anemia (IDA) and zinc deficiency (ZnD) are two serious nutritional problems that affect the health and economic sector of the country. Since rice dominates the Filipino diet by default, biofortification of rice will help improve the micronutrient status. The Philippine government has proactively initiated various programs and policies to address micronutrient deficiencies, particularly through fortification of basic food commodities. Biofortification, the fortification of rice with micronutrients through breeding, is considered the most sustainable and cost-effective strategy that can benefit large vulnerable populations. However, developing promising genotypes with micronutrient-enriched grains should be coupled with improving micronutrient bioavailability in the soil in order to optimize biofortification. This review documents the prevailing soil Zn-deficiency problems in the major rice production areas in the Philippines that may influence the Zn nutritional status of the population. The article also reports on the biofortification efforts that have resulted in the development of two biofortified varieties approved for commercial release in the Philippines. As nutritional security is increasingly recognized as a priority area, greater efforts are required to develop biofortified rice varieties that suit both farmers' and consumers' preferences, and that can address these critical needs for human health in a sustainable and cost-effective manner.

A novel resistance gene for bacterial blight in rice, Xa43(t) identified by GWAS, confirmed by QTL mapping using a bi-parental population.

Bacterial blight (BB) caused by the Xanthomonas oryzae pv. oryzae (Xoo) pathogen is a significant disease in most rice cultivation areas. The disease is estimated to cause annual rice production losses of 20-30 percent throughout rice-growing countries in Asia. The discovery and deployment of durable resistance genes for BB is an effective and sustainable means of mitigating production losses. In this study QTL analysis and fine mapping were performed using an F2 and a BC2F2 population derived from a cross with a new R-donor having broad spectrum resistance to Korean BB races. The QTL qBB11 was identified by composite interval mapping and explained 31.25% of the phenotypic variation (R2) with LOD values of 43.44 harboring two SNP markers. The single major R-gene was designated Xa43 (t). Through dissection of the target region we were able to narrow the region to within 27.83-27.95 Mbp, a physical interval of about 119-kb designated by the two flanking markers IBb27os11_14 and S_BB11.ssr_9. Of nine ORFs in the target region two ORFs revealed significantly different expression levels of the candidate genes. From these results we developed a marker specific to this R-gene, which will have utility for future BB resistance breeding and/or R-gene pyramiding using marker assisted selection. Further characterization of the R-gene would be helpful to enhance understanding of mechanisms of BB resistance in rice.

Genetic Dissection of Grain Nutritional Traits and Leaf Blight Resistance in Rice.

Colored rice is rich in nutrition and also a good source of valuable genes/quantitative trait loci (QTL) for nutrition, grain quality, and pest and disease resistance traits for use in rice breeding. Genome-wide association analysis using high-density single nucleotide polymorphism (SNP) is useful in precisely detecting QTLs and genes. We carried out genome-wide association analysis in 152 colored rice accessions, using 22,112 SNPs to map QTLs for nutritional, agronomic, and bacterial leaf blight (BLB) resistance traits. Wide variations and normal frequency distributions were observed for most of the traits except anthocyanin content and BLB resistance. The structural and principal component analysis revealed two subgroups. The linkage disequilibrium (LD) analysis showed 74.3% of the marker pairs in complete LD, with an average LD distance of 1000 kb and, interestingly, 36% of the LD pairs were less than 5 Kb, indicating high recombination in the panel. In total, 57 QTLs were identified for ten traits at p < 0.0001, and the phenotypic variance explained (PVE) by these QTLs varied from 9% to 18%. Interestingly, 30 (53%) QTLs were co-located with known or functionally-related genes. Some of the important candidate genes for grain Zinc (Zn) and BLB resistance were OsHMA9, OsMAPK6, OsNRAMP7, OsMADS13, and OsZFP252, and Xa1, Xa3, xa5, xa13 and xa26, respectively. Red rice genotype, Sayllebon, which is high in both Zn and anthocyanin content, could be a valuable material for a breeding program for nutritious rice. Overall, the QTLs identified in our study can be used for QTL pyramiding as well as genomic selection. Some of the novel QTLs can be further validated by fine mapping and functional characterization. The results show that pigmented rice is a valuable resource for mineral elements and antioxidant compounds; it can also provide novel alleles for disease resistance as well as for yield component traits. Therefore, large opportunities exist to further explore and exploit more colored rice accessions for use in breeding.

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.

Identification of genomic regions associated with agronomic and biofortification traits in DH populations of rice.

Rice provides energy and nutrition to more than half of the world's population. Breeding rice varieties with the increased levels of bioavailable micronutrients is one of the most sustainable approaches to tackle micronutrient malnutrition. So, high zinc and iron content in the grain are primary targets in rice biofortification breeding. In this study, we conducted QTL mapping using doubled haploid (DH) populations, PSBRc82 x Joryeongbyeo and PSBRc82 x IR69428, phenotyped for agronomic traits and micronutrients during two growing seasons and using genotypic information from analysis with the 6K SNP chip. A number of DH lines were identified as having high grain Zn and Fe content in polished rice. Importantly, we identified 20 QTLs for agronomic traits and 59 QTLs for a number of biofortification traits. Of the 79 QTLs, 12 were large-effect QTLs (>25% PVE), nine QTLs were consistent across seasons in either population, and one QTL was identified in both populations. Moreover, at least two QTLs were clustered in defined regions of chromosomes 1, 2, 3, 4, 5, 7 and 9. Eight epistatic interactions were detected for Cu, Mg, Na, and Zn in population 1. Furthermore, we identified several candidate genes near QTLs for grain Zn (OsNRAMP, OsNAS, OsZIP, OsYSL, OsFER, and OsZIFL family) and grain yield (OsSPL14 and OsSPL16). These new QTLs and candidate genes help to further elucidate the genetic basis for grain micronutrient concentration, and may prove useful for marker assisted breeding for this important trait.

Correction to: Developing japonica rice introgression lines with multiple resistance genes for brown planthopper, bacterial blight, rice blast, and rice stripe virus using molecular breeding.

In the original publication.

Developing japonica rice introgression lines with multiple resistance genes for brown planthopper, bacterial blight, rice blast, and rice stripe virus using molecular breeding.

Yield losses as a result of biotic stresses by fungi, bacteria, viruses, and insects are a key challenge in most rice cultivation areas. The development of resistant cultivars is considered an efficient and sustainable approach to mitigate rice yield reduction. In the present study, we describe the development of japonica rice introgression lines with multiple resistance genes (MR lines), resistant to four different types of biotic stresses, and compare the agronomic performance, yield, and grain quality parameters of these lines with those of the recurrent parent. A total of nine MR lines were developed by marker-assisted backcrossing, which combined five single-R genes in a japonica background with a minimum of linkage drag. All the MR lines harbored the R genes Bph18 and qSTV11SG and two Pi genes (Pib + Pik) in common, offering resistance to brown planthopper (BPH), rice stripe virus (RSV), and rice blast disease, respectively. In the case of bacterial blight (BB), Xa40 was detected in only five out of the nine and Xa3 was validated in the others. In particular, the five MR lines pyramiding the R genes (Bph18 + qSTV11SG + Pib + Pik) in combination with Xa40 showed stable resistance to all bioassays for BPH, BB, blast, and RSV. The MR lines did not show any negative effects on the main agronomic traits, including yield production and rice grain quality. The lines have significant potential to stabilize rice yield and minimize production costs in disease and pest-prone areas in Korea, through the pyramiding of five R genes using a marker-assisted backcrossing strategy.

Genome-wide association study of seedling stage salinity tolerance in temperate japonica rice germplasm.

BACKGROUND: Salinity has a significant impact on rice production in coastal, arid and semi-arid areas in many countries, including countries growing temperate rice, such as Kazakhstan. Recently, the complete genomes of 3000 rice accessions were sequenced through the 3 K rice genome project, and this set included 203 temperate japonica rice accessions. To identify salinity-tolerant germplasm and related genes for developing new salinity-tolerant breeding lines for the temperate japonica rice growing regions, we evaluated the seedling stage salinity tolerance of these sequenced temperate japonica rice accessions, and conducted genome-wide association studies (GWAS) for a series of salinity tolerance related traits. RESULTS: There were 27 accessions performed well (SES < 5.0) under moderate salinity stress (EC12), and 5 accessions were tolerant under both EC12 and EC18. A total of 26 QTLs were identified for 9 measured traits. Eleven of these QTLs were co-located with known salinity tolerance genes. QTL/gene clusters were observed on chromosome 1, 2, 3, 6, 8 and 9. Six candidate genes were identified for five promising QTLs. The alleles of major QTL Saltol and gene O S HKT1;5 (SKC1) for Na+/K+ ratio identified in indica rice accessions were different from those in the temperate japonica rice accessions used in this study. CONCLUSION: Salinity tolerant temperate japonica rice accessions were identified in this study, these accessions are important resources for breeding programs. SNPs located in the promising QTLs and candidate genes could be used for future gene validation and marker assisted selection. This study provided useful information for future studies on genetics and breeding of salinity tolerance in temperate japonica rice.

RiceAtlas, a spatial database of global rice calendars and production.

Knowing where, when, and how much rice is planted and harvested is crucial information for understanding the effects of policy, trade, and global and technological change on food security. We developed RiceAtlas, a spatial database on the seasonal distribution of the world's rice production. It consists of data on rice planting and harvesting dates by growing season and estimates of monthly production for all rice-producing countries. Sources used for planting and harvesting dates include global and regional databases, national publications, online reports, and expert knowledge. Monthly production data were estimated based on annual or seasonal production statistics, and planting and harvesting dates. RiceAtlas has 2,725 spatial units. Compared with available global crop calendars, RiceAtlas is nearly ten times more spatially detailed and has nearly seven times more spatial units, with at least two seasons of calendar data, making RiceAtlas the most comprehensive and detailed spatial database on rice calendar and production.

Advances in breeding for high grain Zinc in Rice.

Zinc (Zn) is one of the most essential micronutrients required for the growth and development of human beings. More than one billion people, particularly children and pregnant women suffer from Zn deficiency related health problems in Asia. Rice is the major staple food for Asians, but the presently grown popular high yielding rice varieties are poor supplier of Zn in their polished form. Breeding rice varieties with high grain Zn has been suggested to be a sustainable, targeted, food-based and cost effective approach in alleviating Zn deficiency. The physiological, genetic and molecular mechanisms of Zn homeostasis have been well studied, but these mechanisms need to be characterized from a biofortification perspective and should be well integrated with the breeding processes. There is a significant variation for grain Zn in rice germplasm and efforts are being directed at exploiting this variation through breeding to develop high Zn rice varieties. Several QTLs and gene specific markers have been identified for grain Zn and there is a great potential to use them in Marker-Assisted Breeding. A thorough characterization of genotype and environmental interactions is essential to identify key environmental factors influencing grain Zn. Agronomic biofortification has shown inconsistent results, but a combination of genetic and agronomic biofortification strategies may be more effective. Significant progress has been made in developing high Zn rice lines for release in target countries. A holistic breeding approach involving high Zn trait development, high Zn product development, product testing and release, including bioefficacy and bioavailability studies is essential for successful Zn biofortification.

Identification of stable QTLs causing chalk in rice grains in nine environments.

KEY MESSAGE: A novel QTL cluster for chalkiness on Chr04 was identified using single environment analysis and joint mapping across 9 environments in Asia and South American. QTL NILs showed that each had a significant effect on chalk. Chalk in rice grains leads to a significant loss in the proportion of marketable grains in a harvested crop, leading to a significant financial loss to rice farmers and traders. To identify the genetic basis of chalkiness, two sets of recombinant inbred lines (RILs) derived from reciprocal crosses between Lemont and Teqing were used to find stable QTLs for chalkiness. The RILs were grown in seven locations in Asia and Latin American and in two controlled environments in phytotrons. A total of 32 (21) and 46 (22) QTLs for DEC and PGWC, most of them explaining more than 10% of phenotypic variation, were detected based on single environment analysis in T/L (L/T) population, respectively. Seven (2) and 7 (3) QTLs for DEC and PGWC were identified in the T/L (L/T) population using joined analysis across all environments, respectively. Six major QTLs clusters were found on five chromosomes: 1, 2, 4, 5 and 11. The biggest cluster at id4007289-RM252 on Chr04 was a novelty, including 16 and 4 QTLs detected by single environment analysis and joint mapping across all environments, respectively. The detected digenic epistatic QTLs explained up to 13% of phenotypic variation, suggesting that epistasis play an important role in the genetic control of chalkiness in rice. QTL NILs showed that each QTL cluster had a significant effect on chalk. These chromosomal regions could be targets for MAS, fine mapping and map-based cloning for low chalkiness breeding.

Identification and fine-mapping of a new resistance gene, Xa40, conferring resistance to bacterial blight races in rice (Oryza sativa L.).

KEY MESSAGE: A new bacterial blight resistance gene has been identified through fine-mapping, which confers high levels of resistance to all Korean Xanthomonas oryzae pv. oryzae (Xoo) races, including the new Xoo race K3a. Rice bacterial leaf blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) is a serious constraint to rice production in Asia and Africa. The japonica advanced backcross breeding lines derived from the indica line IR65482-7-216-1-2 in the background of cultivar Junam are resistant to all Korean BB races, including K3a. To identify the gene(s) involved in resistance to Korean Xoo races, the association of genotypic and phenotypic variations was examined in two F2 populations derived from the crosses between 11325 (IR83261-3-7-23-6-2-1-1-2-1-2)/Anmi and 11325/Ilpum. The segregation ratios of F2 individuals from the crosses of 11325/Anmi and 11325/Ilpum were 578 resistant:209 susceptible and 555 resistant:241 susceptible, respectively, which is consistent with the expected allelic frequency of a 3:1 ratio. Genetic analysis using graphical mapping indicated that resistance (R) was controlled by a new resistance gene linked with the flanking markers RM27320 and ID55.WA18-5 within an approximately 80-kb region between 28.14 and 28.22 Mbp on chromosome 11. The eight candidate genes functionally predicted were included in the target region. Examination of the candidate genes by RT-PCR analysis only corroborated with the significant difference in transcript levels of the WAK3 gene in the presence or absence of pathogen infection. Allelism tests performed with other known BB R-genes revealed that the allele was distinct from others having a similar chromosomal location.