Identification of reliable QTLs and designed QTL breeding for grain shape and milling quality in the reciprocal introgression lines in rice.

Milling quality (MQ) and grain shape (GS) of rice (Oryza sativa L.) are correlated traits, both determine farmers' final profit. More than one population under multiple environments may provide valuable information for breeding selection on these MQ-GS correlations. However, suitable analytical methods for reciprocal introgression lines with linkage map for this kind of correlation remains unclear. In this study, our major tasks were (1) to provide a set of reciprocal introgression lines (composed of two BC2RIL populations) suitable for mapping by linkage mapping using markers/bins with physical positions; (2) to test the mapping effects of different methods by using MQ-GS correlation dissection as sample case; (3) to perform genetic and breeding simulation on pyramiding favorite alleles of QTLs for representative MQ-GS traits. Finally, with four analysis methods and data collected under five environments, we identified about 28.4 loci on average for MQ-GS traits. Notably, 52.3% of these loci were commonly detected by different methods and eight loci were novel. There were also nine regions harboring loci for different MQ-GS traits which may be underlying the MQ-GS correlations. Background independent (BI) loci were also found for each MQ and GS trait. All these information may provide useful resources for rice molecular breeding.

Genetic Dissection of Panicle Morphology Traits in Super High-Yield Hybrid Rice Chaoyou 1000.

The morphological characteristics of the rice panicle play a pivotal role in influencing yield. In our research, we employed F2 and F2:3 populations derived from the high-yielding hybrid rice variety Chaoyou 1000. We screened 123 pairs of molecular markers, which were available, to construct the genetic linkage map. Subsequently, we assessed the panicle morphology traits of F2 populations in Lingshui County, Hainan Province, in 2017, and F2:3 populations in Hangzhou City, Zhejiang Province, in 2018. These two locations represent two types of ecology. Hangzhou's climate is characterized by high temperatures and humidity, while Lingshui's climate is characterized by a tropical monsoon climate. In total, 33 QTLs were identified, with eight of these being newly discovered, and two of them were consistently detected in two distinct environments. We identified fourteen QTL-by-environment interactions (QEs), which collectively explained 4.93% to 59.95% of the phenotypic variation. While most of the detected QTLs are consistent with the results of previous tests, the novel-detected QTLs will lay the foundation for rice yield increase and molecular breeding.

Deciphering the Genetic Architecture of Color Variation in Whole Grain Rice by Genome-Wide Association.

Whole grain rice is recommended in a natural healthy diet because of its high nutritional and healthful benefits compared to polished or white rice. The whole grain contains the pericarp with many assorted colors (such as brown, red, and black) associated with taste and commercial quality. The color attributes of whole grain or brown rice are usually undesirable and need to be improved. To decipher the genetic basis of color variation in the whole grain rice, we conducted a genome-wide association analysis of three parameters of grain colors (brightness, redness, and yellowness) in a panel of 682 rice accessions. Twenty-six loci were identified for the color parameters, implying that grain color is under polygenic control. Among them, some major-effect loci were co-localized with the previously identified genes such as Rc and Rd. To eliminate the possible mask of Rc on other loci influencing grain color, we performed the association analysis in a subset of the panel that excluded the pigmented (red and black) rice. Eighteen loci or SNPs were detected to be associated with grain color in the subpopulation, many of which were not reported before. Two significant peak SNP regions on chromosomes 1 and 9 were validated using near-isogenic lines. Based on differential expression analysis of annotated genes within the SNP regions and metabolic analysis of pooled extreme samples, we found at least three annotated genes as potential candidates involved in the flavonoid metabolic pathway related to pericarp color. These results provide insights into the genetic basis of rice grain color and facilitate genomic breeding to improve appearance and commercial quality of whole grain rice.

A prolific and robust whole-genome genotyping method using PCR amplification via primer-template mismatched annealing.

Whole-genome genotyping methods are important for breeding. However, it has been challenging to develop a robust method for simultaneous foreground and background genotyping that can easily be adapted to different genes and species. In our study, we accidently discovered that in adapter ligation-mediated PCR, the amplification by primer-template mismatched annealing (PTMA) along the genome could generate thousands of stable PCR products. Based on this observation, we consequently developed a novel method for simultaneous foreground and background integrated genotyping by sequencing (FBI-seq) using one specific primer, in which foreground genotyping is performed by primer-template perfect annealing (PTPA), while background genotyping employs PTMA. Unlike DNA arrays, multiple PCR, or genome target enrichments, FBI-seq requires little preliminary work for primer design and synthesis, and it is easily adaptable to different foreground genes and species. FBI-seq therefore provides a prolific, robust, and accurate method for simultaneous foreground and background genotyping to facilitate breeding in the post-genomics era.

Distribution characteristics of selenium, cadmium and arsenic in rice grains and their genetic dissection by genome-wide association study.

High selenium (Se) and low cadmium (Cd) and arsenic (As) contents in rice grains were good for human health. The genetic basis and relationship of Se, Cd and As concentrations in rice grains are still largely unknown. In the present study, large variations were observed in Se, Cd and As concentrations in brown and milled rice in normal and Se treatment conditions in 307 rice accessions from 3K Rice Genomes Project. Se fertilizer treatment greatly increased Se concentrations but had no obvious changes in concentrations of Cd and As both in brown and milled rice. Total of 237 QTL were identified for Se, Cd and As concentrations in brown and milled rice in normal and Se treatment conditions as well as ratio of concentrations under Se treatment to normal conditions. Only 19 QTL (13.4%) were mapped for concentrations of Se and Cd, Se and As, and Se, Cd and As in the same or adjacent regions, indicating that most Se concentration QTL are independent of Cd and As concentration QTL. Forty-three favorable alleles were identified for 40 candidate genes by gene-based association study and haplotype analysis in 14 important QTL regions. Se-enriched rice variety will be developed by pyramiding favorable alleles at different Se QTL and excluding undesirable alleles at Cd and As QTL, or combining favorable alleles at Se QTL with the alleles at Se-sensitive QTL by marker-assisted selection.

Verifying the Breeding Value of A Rare Haplotype of Chalk7, GS3, and Chalk5 to Improve Grain Appearance Quality in Rice.

Grain quality is a key determinant of commercial value in rice. Efficiently improving grain quality, without compromising grain yield, is a challenge in rice breeding programs. Here we report on the identification and application of a grain quality gene, Chalk7, which causes a slender shape and decreases grain chalkiness in rice. Three allele-specific markers for Chalk7, and two other grain genes (GS3 and Chalk5) were developed, and used to stack the desirable alleles at these loci. The effects of individual or combined alleles at the loci were evaluated using a set of near-isogenic lines, each containing one to three favorable alleles in a common background of an elite variety. We found that the favorable allele combination of the three loci, which rarely occurs in natural rice germplasm, greatly reduces chalky grains without negatively impacting on grain yield. The data for newly developed allele-specific markers and pre-breeding lines will facilitate the improvement of grain appearance quality in rice.

Genetic Dissection of Rice Ratooning Ability Using an Introgression Line Population and Substitution Mapping of a Pleiotropic Quantitative Trait Locus qRA5.

Ratooning ability is a key factor that influences ratoon rice yield, in the area where light and temperature are not enough for second season rice. In the present study, an introgression line population derived from Minghui 63 as the recipient parent and 02428 as the donor parent was developed, and a high-density bin map containing 4568 bins was constructed. Nine ratooning-ability-related traits were measured, including maximum tiller number, panicle number, and grain yield per plant in the first season and ratoon season, as well as three secondary traits, maximum tiller number ratio, panicle number ratio, and grain yield ratio. A total of 22 main-effect QTLs were identified and explained for 3.26-18.63% of the phenotypic variations in the introgression line population. Three genomic regions, including 14.12-14.65 Mb on chromosome 5, 4.64-5.76 Mb on chromosome 8, and 10.64-15.52 Mb on chromosome 11, were identified to simultaneously control different ratooning-ability-related traits. Among them, qRA5 in the region of 14.12-14.65 Mb on chromosome 5 was validated for its pleiotropic effects on maximum tiller number and panicle number in the first season, as well as its maximum tiller number ratio, panicle number ratio, and grain yield ratio. Moreover, qRA5 was independent of genetic background and delimited into a 311.16 kb region by a substitution mapping approach. These results will help us better understand the genetic basis of rice ratooning ability and provide a valuable gene resource for breeding high-yield ratoon rice varieties.

Introgression Lines: Valuable Resources for Functional Genomics Research and Breeding in Rice (Oryza sativa L.).

The narrow base of genetic diversity of modern rice varieties is mainly attributed to the overuse of the common backbone parents that leads to the lack of varied favorable alleles in the process of breeding new varieties. Introgression lines (ILs) developed by a backcross strategy combined with marker-assisted selection (MAS) are powerful prebreeding tools for broadening the genetic base of existing cultivars. They have high power for mapping quantitative trait loci (QTLs) either with major or minor effects, and are used for precisely evaluating the genetic effects of QTLs and detecting the gene-by-gene or gene-by-environment interactions due to their low genetic background noise. ILs developed from multiple donors in a fixed background can be used as an IL platform to identify the best alleles or allele combinations for breeding by design. In the present paper, we reviewed the recent achievements from ILs in rice functional genomics research and breeding, including the genetic dissection of complex traits, identification of elite alleles and background-independent and epistatic QTLs, analysis of genetic interaction, and genetic improvement of single and multiple target traits. We also discussed how to develop ILs for further identification of new elite alleles, and how to utilize IL platforms for rice genetic improvement.

Integrating GWAS and transcriptomics to identify candidate genes conferring heat tolerance in rice.

Introduction: Rice (Oryza sativa L.) production is being challenged by global warming. Identifying new loci and favorable alleles associated with heat tolerance is crucial to developing rice heat-tolerant varieties. Methods: We evaluated the heat tolerance at the seedling stage using 620 diverse rice accessions. A total of six loci associated with heat tolerance were identified by a genome-wide association study (GWAS) with ~2.8 million single nucleotide polymorphisms (SNPs). Results: Among the six detected loci, qHT7 harbored the strongest association signal and the most associated SNPs. By comparing the transcriptomes of two representative accessions with contrasting heat tolerance, LOC_Os07g48710 (OsVQ30) was selected as a promising candidate gene in qHT7 due to the significant difference in its expression level between the two accessions. Haplotype 4 (Hap4) of LOC_Os07g48710 was determined as the favorable haplotype for heat tolerance via the gene-based haplotype analysis. The heat-tolerant haplotype LOC_Os07g48710Hap4 is highly enriched in the tropical Geng/Japonica accessions, and its frequency has decreased significantly during the improvement process of rice varieties. Discussion: Based on the GWAS and transcriptomics integrated results, a hypothetical model modulated by qHT7 in response to heat stress was proposed. Our results provide valuable candidate genes for improving rice heat tolerance through molecular breeding.

Identification of Heterotic Loci with Desirable Allelic Interaction to Increase Yield in Rice.

Heterosis denotes the superiority of a hybrid plant over its parents. The use of heterosis has contributed significantly to yield improvement in crops. However, the genetic and molecular bases on heterosis are not fully understood. A large number of heterotic loci were identified for 12 yield-related traits in one parental population of chromosome segment substitution lines (CSSLs) and two test populations, which were interconnected by CSSLs derived from two rice genome-sequenced cultivars, Nipponbare and Zhenshan 97. Seventy-five heterotic loci were identified in both homozygous background of Zhenshan 97 and heterogeneous background of an elite hybrid cultivar Shanyou 63. Among the detected loci, at least 11 were colocalized in the same regions encompassing previously reported heterosis-associated genes. Furthermore, a heterotic locus Ghd8NIP for yield advantage was verified using transgenic experiments. Various allelic interaction at Ghd8 exhibited different heterosis levels in hetero-allelic combinations of five near-isogenic lines that contain a particular allele. The significant overdominance effects from some hetero-allelic combinations were found to improve yield heterosis in hybrid cultivars. Our findings support the role of allelic interaction at heterotic loci in the improvement of yield potential, which will be helpful for dissecting the genetic basis of heterosis and provide an optional strategy for the allele replacement in molecular breeding programs in hybrid rice.

Identification of genes for salt tolerance and yield-related traits in rice plants grown hydroponically and under saline field conditions by genome-wide association study.

BACKGROUND: Soil salinity is one of the main environmental conditions that affects rice production. Identifying the genetic loci that affect rice salt tolerance (ST)-related traits at the seedling stage, especially under saline field conditions, is crucial for ST rice breeding by pyramiding ST genes that act at different developmental stages. RESULTS: Large phenotypic variations were observed in 708 rice accessions, and yield and its related traits were considerably limited when exposed to salt stress. In a genome-wide association study (GWAS), 2255 marker-trait association signals were detected for all measured traits, and the significant SNPs were distributed in 903 genes. Of these, 43 genes processed same functional annotation, and the gene ontology terms "biological processes" and "molecular function" with the known genes responsive to salt stress in rice. Further haplotype analysis detected 15 promising candidates significantly associated with the target traits, including five known genes and 10 novel genes. We identified seven accessions carrying favorable haplotypes of four genes significantly associated with grain yield that performed well under saline stress conditions. CONCLUSIONS: Using high density SNPs within genes to conduct GWAS is an effective way to identify candidate genes for salt tolerance in rice. Five known genes (OsMYB6, OsGAMYB, OsHKT1;4, OsCTR3, and OsSUT1) and two newly identified genes (LOC_Os02g49700, LOC_Os03g28300) significantly associated with grain yield and its related traits under saline stress conditions were identified. These promising candidates provide valuable resources for validating potential ST-related genes and will facilitate rice breeding for salt tolerance through marker-assisted selection.

Examining two sets of introgression lines reveals background-independent and stably expressed QTL that improve grain appearance quality in rice (Oryza sativa L.).

KEY MESSAGE: A novel QTL cluster for appearance quality on Chr07 was identified using reciprocal introgression populations in different locations in China. Two secondary F 2 populations validated QTL with significant effect on appearance quality. Appearance quality (AQ) is the main determinants of market value of rice. Identification of QTL affecting AQ is the prerequisite for efficient improvement of AQ through marker-assisted selection (MAS). Two sets of reciprocal introgression lines derived from indica Minghui 63 and japonica 02428 were used to dissect the stability of QTL affecting five AQ traits, including grain length, grain width, length to width ratio, percentage of grains with chalkiness, and degree of endosperm chalkiness using 4568 bin genotype produced from 58,000 SNPs across five different environments. A total of 41 and 30 main-effect QTL were identified in MH63 and 02428 backgrounds, respectively. Among them, 9 background-independent QTL (BI-QTL) were found. There were also 13 and 10 stable-expressed QTL (SE-QTL) across at least two environments in MH63 and 02428 backgrounds, respectively. Two important BI- and SE-QTL regions (BISERs) including BISER-I harboring qPGWC5, qDEC5, qGW5.1, and qLWR5 on chromosome 5 and BISER-II harboring qGL7, qLWR7, qPGWC7, and qDEC7 on chromosome 7 were identified. The BISER-II was newly reported and validated by two secondary F2 populations in the reciprocal backgrounds. Among 59 epistatic QTL (E-QTL) detected in this study, there were only four SE- but no BI-E-QTL detected in different environments, indicating that genetic background has stronger effect on AQ traits than the environmental factors, especially for percentage of grains with chalkiness (PGWC) and degree of endosperm chalkiness (DEC) with lower heritability. BISER-I and BISER-II harboring many BI- and SE-QTL with favorable alleles from slender grain rice are much important for improvement of rice AQ by MAS.

Genome-Wide Association Study of Grain Appearance and Milling Quality in a Worldwide Collection of Indica Rice Germplasm.

Grain appearance quality and milling quality are the main determinants of market value of rice. Breeding for improved grain quality is a major objective of rice breeding worldwide. Identification of genes/QTL controlling quality traits is the prerequisite for increasing breeding efficiency through marker-assisted selection. Here, we reported a genome-wide association study in indica rice to identify QTL associated with 10 appearance and milling quality related traits, including grain length, grain width, grain length to width ratio, grain thickness, thousand grain weight, degree of endosperm chalkiness, percentage of grains with chalkiness, brown rice rate, milled rice rate and head milled rice rate. A diversity panel consisting of 272 indica accessions collected worldwide was evaluated in four locations including Hangzhou, Jingzhou, Sanya and Shenzhen representing indica rice production environments in China and genotyped using genotyping-by-sequencing and Diversity Arrays Technology based on next-generation sequencing technique called DArTseq™. A wide range of variation was observed for all traits in all environments. A total of 16 different association analysis models were compared to determine the best model for each trait-environment combination. Association mapping based on 18,824 high quality markers yielded 38 QTL for the 10 traits. Five of the detected QTL corresponded to known genes or fine mapped QTL. Among the 33 novel QTL identified, qDEC1.1 (qGLWR1.1), qBRR2.2 (qGL2.1), qTGW2.1 (qGL2.2), qGW11.1 (qMRR11.1) and qGL7.1 affected multiple traits with relatively large effects and/or were detected in multiple environments. The research provided an insight of the genetic architecture of rice grain quality and important information for mining genes/QTL with large effects within indica accessions for rice breeding.

Identification of genomic regions and the isoamylase gene for reduced grain chalkiness in rice.

Grain chalkiness is an important grain quality related to starch granules in the endosperm. A high percentage of grain chalkiness is a major problem because it diminishes grain quality in rice. Here, we report quantitative trait loci identification for grain chalkiness using high-throughput single nucleotide polymorphism genotyping of a chromosomal segment substitution line population in which each line carried one or a few introduced japonica cultivar Nipponbare segments in the genetic background of the indica cultivar ZS97. Ten quantitative trait loci regions were commonly identified for the percentage of grain chalkiness and the degree of endosperm chalkiness. The allelic effects at nine of these quantitative trait loci reduced grain chalkiness. Furthermore, a quantitative trait locus (qPGC8-2) on chromosome 8 was validated in a chromosomal segment substitution line-derived segregation population, and had a stable effect on chalkiness in a multiple-environment evaluation of the near-isogenic lines. Residing on the qPGC8-2 region, the isoamylase gene (ISA1) was preferentially expressed in the endosperm and revealed some nucleotide polymorphisms between two varieties, Nipponbare and ZS97. Transgenic lines with suppression of ISA1 by RNA interference produced grains with 20% more chalkiness than the control. The results support that the gene may underlie qPGC8-2 for grain chalkiness. The multiple-environment trials of the near-isogenic lines also show that combination of the favorable alleles such as the ISA1 gene for low chalkiness and the GS3 gene for long grains considerably improved grain quality of ZS97, which proves useful for grain quality improvement in rice breeding programs.

Natural variation and artificial selection in four genes determine grain shape in rice.

The size of cultivated rice (Oryza sativa) grains has been altered by both domestication and artificial selection over the course of evolutionary history. Several quantitative trait loci (QTLs) for grain size have been cloned in the past 10 yr. To explore the natural variation in these QTLs, resequencing of grain width and weight 2 (GW2), grain size 5 (GS5) and QTL for seed width 5 (qSW5) and genotyping of grain size 3 (GS3) were performed in the germplasms of 127 varieties of rice (O. sativa) and 10-15 samples of wild rice (Oryza rufipogon). Ten, 10 and 15 haplotypes were observed for GW2, GS5 and qSW5. qSW5 and GS3 had the strongest effects on grain size, which have been widely utilized in rice production, whereas GW2 and GS5 showed more modest effects. GS5 showed small sequence variations in O. sativa germplasm and that of its progenitor O. rufipogon. qSW5 exhibited the highest level of nucleotide diversity. GW2 showed signs of purifying selection. The four grain size genes experienced different selection intensities depending on their genetic effects. In the indica population, linkage disequilibrium (LD) was detected among GS3, qSW5 and GS5. The substantial genetic variation in these four genes provides the flexibility needed to design various rice grain shapes. These findings provide insight into the evolutionary features of grain size genes in rice.

Mapping and characterization of the major quantitative trait locus qSS7 associated with increased length and decreased width of rice seeds.

Seed shape in rice (Oryza sativa) is an important factor that determines grain appearance, cooking quality and grain yield. Here, we report a major quantitative trait locus qSS7 on the long arm of chromosome 7 for seed length, seed width and the ratio of seed length to width, identified using a segregating population derived from a cross between an indica variety Zhenshan97 and a chromosomal segment substitution line of a japonica variety Cypress within the genetic background of Zhenshan97. The Cypress allele at qSS7 contributes to an increase in seed length and the ratio of length to width, but a decrease in seed width, without significantly changing seed weight, plant height, heading date or number of spikelets per panicle. Using a large F(2) population generated from a substitution line that carries only a heterozygous single segment surrounding qSS7, we delimited the QTL to a 23-kb region containing two annotated genes. Progeny testing of the informative recombinants suggested that this qSS7 region is a composite QTL in which at least two genes contribute to seed length and width. Sequence comparison and expression analysis of two probable candidate genes revealed differences between the parental lines. These results will facilitate cloning of the gene(s) underlying qSS7 as well as marker-assisted transfer of desirable genes for seed shape in rice improvement.