Identification of Grain Size-Related QTLs in Korean japonica Rice Using Genome Resequencing and High-Throughput Image Analysis

Grain size is a key factor influencing the grain yield in rice. To identify the as-yet-unknown genes regulating grain size in Korean japonica rice, we developed a recombinant inbred line population (n = 162) from a cross between Odae (large-grain) and Joun (small-grain), and measured six traits including the thousand-grain weights of unhulled and hulled seeds, grain area, grain length, grain width and grain length-to-width ratio using high-throughput image analysis at the F8 and F9 generations. A genetic map was constructed using 248 kompetitive allele-specific PCR (KASP) markers that were polymorphic between the parental genotypes, and 29 QTLs affecting the six traits were identified, of which 15 were stable in both F8 and F9 generations. Notably, three QTL clusters affecting multiple traits were detected on chromosomes 6, 7 and 11. We analyzed whole-genome resequencing data of Odae and Joun, and selected candidate genes for the stable QTLs in the identified clusters that have high- or moderate-impact variations between Odae and Joun and encode proteins the families of which have been reported to be related to grain size regulation. These results will facilitate the identification of genes underlying the QTLs and promote molecular breeding of high-yielding Korean japonica rice varieties.

QTL Analysis Based on A Rice Short-Wide Grain CSSL-Z414 and Substitution Mapping of qGL11 and qGW5

Background: Most of rice agronomic traits as grain length etc. are complex traits controlled by multiple genes. Chromosome segment substitution lines (CSSLs) are ideal materials for dissecting and studying of these complex traits. Results: We developed a novel rice short-wide grain CSSL, Z414, deriving from progeny of the recipient parent Xihui 18 (an indica restorer line) and the donor parent Huhan 3 (a japonica cultivar). Z414 contained 4 substitution segments (average length was 3.04 Mb). Compared with Xihui 18, Z414 displayed seven significantly different traits as grain length, width and weight, chalkiness degree, brown rice rate etc. Then, 8 quantitative trait loci (QTLs) were found responding these difference traits by F2 population from Xihui 18/Z414. Among them, 6 QTLs (qPL3, qGW5, qGL11, qRLW5, qRLW11, qGWT5) could be verified by novel developed single segment substitution lines (SSSLs, S1-S6). In addition, 4 QTLs (qGL3, qGL5, qCD3 and qCD5) were novel detected by S1 and S5. Thus, the short–wide grain of Z414 was responded by qGL11, qGL3, qGL5, and qGW5. Then, qGL11 and qGW5 were delimited within intervals of 0.405 and 1.14 Mb on chromosomes 11 and 5, respectively, by substitution mapping. Again by sequencing, qRT-PCR and cell morphology analysis, qGW5 should be a novel allele of GS5 and qGL11 is novel QTL encoding CycT1;3, whose specific function of regulating grain length was still unknown. Finally, pyramid of qGL3 (a=0.22) and qGL11 (a=-0.19) displayed qGL11 epistatic to qGL3. In addition, novel S1 and D2 exhibited different grain sizes and lower chalkiness degree. They are potential to be directly used in breeding hybrid rice varieties.Conclusions: We constructed a novel rice short–wide grain CSSL-Z414 with 4 substitution segments based on the genetic backgrounds of Xihui 18. The broad grain of Z414 was controlled by qGW5, which should be a novel allele of GS5. The short grain of Z414 was controlled by qGL11, qGL3, and qGL5, and qGL11 is a novel QTL encoding CycT1;3, whose specific function of regulating grain length was still unknown, and qGL11 is epistatic to qGL3. Novel S1 and D2 are potential in hybrid rice varieties.

An Adenylate Kinase OsAK3 Involves Brassinosteroid Signaling and Grain Length in Rice (Oryza sativa L.)

Background Grain size is one of the major determinants of cereal crop yield. As a class of plant polyhydroxysteroids, brassinosteroids (BRs) play essential roles in the regulation of grain size and plant architecture in rice. In a previous research, we cloned qGL3/OsPPKL1 encoding a protein phosphatase with Kelch-like repeat domains, which negatively regulates BR signaling and grain length in rice. Results Here, we screened qGL3-interacting proteins (GIPs) via yeast two-hybrid assay and analyzed the phenotypes of the T-DNA insertion mutants of GIPs. Among these mutants, mutant osak3 presents shorter grain length and dwarfing phenotype. OsAK3 encodes an adenylate kinase, which regulates grain size by controlling cell expansion of rice spikelet glume. Overexpression of OsAK3 resulted in longer grain length. OsAK3 interacts with qGL3 in vivo and in vitro. Lamina inclination, coleoptile elongation and root inhibition experiments showed that the osak3 mutant was less sensitive to exogenous brassinolide (BL) treatment. The transcriptional level of OsAK3 was up-regulated under BL induction. In addition, RNA-Seq data indicate that OsAK3 is involved in a variety of biological processes that regulate BR signaling and grain development in rice. Conclusions Our study reveals a novel BR signaling component OsAK3 in the regulation of grain length, and provides novel clues for uncovering the potential functions of OsAK3 in rice growth and development.

Characterization of Functional Genes GS3 and GW2 and their Effect on the Grain Size of Various Landraces of Rice

Grain size is an essential factor in grain quality and yield. In the existing agricultural lands in Pakistan and even all over the world, genetics in rice works better for yield potential and quality improvement. GS3 and GW2 with functional mutation responsible for grain size in rice. In the current study, 17 different Pakistani landraces of various genetic and geographic backgrounds were evaluated for grain phenotypic traits (thousand-grain weight, length, width, and thickness) and characterized genotypes for GS3 gene (grain length) and GW2 (grain width). The two accessions JP5 and Bas370, were used as control. Phenotypic data revealed the range for grain weight from 16.86g (Lateefy) to 26.91g (PS2), grain length ranged from 7.27 mm (JP-5) to 12.18 mm (PS2), grain width ranged from 2.01 mm (Lateefy) to 3.51 mm (JP5), and grain thickness ranged from 1.79 mm to 2.19. Pearson correlation revealed a negative and significant correlation between grain width and length. There was no significant correlation between grain length and 1000-grain weight and grain width. LSD test displayed that the means of three variables grain length, grain width, and 1000-grain weight were statistically different from one another except grain width and grain breadth. GS3 is a negative regulator of grain length. Fifteen accessions GA-5015, PS-2, Swat-1, Swat-2, DR-2, Dilrosh, Malhar-346, Kashmir Basmati, Rachna Basmati, KS-282, Basmati-370, KSK-133, KSK-434, MG-Basmati, and Lateefy, carried the domesticated allele of GS3 while JP5 and Fakhr-e-Malakand carried the dominant allele. Similarly, the GW2 is a negative regulator of grain width. Fifteen accessions, i.e., Bas-370, GA-5015, PS-2, Swat-1, Swat-2, DR-2, Dilrosh, Malhar-346, Kashmir Basmati, Rachna Basmati, KS-282, KSK-133, KSK-434, MG-Basmati, and Lateefy carried the dominant allele while JP-5 and Fakhr-e-Malakand carried the mutant allele. The current phenotypic evaluation of the Germplasm revealed a diverse range of grain size of Pakistani landraces and also suggests that the selection of grain length in Pakistani landraces was independent of 1000-grain weight. The accessions with genotypic characterization will aid in marker-assisted breeding programs to break the stagnant yield prevail for the last few decades in Pakistan.

Identification of Genomic Regions Controlling Chalkiness and Grain Characteristics in a Recombinant Inbred Line Rice Population Based on High-Throughput SNP Markers

Rice (Oryza sativa L.) is the primary food for half of the global population. Recently, there has been increasing concern in the rice industry regarding the eating and milling quality of rice. This study was conducted to identify genetic information for grain characteristics using a recombinant inbred line (RIL) population from a japonica/indica cross based on high-throughput SNP markers and to provide a strategy for improving rice quality. The RIL population used was derived from a cross of “Kaybonnet (KBNT lpa)” and “ZHE733” named the K/Z RIL population, consisting of 198 lines. A total of 4133 SNP markers were used to identify quantitative trait loci (QTLs) with higher resolution and to identify more accurate candidate genes. The characteristics measured included grain length (GL), grain width (GW), grain length to width ratio (RGLW), hundred grain weight (HGW), and percent chalkiness (PC). QTL analysis was performed using QTL IciMapping software. Continuous distributions and transgressive segregations of all the traits were observed, suggesting that the traits were quantitatively inherited. A total of twenty-eight QTLs and ninety-two candidate genes related to rice grain characteristics were identified. This genetic information is important to develop rice varieties of high quality.

GWAS identifies an ortholog of the rice D11 gene as a candidate gene for grain size in an international collection of hexaploid wheat

Grain size is a key agronomic trait that contributes to grain yield in hexaploid wheat. Grain length and width were evaluated in an international collection of 157 wheat accessions. These accessions were genetically characterized using a genotyping-by-sequencing (GBS) protocol that produced 73,784 single nucleotide polymorphism (SNP) markers. GBS-derived genotype calls obtained on Chinese Spring proved extremely accurate when compared to the reference (> 99.9%) and showed > 95% agreement with calls made at SNP loci shared with the 90 K SNP array on a subset of 71 Canadian wheat accessions for which both types of data were available. This indicates that GBS can yield a large amount of highly accurate SNP data in hexaploid wheat. The genetic diversity analysis performed using this set of SNP markers revealed the presence of six distinct groups within this collection. A GWAS was conducted to uncover genomic regions controlling variation for grain length and width. In total, seven SNPs were found to be associated with one or both traits, identifying three quantitative trait loci (QTLs) located on chromosomes 1D, 2D and 4A. In the vicinity of the peak SNP on chromosome 2D, we found a promising candidate gene (TraesCS2D01G331100), whose rice ortholog (D11) had previously been reported to be involved in the regulation of grain size. These markers will be useful in breeding for enhanced wheat productivity.

Identification and Pyramid of QTLs Based on Rice Short-wide Grain CSSL-Z414, SSSL, DSSL and Candidate Gene Analysis of qGL11 and qGW5

BackgroundMost of rice agronomic traits as grain length etc. are complex traits controlled by multiple genes. Chromosome segment substitution lines (CSSLs) are ideal materials for dissecting and studying of these complex traits. ResultsA rice short-wide grain CSSL Z414 was identified among progeny of the recipient parent Xihui 18 (an indica restorer line) and the donor parent Huhan 3 (a japonica cultivar). Z414 carried 4 substitution segments (average length was 3.04 Mb), and displayed shorter panicle length and less number of primary branches, shorter, wider and larger grain, higher brown rice rate and chalkiness degree when compared with Xihui 18. Then, 9 quantitative trait loci (QTLs) for associated traits were identified using the secondary F2 population from Xihui 18 / Z414. Among them, 6 QTLs (qPL3, qGW5, qGL11, qRLW5, qRLW11, qGWT5) could be verified by corresponding single segment substitution lines (SSSLs, S1-S6). In addition, 4 QTLs (qGL3, qGL5, qCD3 and qCD5) were detected by S1 and S5, which was not detected by the F2 population. Thus, the grain length of Z414 was controlled by qGL11, qGL3 and qGL5, and the grain width of Z414 was answered by qGW5. Then by substitution mapping, qGL11 and qGW5 were delimited within the estimated substitution length of 1.42 and 1.14 Mb on chromosomes 11 and 5, and 4 and 2 candidate genes were found respectively for qGL11 and qGW5 by sequencing. However, only two had expression differences by qRT-PCR analysis. Finally, Analysis of QTL epistatic effects revealed that pyramid of qGL3 (a= 0.22) and qGL11 (a=-0.19) caused grain length of double segment substitution line (DSSL, D2) shorter than that of S5 (qGL11).ConclusionsWe developed a rice short –wide grain CSSL with 4 substitution segments from Huhan 3 based on the genetic backgrounds of Xihui 18. The grain width of Z414 was controlled by qGW5, and GS5 should be the candidate gene for qGW5 by sequencing and qRT-PCR analysis. The grain length of Z414 was controlled by qGL11, qGL3, and qGL5, and CycT1;3 should be the best candidate gene of qGL11, whose specific function of regulating grain length was still unknown, and qGL11 is epistatic to qGL3.

Genetic editing of GS3 via CRISPR/Cas9 Accelerates the Breeding of Three-line Hybrid Rice With Superior Yield and Grain Quality

Grain size is one of the major traits that control rice grain yield and quality. The GS3 gene, a major QTL regulating grain length and weight, is the first one been identified. A mutation occurred in its N-terminal organ size regulating domain (OSR)results in a loss of function of the mutant allele gs3 and rice varieties carry this gs3 allele often produce longer grains. In this study, we exploited the CRISPR/Cas9 gene editing technology to introduce an edited gs3 allele, two guide RNAs(gRNA) targeted at the OSR of the GS3 gene were transformed into one of our indica maintainer lines, GM1B, for its grain yield and quality improvement. Through molecular analysis and sequencing, in T1 generation, a homologous edited-gs3 mutant line without tansgene was obtained and name as GM2B, then converted to CMS line GM1A by backcrossing to obtain another superior male sterile line GM2A for further tests. GM2B showed improved grain quality and yield compared to the WT GM1B, with grain length increased by 7.9%, length/width ratio increased from 3.89 to 4.19, TGW increased by 6.7%, and grain yield per plant increased by 14.9%. Meanwhile, genetic improvement of other quality traits including rice length (6.83mm), rice grain length/width ratio (3.61), matched the appearance standards set for traditional Simiao (silk seedling) type cultivars. Two restorer lines were outcrossed to both GM1A and GM2A to produce hybrid rice. Compared to GM1A’s two hybrids, the hybrids of GM2A had longer grains, higher length/width ratio, higher TGW and yield per plant. In addition, hybrids of GM2A showed better performance on grain appearance including better translucency, lower chalky rice rate and chalkiness degree than hybrids of GM1A.These data strongly demonstrate that the introduction of an elite gs3 allele into GM1A via CRISPR/Cas9 gene editing technology leads to significant genetic improvement. The resultant CMS line GM2A(gs3) performs much better than the original GM1A on grain quality and yield. Thus, our study proves that targeted genetic improvement using gene editing technology can expedite rice breeding, especially the breeding of three-line hybrid rice.

Evaluation of advanced bread wheat lines for yield components during season 2019-2020

ABSTRACT A study was conducted with seven advanced bread wheat lines to determine grain yield, a thousand grain weight, grain length, grain weight per spike, number of grains per spike, spike length and spike weight, at the Norman E. Borlaug Experimental Station in the Yaqui Valley, Sonora, Mexico, during the 2019-2020 crop season. Plots were 100 m long on three beds, 0.80 m apart, with two rows, and three replications; seed density was 100 kg/ha. Sowing date was December 14, 2019. Experimental lines were generated by the Global Wheat Program from the International Maize and Wheat Improvement Center, and they are candidates for commercial release by the National Institute for Forestry, Agriculture, and Livestock Research in Mexico. Genotypes BORL14*2/5/ATTILA/3*BCN*2//BAV92/3/ KIRITATI/WBLL1/4/DANPHE, BORL14*2//MUNAL#1/FRANCOLIN#1, and PREMIO /4/CROC_1/AE.SQUARROSA(205)//KAUZ/3/PIFED/5/BORL14, showed the highest grain yield with 7.1, 7, and 6.9 t/ha, respectively, which can compete with current bread wheat cultivars in southern Sonora, Mexico, like Borlaug 100. The third line also showed the highest a thousand grain weight, grain length, grain weight per spike, and spike weight. RESUMEN Se realizó un estudio con siete líneas avanzadas de trigo pan para determinar el rendimiento de grano, peso de mil granos, longitud de grano, peso de grano por espiga, número de granos por espiga, longitud de espiga y peso de espiga, en la Estación Experimental Norman E. Borlaug en el Valle del Yaqui, Sonora, México, durante la temporada de cultivo 2019-2020. Las parcelas tuvieron una longitud de 100 m en tres camas, separadas 0.80 m, con dos hileras y tres repeticiones; la densidad de semilla fue de 100 kg/ha. La fecha de siembra fue el 14 de diciembre de 2019. Las líneas experimentales fueron generadas por el Programa Global de Trigo del Centro Internacional de Mejoramiento de Maíz y Trigo, y son candidatas a ser liberadas comercialmente por el Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias de México. Los genotipos BORL14*2/5/ATTILA/3*BCN*2//BAV92/3/ KIRITATI/WBLL1/4/DANPHE, BORL14*2//MUNAL#1/FRANCOLIN#1, y PREMIO /4/CROC_1/AE.SQUARROSA(205)//KAUZ/3/PIFED/5/BORL14, mostraron el mayor rendimiento de grano con 7. 1, 7 y 6.9 t/ha, respectivamente, que pueden competir con los cultivares actuales de trigo pan en el sur de Sonora, México, como Borlaug 100. La tercera línea también mostró el mayor peso de mil granos, longitud de grano, peso de grano por espiga y peso de espiga.