De-lin Hong

Time-Course Association Mapping of the Grain-Filling Rate in Rice (Oryza sativa L.)

Detecting quantity trait locus (QTLs) and elite alleles that are associated with grain-filling rate (GFR) in rice is essential for promoting the utilization of hybrid japonica rice and improving rice yield. Ninety-five varieties including 58 landraces and 37 elite varieties from the core germplasm collection were genotyped with 263 simple sequence repeat (SSR) markers. The GFR of the 95 varieties was evaluated at five stages, 7, 14, 21, 28 and 35 days after flowering (DAF) both in 2011 and 2012. We found abundant phenotypic and genetic diversity in the studied population. A population structure analysis identified seven subpopulations. A linkage disequilibrium (LD) analysis indicated that the levels of LD ranged from 60.3 cM to 84.8 cM and artificial selection had enhanced the LD. A time-course association analysis detected 31 marker-GFR associations involving 24 SSR markers located on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, 11 and 12 of rice at five stages. The elite alleles for high GFR at each stage were detected. Fifteen excellent parental combinations were predicted, and the best parental combination ‘Nannongjing62401×Laolaihong’ could theoretically increase 4.086 mg grain-1 d-1 at the five stages. Our results demonstrate that the time-course association mapping for GFR in rice could detect elite alleles at different filling stages and that these elite alleles could be used to improve the GFR via pyramiding breeding.

Genetic Diversity and Elite Allele Mining for Grain Traits in Rice (Oryza sativa L.) by Association Mapping

Mining elite alleles for grain size and weight is of importance for the improvement of cultivated rice and selection for market demand. In this study, association mapping for grain traits was performed on a selected sample of 628 rice cultivars using 262 SSRs. Grain traits were evaluated by grain length (GL), grain width (GW), grain thickness (GT), grain length to width ratio (GL/GW), and 1000-grain weight (TGW) in 2013 and 2014. Our result showed abundant phenotypic and genetic diversities found in the studied population. In total, 2953 alleles were detected with an average of 11.3 alleles per locus. The population was divided into seven subpopulations and the levels of linkage disequilibrium (LD) ranged from 34 to 84 cM. Genome-wide association mapping detected 10 marker trait association (MTAs) loci for GL, 1MTAs locus for GW, 7 MTAs loci for GT, 3 MTAs loci for GL/GW, and 1 MTAs locus for TGW. Twenty-nine, 2, 10, 5, and 3 elite alleles were found for the GL, GW, GT, GL/GW, and TGW, respectively. Optimal cross designs were predicted for improving the target traits. The accessions containing elite alleles for grain traits mined in this study could be used for breeding rice cultivars and cloning the candidate genes.

Identification of a Candidate Gene for Panicle Length in Rice (Oryza sativa L.) Via Association and Linkage Analysis

Panicle length (PL) is an important trait for improving panicle architecture and grain yield in rice (Oryza sativa L.). Three populations were used to identify QTLs and candidate genes associated with PL. Four QTLs for PL were detected on chromosomes 4, 6, and 9 through linkage mapping in the recombinant inbred line population derived from a cross between the cultivars Xiushui79 (short panicle) and C-bao (long panicle). Ten SSR markers associated with PL were detected on chromosomes 2, 3, 5, 6, 8, 9, and 10 in the natural population consisting of 540 accessions collected from East and Southeast Asia. A major locus on chromosome 9 with the largest effect was identified via both linkage and association mapping. LONG PANICLE 1 (LP1) locus was delimited to a 90-kb region of the long arm of chromosome 9 through fine mapping using a single segment segregating F2 population. Two single nucleotide polymorphisms (SNPs) leading to amino acid changes were detected in the third and fifth exons of LP1. LP1 encodes a Remorin_C-containing protein of unknown function with homologs in a variety of species. Sequencing analysis of LP1 in two parents and 103 rice accessions indicated that SNP1 is associated with panicle length. The LP1 allele of Xiushui79 leads to reduced panicle length, whereas the allele of C-bao relieves the suppression of panicle length. LP1 and the elite alleles can be used to improve panicle length in rice.

QTL Detection and Elite Alleles Mining for Stigma Traits in Oryza sativa by Association Mapping.

Stigma traits are very important for hybrid seed production in Oryza sativa, which is a self-pollinated crop; however, the genetic mechanism controlling the traits is poorly understood. In this study, we investigated the phenotypic data of 227 accessions across 2 years and assessed their genotypic variation with 249 simple sequence repeat (SSR) markers. By combining phenotypic and genotypic data, a genome-wide association (GWA) map was generated. Large phenotypic variations in stigma length (STL), stigma brush-shaped part length (SBPL) and stigma non-brush-shaped part length (SNBPL) were found. Significant positive correlations were identified among stigma traits. In total, 2072 alleles were detected among 227 accessions, with an average of 8.3 alleles per SSR locus. GWA mapping detected 6 quantitative trait loci (QTLs) for the STL, 2 QTLs for the SBPL and 7 QTLs for the SNBPL. Eleven, 5, and 12 elite alleles were found for the STL, SBPL, and SNBPL, respectively. Optimal cross designs were predicted for improving the target traits. The detected genetic variation in stigma traits and QTLs provides helpful information for cloning candidate STL genes and breeding rice cultivars with longer STLs in the future.

QTL Analysis for Seven Quality Traits of RIL Population in Japonica Rice Based on Three Genetic Statistical Models

QTL mapping for seven quality traits was conducted by using 254 recombinant inbred lines (RIL) derived from a japonica-japonica rice cross of Xiushui 79/C Bao. The seven traits investigated were grain length (GL), grain length to width ratio (LWR), chalk grain rate (CGR), chalkiness degree (CD), gelatinization temperature (GT), amylose content (AC) and gel consistency (GC) of head rice. Three mapping methods employed were composite interval mapping in QTLMapper 2.0 software based on mixed linear model (MCIM), inclusive composite interval mapping in QTL IciMapping 3.0 software based on stepwise regression linear model (ICIM) and multiple interval mapping with regression forward selection in Windows QTL Cartographer 2.5 based on multiple regression analysis (MIMR). Results showed that five QTLs with additive effect (A-QTLs) were detected by all the three methods simultaneously, two by two methods simultaneously, and 23 by only one method. Five A-QTLs were detected by MCIM, nine by ICIM and 28 by MIMR. The contribution rates of single A-QTL ranged from 0.89% to 38.07%. All the QTLs with epistatic effect (E-QTLs) detected by MIMR were not detected by the other two methods. Fourteen pairs of E-QTLs were detected by both MCIM and ICIM, and 142 pairs of E-QTLs were detected by only one method. Twenty-five pairs of E-QTLs were detected by MCIM, 141 pairs by ICIM and four pairs by MIMR. The contribution rates of single pair of E-QTL were from 2.60% to 23.78%. In the Xiu-Bao RIL population, epistatic effect played a major role in the variation of GL and CD, and additive effect was the dominant in the variation of LWR, while both epistatic effect and additive effect had equal importance in the variation of CGR, AC, GT and GC. QTLs detected by two or more methods simultaneously were highly reliable, and could be applied to improve the quality traits in japonica hybrid rice.

Construction of SSR Linkage Map and Analysis of QTLs for Rolled Leaf in Japonica Rice

Abstract Genetic analysis of rolled leaf is important to rice ideotype breeding. To detect loci controlling rolled leaf of japonica restorer lines, SSR marker genotypes and phenotypes of flag leaf rolling index (LRI) were investigated in Xiushui 79 (P 1 , a japonica rice variety), C Bao (P 2 , a japonica restorer line) and 254 recombinant inbred lines derived from the cross between P 1 and P 2, and in two environments. A genetic map of this cross was constructed, QTLs for LRI were detected and their interactions with environments were analyzed. Among 818 pairs of SSR primers, 90 primers showed polymorphism between P 1 and P 2 , and 12 markers showed highly significant correlation with LRI in both environments based on single marker regression analysis. The genetic map containing 74 information loci has a total distance of 744.6 cM, with an average of 10.1 cM between two adjacent loci. Three QTLs ( qRL-1 , qRL-7 and qRL-8-1 ) were detected with two softwares: WinQTLCart 2.5 and QTLNetwork2.0. qRL-8-1 was a new locus, accounting for 15.5% and 12.8% of phenotypic variations in the two environments, respectively. The phenotypic variation explained by additive effect was 6.6%. No interaction was found between qRL-8-1 genotype and environments.

Fine Mapping and Candidate Gene Analysis of qSTL3, a Stigma Length-Conditioning Locus in Rice (Oryza sativa L.).

The efficiency of hybrid seed production can be improved by increasing the percentage of exserted stigma, which is closely related to the stigma length in rice. In the chromosome segment substitute line (CSSL) population derived from Nipponbare (recipient) and Kasalath (donor), a single CSSL (SSSL14) was found to show a longer stigma length than that of Nipponbare. The difference in stigma length between Nipponbare and SSSL14 was controlled by one locus (qSTL3). Using 7,917 individuals from the SSSL14/Nipponbare F2 population, the qSTL3 locus was delimited to a 19.8-kb region in the middle of the short arm of chromosome 3. Within the 19.8-kb chromosome region, three annotated genes (LOC_Os03g14850, LOC_Os03g14860 and LOC_Os03g14880) were found in the rice genome annotation database. According to gene sequence alignments in LOC_Os03g14850, a transition of G (Nipponbare) to A (Kasalath) was detected at the 474-bp site in CDS. The transition created a stop codon, leading to a deletion of 28 amino acids in the deduced peptide sequence in Kasalath. A T-DNA insertion mutant (05Z11CN28) of LOC_Os03g14850 showed a longer stigma length than that of wild type (Zhonghua 11), validating that LOC_Os03g14850 is the gene controlling stigma length. However, the Kasalath allele of LOC_Os03g14850 is unique because all of the alleles were the same as that of Nipponbare at the 474-bp site in the CDS of LOC_Os03g14850 among the investigated accessions with different stigma lengths. A gene-specific InDel marker LQ30 was developed for improving stigma length during rice hybrid breeding by marker-assisted selection.

Distribution Characterization of Leaf and Hull Pubescences and Genetic Analysis of Their Numbers in japonica Rice (Oryza sativa)

Distributions of pubescences on leaf blade and hull in japonica rice were observed under an optical microscope. Numbers of leaf and hull pubescences in P1, P2, F1, B1, B2 and F2 generations were investigated in three combinations of japonica rice (Sidao 10A/Wuyujing 3R, Wuyujing 3A/Sidao 10R and Liuyan 189A/HR-122), and genetic analysis for these two traits were conducted by using the joint analysis method of P1, P2, F1, B1, B2 and F2 generations with the mixed major gene plus polygene inheritance models. Leaf pubescences characterized by swollen base and fine tip distributed regularly on the boundary between dark green stripe and light green stripe of leaf blade. Hull pubescences with various lengths distributed irregularly on the whole hull. Numbers of leaf pubescences in the reciprocal combinations of Sidao 10A/Wuyujing 3R and Wuyujing 3A/Sidao 10R and numbers of hull pubescences in all the three combinations were controlled by one pair of additive major genes plus additive-dominant polygenes. In the combination of Liuyan 189A/HR-122, number of leaf pubescences was controlled by one pair of additive-dominant major genes plus additive-dominant polygenes. Both numbers of leaf and hull pubescences were mainly governed by major genes.

Genetic Analysis and QTL Mapping of Large Flag Leaf Angle Trait in Japonica Rice

Genetic segregation analysis for flag leaf angle was conducted using six generations of P1, P2, F1, B1, B2 and F2 derived from a cross of 863B (a maintainer line of japonica rice) and A7444 (a germplasm with large flag leaf angle). Genotypes and phenotypes of flag leaf angle were investigated in 863B (P1), A7444 (P2) and 141 plants in BC1F1 (863B/A7444//863B) population. An SSR genetic linkage map was constructed and QTLs for flag leaf angle were detected. The genetic map containing 79 information loci was constructed, which covers a total distance of 441.6 cM, averaging 5.6 cM between two neighboring loci. Results showed that the trait was controlled by two major genes plus polygene and the major genes were more important. Fifteen markers showed highly significant correlations with flag leaf angle based on single marker regression analysis. Two QTLs (qFLA2 and qFLA8) for flag leaf angle were detected by both composite interval method in software WinQTLCart 2.5 and composite interval method based on mixed linear model in QTL Network 2.0. The qFLA2 explained 10.50% and 13.28% of phenotypic variation, respectively, and was located at the interval of RM300 and RM145 on the short arm of chromosome 2. The qFLA8 explained 9.59% and 7.64% of phenotypic variation, respectively, and was located at the interval flanking RM6215 and RM8265 on the long arm of chromosome 8. The positive alleles at the two QTLs were both contributed from A7444.

Mining Applicable Elite Alleles of Growth Duration, Plant Height and Panicle Number per Plant by Conditional QTL Mapping in Japonica Rice

Unconditional and conditional QTL mapping were conducted for growth duration (GD), plant height (PH) and effective panicle number per plant (PN) using a recombinant inbred line (RIL) population derived from a cross between two japonica rice varieties Xiushui 79 and C Bao. The RIL population consisted of 254 lines was planted in two environments, Nanjing and Sihong, Jiangsu Province, China. Results showed that additive effects were major in all of QTLs for GD, PH and PN detected by the two methods, and the epistatic effects explained a small proportion of phenotypic variation. No interactions were detected between additive QTL and environment, and between epistatic QTL pairs and environment. After growth duration was adjusted to an identical level, RM80-160bp was detected as an applicable elite allele for PN, with an additive effect of 0.71. When effective panicle number per plant was adjusted to an identical level, RM448-240bp was detected as an applicable elite allele for GD, with an additive effect of 4.64. After plant height was adjusted to an identical level, RM80-160bp was detected as an applicable elite allele for PN, with an additive effect of 0.62, and RM448-240bp was detected as an applicable elite allele for GD, with an additive effect of 3.89. These applicable elite alleles could be used to improve target traits without influencing the other two traits.