Erbao Liu

Population genetic structure of Oryza sativa in East and Southeast Asia and the discovery of elite alleles for grain traits

We investigated the nuclear simple sequence repeat (SSR) genotypes of 532 rice (Oryza sativa L.) accessions collected from East and Southeast Asia and detected abundant genetic diversity within the population. We identified 6 subpopulations and found a tendency towards directional evolution in O. sativa from low to high latitudes, with levels of linkage disequilibrium (LD) in the 6 subpopulations ranging from 10 to 30 cM. We then investigated the phenotypic data for grain length, grain width, grain thickness and 1,000-grain weight over 4 years. Using a genome-wide association analysis, we identified 17 marker-trait associations involving 14 SSR markers on 12 chromosome arms, and 8 of the 17 associations were novel. The elite alleles were mined based on the phenotypic effects of the detected quantitative trait loci (QTLs). These elite alleles could be used to improve target traits through optimal cross designs, with the expected results obtained by pyramiding or substituting the elite alleles per QTL (independent of possible epistatic effects). Together, these results provide an in-depth understanding of the genetic diversity pattern among rice-grain traits across a broad geographic scale, which has potential use in future research work, including studies related to germplasm conservation and molecular breeding by design.

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.

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.

Favorable Marker Alleles for Panicle Exsertion Length in Rice (Oryza sativa L.) Mined by Association Mapping and the RSTEP-LRT Method

The panicle exsertion length (PEL) in rice (Oryza sativa L.) is an important trait for hybrid seed production. We investigated the PEL in a chromosome segment substitution line (CSSL) population consisting of 66 lines and a natural population composed of 540 varieties. In the CSSL population, a total of seven QTLs for PEL were detected across two environments. The percentage of phenotypic variance explained (PVE) ranged from 10.22 to 50.18%, and the additive effect ranged from −1.77 to 6.47 cm. Among the seven QTLs, qPEL10.2 had the largest PVE, 44.05 and 50.18%, with an additive effect of 5.91 and 6.47 cm in 2015 and in 2016, respectively. In the natural population, 13 SSR marker loci were detected that were associated with PEL in all four environments, with the PVE ranging from 1.20 to 6.26%. Among the 13 loci, 7 were novel. The RM5746-170 bp allele had the largest phenotypic effect (5.11 cm), and the typical carrier variety was Qiaobinghuang. An RM5620-RM6100 region harboring the EUI2 locus on chromosome 10 was detected in both populations. The sequencing results showed that the accessions with a shorter PEL contained the A base, while the accessions with a longer PEL contained the G base at the 1,475 bp location of the EUI2 gene.

Genome-Wide Single-Nucleotide Polymorphisms in CMS and Restorer Lines Discovered by Genotyping Using Sequencing and Association with Marker-Combining Ability for 12 Yield-Related Traits in Oryza sativa L. subsp. Japonica

Heterosis or hybrid vigor is closely related with general combing ability (GCA) of parents and special combining ability (SCA) of combinations. The evaluation of GCA and SCA facilitate selection of parents and combinations in heterosis breeding. In order to improve combining ability (CA) by molecular marker assist selection, it is necessary to identify marker loci associated with the CA. To identify the single nucleotide polymorphisms (SNP) loci associated with CA in the parental genomes of japonica rice, genome-wide discovered SNP loci were tested for association with the CA of 18 parents for 12 yield-related traits. In this study, 81 hybrids were created and evaluated to calculate the CA of 18 parents. The parents were sequenced by genotyping by sequencing (GBS) method for identification of genome-wide SNPs. The analysis of GBS indicated that the successful mapping of 9.86 × 106 short reads in the Nipponbare reference genome consists of 39,001 SNPs in parental genomes at 11,085 chromosomal positions. The discovered SNPs were non-randomly distributed within and among the 12 chromosomes of rice. Overall, 20.4% (8026) of the discovered SNPs were coding types, and 8.6% (3344) and 9.9% (3951) of the SNPs revealed synonymous and non-synonymous changes, which provide valuable knowledge about the underlying performance of the parents. Furthermore, the associations between SNPs and CA indicated that 362 SNP loci were significantly related to the CA of 12 parental traits. The identified SNP loci of CA in our study were distributed genome wide and caused a positive or negative effect on the CA of traits. For the yield-related traits, such as grain thickness, days to heading, panicle length, grain length and 1000-grain weight, a maximum number of positive SNP loci of CA were found in CMS A171 and in the restorers LC64 and LR27. On an individual basis, some of associated loci that resided on chromosomes 2, 5, 7, 9, and 11 recorded maximum positive values for the CA of traits. From our results, we suggest that heterosis in japonica rice would be improved by pyramiding the favorable SNP loci of CA and eliminating the unfavorable loci from parental genomes.