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OsLG3 contributing to rice grain length and yield was mined by Ho-LAMap

View Article: PubMed Central - PubMed

ABSTRACT

Background: Most agronomic traits in rice are complex and polygenic. The identification of quantitative trait loci (QTL) for grain length is an important objective of rice genetic research and breeding programs.

Results: Herein, we identified 99 QTL for grain length by GWAS based on approximately 10 million single nucleotide polymorphisms from 504 cultivated rice accessions (Oryza sativa L.), 13 of which were validated by four linkage populations and 92 were new loci for grain length. We scanned the Ho (observed heterozygosity per locus) index of coupled-parents of crosses mapping the same QTL, based on linkage and association mapping, and identified two new genes for grain length. We named this approach as Ho-LAMap. A simulation study of six known genes showed that Ho-LAMap could mine genes rapidly across a wide range of experimental variables using deep-sequencing data. We used Ho-LAMap to clone a new gene, OsLG3, as a positive regulator of grain length, which could improve rice yield without influencing grain quality. Sequencing of the promoter region in 283 rice accessions from a wide geographic range identified four haplotypes that seem to be associated with grain length. Further analysis showed that OsLG3 alleles in the indica and japonica evolved independently from distinct ancestors and low nucleotide diversity of OsLG3 in indica indicated artificial selection. Phylogenetic analysis showed that OsLG3 might have much potential value for improvement of grain length in japonica breeding.

Conclusions: The results demonstrated that Ho-LAMap is a potential approach for gene discovery and OsLG3 is a promising gene to be utilized in genomic assisted breeding for rice cultivar improvement.

Electronic supplementary material: The online version of this article (doi:10.1186/s12915-017-0365-7) contains supplementary material, which is available to authorized users.

No MeSH data available.


Functional variation, the haplotypes and origin of OsLG3. a Haplotype analysis of OsLG3. Gene structure and natural variation between alleles from SLG and Nipponbare (top). Natural variation in OsLG3 among 283 rice accessions of a mini-core collection compared with the NILs (bottom). b Cladogram of four haplotypes. c Grain lengths of accessions for the two classes in MCC1; raw data are provided in Additional file 32: Table S1; n, is the number of accessions. P values were generated by two-tailed t tests. Error bars, SEM. d Relative expressions of OsLG3 in two haplotypes (SLG-Hap, n = 17; Nip-Hap, n = 16). e Transient expression assays of the effect of the four consensus SNPs and one indel on gene expression. Relative firefly luciferase activities in Arabidopsis protoplasts, with data normalized to activity of co-transformed constitutively expressed Renilla luciferase. Data are shown as means ± SEM (n = 5 technical replicates). f Geographic origin of rice containing the SLG-type OsLG3. Hap1–4 are represented by red, green, purple, and blue circles, respectively. The indica and japonica cultivars are denoted by solid and dashed circles, respectively. g Nucleotide diversity analysis in OsLG3 and flanking regions (~400 kb). Representative samples included 99 indica accessions, 68 temperate japonica accessions, 27 tropical japonica accessions, and 10 wild rice accessions (Additional file 32: Table S1). Tej, temperate japonica; trj, tropical japonica. Y-axis, average π value; x-axis, Nipponbare TIGR v7.0 genome coordinate of chromosome 3. h Averaged nucleotide diversity of the 20 kb surrounding OsLG3. i A minimum-spanning tree for the OsLG3 region. Each haplotype group is represented by a circle, and circle size represents the number of lines within the haplotype, as in Fig. 4 f. Orange, brown, yellow, red, blue and pink represent O. rufipogon in China, O. rufipogon in Southeast Asia, indica, tropical japonica, temperate japonica, and template japonica with glutinous varieties from the Yunnan-Guizhou Plateau or mixtures with recently improved indica or tropical japonica. Grid corresponds to oslg3/oslg3
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Fig4: Functional variation, the haplotypes and origin of OsLG3. a Haplotype analysis of OsLG3. Gene structure and natural variation between alleles from SLG and Nipponbare (top). Natural variation in OsLG3 among 283 rice accessions of a mini-core collection compared with the NILs (bottom). b Cladogram of four haplotypes. c Grain lengths of accessions for the two classes in MCC1; raw data are provided in Additional file 32: Table S1; n, is the number of accessions. P values were generated by two-tailed t tests. Error bars, SEM. d Relative expressions of OsLG3 in two haplotypes (SLG-Hap, n = 17; Nip-Hap, n = 16). e Transient expression assays of the effect of the four consensus SNPs and one indel on gene expression. Relative firefly luciferase activities in Arabidopsis protoplasts, with data normalized to activity of co-transformed constitutively expressed Renilla luciferase. Data are shown as means ± SEM (n = 5 technical replicates). f Geographic origin of rice containing the SLG-type OsLG3. Hap1–4 are represented by red, green, purple, and blue circles, respectively. The indica and japonica cultivars are denoted by solid and dashed circles, respectively. g Nucleotide diversity analysis in OsLG3 and flanking regions (~400 kb). Representative samples included 99 indica accessions, 68 temperate japonica accessions, 27 tropical japonica accessions, and 10 wild rice accessions (Additional file 32: Table S1). Tej, temperate japonica; trj, tropical japonica. Y-axis, average π value; x-axis, Nipponbare TIGR v7.0 genome coordinate of chromosome 3. h Averaged nucleotide diversity of the 20 kb surrounding OsLG3. i A minimum-spanning tree for the OsLG3 region. Each haplotype group is represented by a circle, and circle size represents the number of lines within the haplotype, as in Fig. 4 f. Orange, brown, yellow, red, blue and pink represent O. rufipogon in China, O. rufipogon in Southeast Asia, indica, tropical japonica, temperate japonica, and template japonica with glutinous varieties from the Yunnan-Guizhou Plateau or mixtures with recently improved indica or tropical japonica. Grid corresponds to oslg3/oslg3

Mentions: We next sequenced the OsLG3 promoter regions and measured grain length in 283 rice accessions, including 247 cultivated, worldwide varieties of O. sativa in MCC1, and 36 wild accessions originating from a wide geographic range across Asia (Additional file 32: Table S1). On the basis of the nucleotide polymorphisms identified by Ho-LAMap, we could divide the sequences of the cultivated varieties into four haplotypes that were placed into two classes by phylogenetic analysis: haplotypes 1 and 3 in class A and haplotypes 2 and 4 in class B (Fig. 4a, b). Cultivars having class A haplotypes showed significantly higher OsLG3 expression levels and a longer grain phenotype than those with class B haplotypes by qRT-PCR analysis (Fig. 4c, d). Furthermore, transient assays indicated that the four consensus SNPs, rather than indel1, played a significant role in regulating OsLG3 expression differences between genotypes, resulting in diversity on grain length (Fig. 4a, e).Fig. 4


OsLG3 contributing to rice grain length and yield was mined by Ho-LAMap
Functional variation, the haplotypes and origin of OsLG3. a Haplotype analysis of OsLG3. Gene structure and natural variation between alleles from SLG and Nipponbare (top). Natural variation in OsLG3 among 283 rice accessions of a mini-core collection compared with the NILs (bottom). b Cladogram of four haplotypes. c Grain lengths of accessions for the two classes in MCC1; raw data are provided in Additional file 32: Table S1; n, is the number of accessions. P values were generated by two-tailed t tests. Error bars, SEM. d Relative expressions of OsLG3 in two haplotypes (SLG-Hap, n = 17; Nip-Hap, n = 16). e Transient expression assays of the effect of the four consensus SNPs and one indel on gene expression. Relative firefly luciferase activities in Arabidopsis protoplasts, with data normalized to activity of co-transformed constitutively expressed Renilla luciferase. Data are shown as means ± SEM (n = 5 technical replicates). f Geographic origin of rice containing the SLG-type OsLG3. Hap1–4 are represented by red, green, purple, and blue circles, respectively. The indica and japonica cultivars are denoted by solid and dashed circles, respectively. g Nucleotide diversity analysis in OsLG3 and flanking regions (~400 kb). Representative samples included 99 indica accessions, 68 temperate japonica accessions, 27 tropical japonica accessions, and 10 wild rice accessions (Additional file 32: Table S1). Tej, temperate japonica; trj, tropical japonica. Y-axis, average π value; x-axis, Nipponbare TIGR v7.0 genome coordinate of chromosome 3. h Averaged nucleotide diversity of the 20 kb surrounding OsLG3. i A minimum-spanning tree for the OsLG3 region. Each haplotype group is represented by a circle, and circle size represents the number of lines within the haplotype, as in Fig. 4 f. Orange, brown, yellow, red, blue and pink represent O. rufipogon in China, O. rufipogon in Southeast Asia, indica, tropical japonica, temperate japonica, and template japonica with glutinous varieties from the Yunnan-Guizhou Plateau or mixtures with recently improved indica or tropical japonica. Grid corresponds to oslg3/oslg3
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Fig4: Functional variation, the haplotypes and origin of OsLG3. a Haplotype analysis of OsLG3. Gene structure and natural variation between alleles from SLG and Nipponbare (top). Natural variation in OsLG3 among 283 rice accessions of a mini-core collection compared with the NILs (bottom). b Cladogram of four haplotypes. c Grain lengths of accessions for the two classes in MCC1; raw data are provided in Additional file 32: Table S1; n, is the number of accessions. P values were generated by two-tailed t tests. Error bars, SEM. d Relative expressions of OsLG3 in two haplotypes (SLG-Hap, n = 17; Nip-Hap, n = 16). e Transient expression assays of the effect of the four consensus SNPs and one indel on gene expression. Relative firefly luciferase activities in Arabidopsis protoplasts, with data normalized to activity of co-transformed constitutively expressed Renilla luciferase. Data are shown as means ± SEM (n = 5 technical replicates). f Geographic origin of rice containing the SLG-type OsLG3. Hap1–4 are represented by red, green, purple, and blue circles, respectively. The indica and japonica cultivars are denoted by solid and dashed circles, respectively. g Nucleotide diversity analysis in OsLG3 and flanking regions (~400 kb). Representative samples included 99 indica accessions, 68 temperate japonica accessions, 27 tropical japonica accessions, and 10 wild rice accessions (Additional file 32: Table S1). Tej, temperate japonica; trj, tropical japonica. Y-axis, average π value; x-axis, Nipponbare TIGR v7.0 genome coordinate of chromosome 3. h Averaged nucleotide diversity of the 20 kb surrounding OsLG3. i A minimum-spanning tree for the OsLG3 region. Each haplotype group is represented by a circle, and circle size represents the number of lines within the haplotype, as in Fig. 4 f. Orange, brown, yellow, red, blue and pink represent O. rufipogon in China, O. rufipogon in Southeast Asia, indica, tropical japonica, temperate japonica, and template japonica with glutinous varieties from the Yunnan-Guizhou Plateau or mixtures with recently improved indica or tropical japonica. Grid corresponds to oslg3/oslg3
Mentions: We next sequenced the OsLG3 promoter regions and measured grain length in 283 rice accessions, including 247 cultivated, worldwide varieties of O. sativa in MCC1, and 36 wild accessions originating from a wide geographic range across Asia (Additional file 32: Table S1). On the basis of the nucleotide polymorphisms identified by Ho-LAMap, we could divide the sequences of the cultivated varieties into four haplotypes that were placed into two classes by phylogenetic analysis: haplotypes 1 and 3 in class A and haplotypes 2 and 4 in class B (Fig. 4a, b). Cultivars having class A haplotypes showed significantly higher OsLG3 expression levels and a longer grain phenotype than those with class B haplotypes by qRT-PCR analysis (Fig. 4c, d). Furthermore, transient assays indicated that the four consensus SNPs, rather than indel1, played a significant role in regulating OsLG3 expression differences between genotypes, resulting in diversity on grain length (Fig. 4a, e).Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Most agronomic traits in rice are complex and polygenic. The identification of quantitative trait loci (QTL) for grain length is an important objective of rice genetic research and breeding programs.

Results: Herein, we identified 99 QTL for grain length by GWAS based on approximately 10 million single nucleotide polymorphisms from 504 cultivated rice accessions (Oryza sativa L.), 13 of which were validated by four linkage populations and 92 were new loci for grain length. We scanned the Ho (observed heterozygosity per locus) index of coupled-parents of crosses mapping the same QTL, based on linkage and association mapping, and identified two new genes for grain length. We named this approach as Ho-LAMap. A simulation study of six known genes showed that Ho-LAMap could mine genes rapidly across a wide range of experimental variables using deep-sequencing data. We used Ho-LAMap to clone a new gene, OsLG3, as a positive regulator of grain length, which could improve rice yield without influencing grain quality. Sequencing of the promoter region in 283 rice accessions from a wide geographic range identified four haplotypes that seem to be associated with grain length. Further analysis showed that OsLG3 alleles in the indica and japonica evolved independently from distinct ancestors and low nucleotide diversity of OsLG3 in indica indicated artificial selection. Phylogenetic analysis showed that OsLG3 might have much potential value for improvement of grain length in japonica breeding.

Conclusions: The results demonstrated that Ho-LAMap is a potential approach for gene discovery and OsLG3 is a promising gene to be utilized in genomic assisted breeding for rice cultivar improvement.

Electronic supplementary material: The online version of this article (doi:10.1186/s12915-017-0365-7) contains supplementary material, which is available to authorized users.

No MeSH data available.