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Identification of qGL3.5, a Novel Locus Controlling Grain Length in Rice Through Bulked Segregant Analysis and Fine Mapping.

ABSTRACT: Grain length (GL) directly affects the yield and quality of rice. Very few cloned GL-related genes are applied in production because their yield-increasing effects are not obvious, and the overall regulatory networks underlying the associated processes remain poorly understood. DNA samples from two bulk DNA pools (L-pool and S-pool) and their parents (KJ01 and Huaye 3) were subjected to high-throughput sequencing. Using bulked segregant analysis (BSA), qGL3.5 was mapped to a 0.34-Mb "hotspot" region on chromosome 3 that contains 37 genes related to various traits. Then, qGL3.5 was mapped to the genomic interval between the flanking markers M2 and M3 using 2786 BC₄F₂ individuals. Because the region from B5 to B6 was not the associated region under BSA-seq analysis, qGL3.5 was narrowed down to the interval between B6 and M3, which spanned 24.0-kb. Of all 37 genes with non-synonymous single-nucleotide polymorphisms (SNPs) between KJ01 and Huaye 3 based on BSA-seq analysis, only one complete annotated gene, ORF18 (Gene ID: LOC_Os03g42790.1) was found. ORF18 encodes an IBR-RING zinc-finger-related protein, with one really interesting new gene (RING) and two in between ring finger (IBR) domains. The knockout of ORF18 derived from Huaye 3 using clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) editing technology increased the GL of the mutant by approximately 2.2 mm. The novel locus qGL3.5 negatively regulated GL by promoting elongation of the longitudinal cell of the grain outer glume. These results provide a new genetic resource for rice grain shape breeding and a starting point for the functional characterization of the wild rice GL gene.

SUBMITTER: Wang L

PROVIDER: S-EPMC9240483 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Grain length is a prominent determinant for the grain weight and appearance quality of rice. To elucidate the genetic basis of the control of grain length, we conducted quantitative trait locus (QTL) mapping to determine a genomic interval responsible for the a long-grain phenotype observed in a japonica cultivar HD385 by using an F2:3 population and recombinant inbred line (RIL) population, which is are derived from a cross between the long-grain japonica cultivar HD385 and short-grain japonica cultivar Nipponbare (NIP). This led to the identification of a novel QTL, which is located on chromosome 3, for grain length, named qIGL1 (for Increased Grain Length 1); the HD385-derived allele showed enhancement effects on grain length, and such an allele as well as NIP-derived allele are were thus designated qigl1 HD385 and qIGL1NIP, respectively. Genetic analysis revealed that qigl1HD385 displayed semi-dominant effects on grain length. To further narrow down the qIGL1 region, we developed a few advanced backcross populations to determine the precise location of qIGL1. Eventually, qIGL1 was delimited to a 70.8-kb region containing 9 open reading frames (ORFs). A comprehensive analysis indicated that ORF6 and ORF9, separately corresponding to LOC_Os03g30530 and LOC_Os03g30550, are were found to carry base substitutions and/or deletions, which resulted in changes or losses of amino acid residues. However, these alterations in coding sequences does did not significantly change the expression levels of both ORFs. To further elucidate the mechanisms behind the increases of grain length conferred by qigl1HD385, we generated a pair of near-isogenic lines (NILs), termed NIL-qigl1HD385 and NIL-qIGL1NIP, and discovered that introduction of qigl1HD385 into the NIP background significantly lead resulted into the elevations of grain length and 1000-grain weight. Closer inspection of outer epidermal cells of grain surfaces of both lines revealed that the cell length and width in the longitudinal direction are were significantly longer in NIL-qigl1HD385 compared with in NIL-qIGL1NIP, which may might stem partly from the downregulation of several cell cycle-related genes. Hence, our study studies identify identified a new semi-dominant natural allele contributing to the increase of grain length, and further shed light on the regulatory mechanisms of grain length.

Project description:BackgroundRice growth is frequently affected by salinity. When exposed to high salinity, rice seed germination and seedling establishment are significantly inhibited. With the promotion of direct-seeding in Asia, improving rice seed germination under salt stress is crucial for breeding.ResultsIn this study, an indica landrace Wujiaozhan (WJZ) was identified with high germinability under salt stress. A BC1F2 population derived from the crossing WJZ/Nip (japonica, Nipponbare)//Nip, was used to quantitative trait loci (QTL) mapping for the seed germination rate (GR) and germination index (GI) under H2O and 300 mM NaCl conditions. A total of 13 QTLs were identified, i.e. ten QTLs under H2O conditions and nine QTLs under salt conditions. Six QTLs, qGR6.1, qGR8.1, qGR8.2, qGR10.1, qGR10.2 and qGI10.1 were simultaneously identified under two conditions. Under salt conditions, three QTLs, qGR6.2, qGR10.1 and qGR10.2 for GR were identified at different time points during seed germination, which shared the same chromosomal region with qGI6.2, qGI10.1 and qGI10.2 for GI respectively. The qGR6.2 accounted for more than 20% of phenotypic variation under salt stress, as the major effective QTL. Furthermore, qGR6.2 was verified via the BC2F2 population and narrowed to a 65.9-kb region with eleven candidate genes predicted. Based on the microarray database, five candidate genes were found with high transcript abundances at the seed germination stage, of which LOC_Os06g10650 and LOC_Os06g10710 were differentially expressed after seed imbibition. RT-qPCR results showed the expression of LOC_Os06g10650 was significantly up-regulated in two parents with higher levels in WJZ than Nip during seed germination under salt conditions. Taken together, it suggests that LOC_Os06g10650, encoding tyrosine phosphatase family protein, might be the causal candidate gene for qGR6.2.ConclusionsIn this study, we identified 13 QTLs from a landrace WJZ that confer seed germination traits under H2O and salt conditions. A major salt-tolerance-specific QTL qGR6.2 was fine mapped to a 65.9-kb region. Our results provide information on the genetic basis of improving rice seed germination under salt stress by marker-assisted selection (MAS).

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Identification of qGL3.5, a Novel Locus Controlling Grain Length in Rice Through Bulked Segregant Analysis and Fine Mapping.

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