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SNP-based analysis of genetic diversity reveals important alleles associated with seed size in rice.

Tang W, Wu T, Ye J, Sun J, Jiang Y, Yu J, Tang J, Chen G, Wang C, Wan J - BMC Plant Biol. (2016)

Bottom Line: Meanwhile, we identified polymorphic SNPs with large effects on protein-coding and miRNA genes.To validate the effect of the polymorphic SNPs, we further investigated a SNP (chr4:28,894,757) at the miRNA binding site in the 3'-UTR region of the locus Os4g48460, which is associated with rice seed size.Our study has identified the genome-wide SNPs by GBS of the parental varieties of RIL populations and identified CYP704A3, a miRNA-regulated gene that is responsible for rice seed length.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, 210095, Nanjing, China.

ABSTRACT

Background: Single-nucleotide polymorphisms (SNPs) have become the genetic markers of choice in various genetic, ecological, and evolutionary studies. Genotyping-by-sequencing (GBS) is a next-generation-sequencing based method that takes advantage of reduced representation to enable high-throughput genotyping using a large number of SNP markers.

Results: In the present study, the distribution of non-redundant SNPs in the parents of 12 rice recombination line populations was evaluated through GBS. A total of 45 Gigabites of nucleotide sequences conservatively provided satisfactory genotyping of rice SNPs. By assembling to the genomes of reference genomes of japonica Nipponbare, we detected 22,682 polymorphic SNPs that may be utilized for QTL/gene mapping with the Recombinant Inbred Lines (RIL) populations derived from these parental lines. Meanwhile, we identified polymorphic SNPs with large effects on protein-coding and miRNA genes. To validate the effect of the polymorphic SNPs, we further investigated a SNP (chr4:28,894,757) at the miRNA binding site in the 3'-UTR region of the locus Os4g48460, which is associated with rice seed size. Os4g48460 encodes a putative cytochrome P450, CYP704A3. Direct degradation of the 3'-UTR of the CYP704A3 gene by a miRNA (osa-miRf10422-akr) was validated by in planta mRNA degradation assay. We also showed that rice seeds of longer lengths may be produced by downregulating CYP704A3 via RNAi.

Conclusions: Our study has identified the genome-wide SNPs by GBS of the parental varieties of RIL populations and identified CYP704A3, a miRNA-regulated gene that is responsible for rice seed length.

No MeSH data available.


The effects of osa-miRf10422-akrexpression on the accumulation of the CYP704A3 gene. The schematic representation of the reporters and the effectors used in this assay is shown in (a). GFP fluorescence images of the co-expression of osa-miRf10422-akr with the reporter gene EGFP, which was fused with the empty vector control (b) and miRNA target region in the 3′-UTR of the CYP704A3 gene (c). Fluorescence imaging analysis of the agroinfiltrated leaves at 2 dpi under UV illumination. Quantitation of EGFP mRNA as averaged from three leaves from each infiltration treatment (d)
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Fig5: The effects of osa-miRf10422-akrexpression on the accumulation of the CYP704A3 gene. The schematic representation of the reporters and the effectors used in this assay is shown in (a). GFP fluorescence images of the co-expression of osa-miRf10422-akr with the reporter gene EGFP, which was fused with the empty vector control (b) and miRNA target region in the 3′-UTR of the CYP704A3 gene (c). Fluorescence imaging analysis of the agroinfiltrated leaves at 2 dpi under UV illumination. Quantitation of EGFP mRNA as averaged from three leaves from each infiltration treatment (d)

Mentions: To validate that CYP704A3 is regulated by osa-miRf10422-akr, a schematic representation of the reporters and the effectors (Fig. 5a) was used in this assay. To examine the ability of plant expression vectors to produce osa-miRf10422-akr miRNAs in vivo, an Agrobacterium strain harboring pCAMBIA1300-35S:osa-miRf10422-akr or the control pCAMBIA 1300 vector (35S) was infiltrated into N. benthamiana leaves, together with the reporter gene EGFP which was fused with the 3′-UTR of the rice CYP704A3 gene, which contained the putative miRNA target. When the effecter recognizes the miRNA target in the reporter construct, the mRNA level of EGFP and the fluorescence of EGFP are downregulated. The fluorescence of the agroinfiltrated leaves was taken at 2 dpi under UV illumination. Fluorescence imaging showed that EGFP and osa-miRf10422-akr were co-expressed (Fig. 5b) and together with miRNA target region in the 3′-UTR of the CYP704A3 gene (Fig. 5c). Total RNA was extracted at 3 dpi, and quantitative EGFP mRNA analysis using the average measurements of three leaves utilized in each infiltration treatment (Fig. 5d). As expected, osa-miRf10422-akr expression affected the CYP704A3 gene expression both at the transcriptional and protein levels. These findings therefore indicate that osa-miRf10422-akr participates in seed size determination by directly regulating CYP704A3.Fig. 5


SNP-based analysis of genetic diversity reveals important alleles associated with seed size in rice.

Tang W, Wu T, Ye J, Sun J, Jiang Y, Yu J, Tang J, Chen G, Wang C, Wan J - BMC Plant Biol. (2016)

The effects of osa-miRf10422-akrexpression on the accumulation of the CYP704A3 gene. The schematic representation of the reporters and the effectors used in this assay is shown in (a). GFP fluorescence images of the co-expression of osa-miRf10422-akr with the reporter gene EGFP, which was fused with the empty vector control (b) and miRNA target region in the 3′-UTR of the CYP704A3 gene (c). Fluorescence imaging analysis of the agroinfiltrated leaves at 2 dpi under UV illumination. Quantitation of EGFP mRNA as averaged from three leaves from each infiltration treatment (d)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4837510&req=5

Fig5: The effects of osa-miRf10422-akrexpression on the accumulation of the CYP704A3 gene. The schematic representation of the reporters and the effectors used in this assay is shown in (a). GFP fluorescence images of the co-expression of osa-miRf10422-akr with the reporter gene EGFP, which was fused with the empty vector control (b) and miRNA target region in the 3′-UTR of the CYP704A3 gene (c). Fluorescence imaging analysis of the agroinfiltrated leaves at 2 dpi under UV illumination. Quantitation of EGFP mRNA as averaged from three leaves from each infiltration treatment (d)
Mentions: To validate that CYP704A3 is regulated by osa-miRf10422-akr, a schematic representation of the reporters and the effectors (Fig. 5a) was used in this assay. To examine the ability of plant expression vectors to produce osa-miRf10422-akr miRNAs in vivo, an Agrobacterium strain harboring pCAMBIA1300-35S:osa-miRf10422-akr or the control pCAMBIA 1300 vector (35S) was infiltrated into N. benthamiana leaves, together with the reporter gene EGFP which was fused with the 3′-UTR of the rice CYP704A3 gene, which contained the putative miRNA target. When the effecter recognizes the miRNA target in the reporter construct, the mRNA level of EGFP and the fluorescence of EGFP are downregulated. The fluorescence of the agroinfiltrated leaves was taken at 2 dpi under UV illumination. Fluorescence imaging showed that EGFP and osa-miRf10422-akr were co-expressed (Fig. 5b) and together with miRNA target region in the 3′-UTR of the CYP704A3 gene (Fig. 5c). Total RNA was extracted at 3 dpi, and quantitative EGFP mRNA analysis using the average measurements of three leaves utilized in each infiltration treatment (Fig. 5d). As expected, osa-miRf10422-akr expression affected the CYP704A3 gene expression both at the transcriptional and protein levels. These findings therefore indicate that osa-miRf10422-akr participates in seed size determination by directly regulating CYP704A3.Fig. 5

Bottom Line: Meanwhile, we identified polymorphic SNPs with large effects on protein-coding and miRNA genes.To validate the effect of the polymorphic SNPs, we further investigated a SNP (chr4:28,894,757) at the miRNA binding site in the 3'-UTR region of the locus Os4g48460, which is associated with rice seed size.Our study has identified the genome-wide SNPs by GBS of the parental varieties of RIL populations and identified CYP704A3, a miRNA-regulated gene that is responsible for rice seed length.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, 210095, Nanjing, China.

ABSTRACT

Background: Single-nucleotide polymorphisms (SNPs) have become the genetic markers of choice in various genetic, ecological, and evolutionary studies. Genotyping-by-sequencing (GBS) is a next-generation-sequencing based method that takes advantage of reduced representation to enable high-throughput genotyping using a large number of SNP markers.

Results: In the present study, the distribution of non-redundant SNPs in the parents of 12 rice recombination line populations was evaluated through GBS. A total of 45 Gigabites of nucleotide sequences conservatively provided satisfactory genotyping of rice SNPs. By assembling to the genomes of reference genomes of japonica Nipponbare, we detected 22,682 polymorphic SNPs that may be utilized for QTL/gene mapping with the Recombinant Inbred Lines (RIL) populations derived from these parental lines. Meanwhile, we identified polymorphic SNPs with large effects on protein-coding and miRNA genes. To validate the effect of the polymorphic SNPs, we further investigated a SNP (chr4:28,894,757) at the miRNA binding site in the 3'-UTR region of the locus Os4g48460, which is associated with rice seed size. Os4g48460 encodes a putative cytochrome P450, CYP704A3. Direct degradation of the 3'-UTR of the CYP704A3 gene by a miRNA (osa-miRf10422-akr) was validated by in planta mRNA degradation assay. We also showed that rice seeds of longer lengths may be produced by downregulating CYP704A3 via RNAi.

Conclusions: Our study has identified the genome-wide SNPs by GBS of the parental varieties of RIL populations and identified CYP704A3, a miRNA-regulated gene that is responsible for rice seed length.

No MeSH data available.