Limits...
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.


CYP704A3-RNAi transgenic plantsproduced longer grains. a The morphology of wild-type and RNAi-12 transgenic plants. Scale bar: 10 mm. b Relative expression levels of CYP704A3 mRNA of the wild-type RNAi-4 (as a transgenic negative control) and T1CYP704A3-RNAi transgenic plants which were determined by qRT-PCR. c Grains of the wild-type, RNAi-4 (as a transgenic negative control), RNAi-12, RNAi-3 and RNAi-6. Scale bar: 1 mm. d Grain length (n = 100). e Grain length to width ratio (n = 100)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: CYP704A3-RNAi transgenic plantsproduced longer grains. a The morphology of wild-type and RNAi-12 transgenic plants. Scale bar: 10 mm. b Relative expression levels of CYP704A3 mRNA of the wild-type RNAi-4 (as a transgenic negative control) and T1CYP704A3-RNAi transgenic plants which were determined by qRT-PCR. c Grains of the wild-type, RNAi-4 (as a transgenic negative control), RNAi-12, RNAi-3 and RNAi-6. Scale bar: 1 mm. d Grain length (n = 100). e Grain length to width ratio (n = 100)

Mentions: To further confirm the function of CYP704A3, CYP704A3-RNAi transgenic rice plants were generated. Several independently transformed plants showed a reduction in the level of expression of the CYP704A3 gene (Fig. 4a). Three of these were selected to represent the relative expression of CYP704A3 in relation to the transgenic negative control RNAi-4 (Fig. 4b). The reduced expression of CYP704A3 caused various degrees of elongation in grain length (Fig. 4c). Significant difference (P ≤ 0.01) in grain length (Fig. 4d) and length/width ratio (P ≤ 0.01) (Fig. 4d) among the wild-type, RNAi-4, and OsCYP704A3-RNAi transgenic plants were observed. These results further indicated that CYP704A3 negatively regulates grain length in rice.Fig. 4


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)

CYP704A3-RNAi transgenic plantsproduced longer grains. a The morphology of wild-type and RNAi-12 transgenic plants. Scale bar: 10 mm. b Relative expression levels of CYP704A3 mRNA of the wild-type RNAi-4 (as a transgenic negative control) and T1CYP704A3-RNAi transgenic plants which were determined by qRT-PCR. c Grains of the wild-type, RNAi-4 (as a transgenic negative control), RNAi-12, RNAi-3 and RNAi-6. Scale bar: 1 mm. d Grain length (n = 100). e Grain length to width ratio (n = 100)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: CYP704A3-RNAi transgenic plantsproduced longer grains. a The morphology of wild-type and RNAi-12 transgenic plants. Scale bar: 10 mm. b Relative expression levels of CYP704A3 mRNA of the wild-type RNAi-4 (as a transgenic negative control) and T1CYP704A3-RNAi transgenic plants which were determined by qRT-PCR. c Grains of the wild-type, RNAi-4 (as a transgenic negative control), RNAi-12, RNAi-3 and RNAi-6. Scale bar: 1 mm. d Grain length (n = 100). e Grain length to width ratio (n = 100)
Mentions: To further confirm the function of CYP704A3, CYP704A3-RNAi transgenic rice plants were generated. Several independently transformed plants showed a reduction in the level of expression of the CYP704A3 gene (Fig. 4a). Three of these were selected to represent the relative expression of CYP704A3 in relation to the transgenic negative control RNAi-4 (Fig. 4b). The reduced expression of CYP704A3 caused various degrees of elongation in grain length (Fig. 4c). Significant difference (P ≤ 0.01) in grain length (Fig. 4d) and length/width ratio (P ≤ 0.01) (Fig. 4d) among the wild-type, RNAi-4, and OsCYP704A3-RNAi transgenic plants were observed. These results further indicated that CYP704A3 negatively regulates grain length in rice.Fig. 4

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.