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Associative transcriptomics study dissects the genetic architecture of seed glucosinolate content in Brassica napus.

Lu G, Harper AL, Trick M, Morgan C, Fraser F, O'Neill C, Bancroft I - DNA Res. (2014)

Bottom Line: A weighted gene co-expression network analysis provided additional 40 candidate genes.Resequencing of genomic regions revealed six new SNPs in BnaA.GTR2a and four insertions or deletions in BnaC.HAG3b.These deletion polymorphisms were then successfully converted into polymerase chain reaction-based diagnostic markers that can, due to high linkage disequilibrium observed in these regions of the genome, be used for marker-assisted breeding for low seed GS lines.

View Article: PubMed Central - PubMed

Affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York YO10 5DD, UK Oil Crops Research Institute, CAAS, Wuhan 430062, Hubei, China.

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PCR assay for the 11-bp deletions at BnaC.HAG3b locus. PCR primer combination 260F/490R was used, which produced a 226-bp (haplotype I) or 237-bp (haplotype II) band. Numbers on the left and right sides are fragment length in base pair. M, 100-bp DNA ladder.
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DSU024F5: PCR assay for the 11-bp deletions at BnaC.HAG3b locus. PCR primer combination 260F/490R was used, which produced a 226-bp (haplotype I) or 237-bp (haplotype II) band. Numbers on the left and right sides are fragment length in base pair. M, 100-bp DNA ladder.

Mentions: Another unigene of interest is EX043693 (BnaC.HAG3b), which is located within the peak on C9 which coincides with a region of strong and extensive LD (Fig. 3b). In Arabidopsis, HAG3 (MYB29) is a transcription factor modulating many genes in the GS pathway.65 Expression of BnaC.HAG3b is highly associated with GS accumulation (r = 0.41, P < 10−8) and accounts for 16.8% of trait variation (Supplementary Fig. S10b). However, no SNPs have been detected by mRNA-Seq within this gene. Therefore, genomic regions covering the length of EX043693 were amplified from 70 lines and then sequenced to detect potential DNA sequence variations. By comparing sequences of these DNA fragments, four InDels, i.e. InDel3–1 (3-bp insertion), InDel3–2 (3-bp deletion), InDel7 (7-bp deletion), and InDel1 (1-bp deletion), were detected. These InDels formed two haplotypes: haplotype I includes the 3-bp insertion and 11-bp (i.e. 3 plus 7 plus 1) of deletions, while haplotype II has the sequence identical to the reference unigene EX043693 (Fig. 4). A total number of 60 lines were determined as haplotype I at these loci and 10 lines as haplotype II. The average GS concentration of haplotype I (56.3 µmol g−1) was only 55% of that of haplotype II (the wild type), consistent with the net 8-bp (frame-shift) deletion reducing the functional properties of the encoded protein. To facilitate germplasm screening and marker-assisted selection for the low GS content, a pair of specific PCR primers was designed, which only captures the 11-bp deletions so that the polymorphism can be more easily resolved by agarose gel (Fig. 4). An example of PCR amplification is given in Fig. 5; the haplotype I and II lines can be clearly distinguished by the presence of a 226-bp- and 237-bp-specific fragment, respectively. Thus, the polymorphism at BnaC.HAG3b locus has been successfully converted into a PCR-based marker.Figure 4.


Associative transcriptomics study dissects the genetic architecture of seed glucosinolate content in Brassica napus.

Lu G, Harper AL, Trick M, Morgan C, Fraser F, O'Neill C, Bancroft I - DNA Res. (2014)

PCR assay for the 11-bp deletions at BnaC.HAG3b locus. PCR primer combination 260F/490R was used, which produced a 226-bp (haplotype I) or 237-bp (haplotype II) band. Numbers on the left and right sides are fragment length in base pair. M, 100-bp DNA ladder.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263295&req=5

DSU024F5: PCR assay for the 11-bp deletions at BnaC.HAG3b locus. PCR primer combination 260F/490R was used, which produced a 226-bp (haplotype I) or 237-bp (haplotype II) band. Numbers on the left and right sides are fragment length in base pair. M, 100-bp DNA ladder.
Mentions: Another unigene of interest is EX043693 (BnaC.HAG3b), which is located within the peak on C9 which coincides with a region of strong and extensive LD (Fig. 3b). In Arabidopsis, HAG3 (MYB29) is a transcription factor modulating many genes in the GS pathway.65 Expression of BnaC.HAG3b is highly associated with GS accumulation (r = 0.41, P < 10−8) and accounts for 16.8% of trait variation (Supplementary Fig. S10b). However, no SNPs have been detected by mRNA-Seq within this gene. Therefore, genomic regions covering the length of EX043693 were amplified from 70 lines and then sequenced to detect potential DNA sequence variations. By comparing sequences of these DNA fragments, four InDels, i.e. InDel3–1 (3-bp insertion), InDel3–2 (3-bp deletion), InDel7 (7-bp deletion), and InDel1 (1-bp deletion), were detected. These InDels formed two haplotypes: haplotype I includes the 3-bp insertion and 11-bp (i.e. 3 plus 7 plus 1) of deletions, while haplotype II has the sequence identical to the reference unigene EX043693 (Fig. 4). A total number of 60 lines were determined as haplotype I at these loci and 10 lines as haplotype II. The average GS concentration of haplotype I (56.3 µmol g−1) was only 55% of that of haplotype II (the wild type), consistent with the net 8-bp (frame-shift) deletion reducing the functional properties of the encoded protein. To facilitate germplasm screening and marker-assisted selection for the low GS content, a pair of specific PCR primers was designed, which only captures the 11-bp deletions so that the polymorphism can be more easily resolved by agarose gel (Fig. 4). An example of PCR amplification is given in Fig. 5; the haplotype I and II lines can be clearly distinguished by the presence of a 226-bp- and 237-bp-specific fragment, respectively. Thus, the polymorphism at BnaC.HAG3b locus has been successfully converted into a PCR-based marker.Figure 4.

Bottom Line: A weighted gene co-expression network analysis provided additional 40 candidate genes.Resequencing of genomic regions revealed six new SNPs in BnaA.GTR2a and four insertions or deletions in BnaC.HAG3b.These deletion polymorphisms were then successfully converted into polymerase chain reaction-based diagnostic markers that can, due to high linkage disequilibrium observed in these regions of the genome, be used for marker-assisted breeding for low seed GS lines.

View Article: PubMed Central - PubMed

Affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, Heslington, York YO10 5DD, UK Oil Crops Research Institute, CAAS, Wuhan 430062, Hubei, China.

Show MeSH