Figure 1.
Sequence architecture of ZmLOX4.
Similar to the ZmLOX5 gene ZmLOX4 consists of 9 exons (shown in red), and 8 introns (shown in green). The major difference between the architecture of the two genes is the length of the second intron, much larger in ZmLOX4 spanning 11,191 bp (not shown). ZmLOX4 is located on the forward strand of chromosome 1:264,209,651–264,226,078, spanning 16,427 bp (B73 RefGen_v2). Vertical blue lines in the final 3′ exon of the gene are the relative locations of SNPs discovered from Sanger sequencing. Their base pair change, derived state (as compared to Z. perennis), and derived state percentages are shown in the grey boxes.
Figure 2.
Sequence architecture of ZmLOX5.
ZmLOX5 consists of 9 exons (shown in red), and 8 introns (shown in green). ZmLOX5 has a shorter second intron, spanning 511 bp. ZmLOX5 is located on the reverse strand of chromosome 5:12,274,159–12,279,067, spanning 4908 bp (B73 RefGen_V2). Vertical blue lines in the final 3′ exon of the gene are the relative locations of SNPs discovered from Sanger sequencing. An InDel of 28 bp was found in the inbred line Va99 and located in the final exon of ZmLOX5, shown here in light blue. Their base pair change, derived state (as compared to Z. perennis), and derived state percentages are shown in the grey boxes.
Table 1.
SNP locations per B73 RefGen_v2 and their derived state percentages.
Figure 3.
Southern blot of lines that did not PCR amplify for ZmLOX5.
B73, confirmed to have a functioning ZmLOX5, is used as a control for the Southern blot. Five of the inbred lines screened show double banding: Yu796_NS, 4226, I29, HP301 and CI 187-2 and are suspected to have two copies of ZmLOX5. One is also missing a band: CML 247, and is suspected to have no copy of ZmLOX5.
Figure 4.
BamHI cut of ZmLOX5 probe to check for a cut site within the probe.
To rule out a BamHI cut site in the ZmLOX5 probe used for Southern blotting, the probe was PCR amplified and then cut with BamHI. Shown here is an agarose gel with the untreated control (left) and BamHI treated (right) samples of four of the lines that showed two bands in the Southern blot. There is only one band in the BamHI treated lanes, showing that there is no cut site within the probe.
Figure 5.
LD patterns in the C-terminus exon of ZmLOX4 (left) and ZmLOX5 (right).
LD plots of SNPs found via Sanger sequencing (outlined in Figures 1 and 2, and Table 1) based on ZmLOX4 with 6 SNPs and 1 InDel, spanning a total of 663 bp in the final 3′ exon across 260 diverse inbred lines, and ZmLOX5 with 14 SNPs, spanning 709 bp across 203 diverse inbred lines. Both show a rapid (<100 bp) decay of LD (r2>0.1).
Figure 6.
LD patterns across the whole locus of ZmLOX4 (left) and ZmLOX5 (right).
LD plots of SNPs across the entire gene from the Maize HapMap in ZmLOX4 (left) and ZmLOX5 (right) Were based on 45 SNPs in ZmLOX4 spanning 15,756 bp across the 27 NAM parents, and 91 SNPs in ZmLOX5, spanning 4884 bp across the 27 NAM parents. Again, we see a rapid (<100 bp) decay of LD (r2>0.1) though some moderate LD spans 250–300 bp.
Figure 7.
LD plot containing 10 members of the ZmLOX gene family.
The LD plot of all SNPs from the Maize HapMap with 10 members of the ZmLOX gene family (ZmLOX2, ZmLOX4, ZmLOX5, ZmLOX6, ZmLOX7, ZmLOX8, ZmLOX9, ZmLOX10, ZmLOX11, and ZmLOX12) ordered numerically from left to right shows little LD across all of the ZmLOXs except for ZmLOX12, which is located in the lower right-hand corner.
Figure 8.
LD plot containing ZmLOX12 and the 100 kb flanking the gene both upstream and downstream.
The LD plot of SNPs from the Maize HapMap ZmLOX12, and the flaking 100 kb on either side of the gene shows that LD at this locus is very strong and extends farther than expected in maize. Approximately 3,000 bp across the ZmLOX12 gene and extends beyond to approximately 19,000 bp. Further investigation of this locus revealed that there is a predicted gene of unknown function located very near (<100 bp) to ZmLOX12.
Table 2.
Gene specific PCR primers and expected amplicon sizes.