Delimitation of the Earliness per se D1 (Eps-D1) flowering gene to a subtelomeric chromosomal deletion in bread wheat (Triticum aestivum)

Highlight The major flowering time genes cloned to date regulate photoperiod and vernalization response. We identified a deletion containing genes regulating earliness per se, which fine tune flowering in hexaploid wheat.

KASP name and primers Sequence

TaMOT1-D1
TaMOT1-D1_KASP1_F TaMOT1  Wheat orthologues were assigned to chromosome arms by homology to chromosome arm sorted survey sequence as described in Materials and Methods. Of the forty genes, eleven genes, TaBradi2g14730, TaBradi2g14460, Bradi2g14440, Bradi2g14400, Bradi2g14380, Bradi2g14370, Bradi2g14340, Bradi2g14310, Bradi2g14290 and Bradi2g14210, and TaBradi2g14190 were all shown to be part of the segment that has several genes deleted from Spark, and Cadenza and they are shown in red colour (Table S2). Twelve of the forty genes had no matches with wheat group one chromosomes and these are shown in blue ( Table 1).
Out of the twelve genes that do not match group 1 wheat chromosomes, five matched the wheat group three chromosomes and these are Bradi2g14070, Bradi2g13870, Bradi2g13820, Bradi2g13810, and Bradi2g13800 (Table S2).
The gene Bradi2g14770 matched group 3 genes but the match on group 1 was on 1DS. The genes Bradi2g14740, Bradi2g14120 and Bradi2g14110, matched homologues on both group 1 and group3 wheat chromosomes (Table S2) and these were not used to define the deletion because amplification from group 3 would not be distinguishable from group1 in the absence of polymorphism that can be used to differentiate the locations. The genes Bradi2g14780, Bradi2g14750, and Bradi2g14440 (Table S2), matched genes on both group1 and group3 chromosomes but none of the three had sequence match with the group 3 D genome chromosome of "Chinese Spring" and hence Bradi2g14440 was used to define the deletion.
The gene Bradi2g14730 matched both group1 and group 3 but when the genes were aligned, the group 1 genes were sufficiently different from the group 3 genes hence primers were designed to be specific to 1DL and this gene was also found to be among the deleted genes ( Fig. 2). Eleven genes outside the 1DL deletion matched group 1 chromosomes only and all these were used to define the deletion (bold black Table S2).  60  1AL_TaELF3  CCGGGTGGTCCCGCACACAGCGCGCACCGCGTCAGAGTCGGCGGCGCGCATCTTCCGGTC 60  TuELF3  CCGGGTGGTCCCGCACACAGCGCGCACCGCGTCAGAGTCGGCGGCGCGCATCTTCCGGTC 60  AtELF3  CCGGGTGGTACCGCACACGGCGCGCACCGCGTCGGAGTCGGCAGCGCGCATCTTCCGGTC 60  1DL_TaELF3  CCGGGTGGTACCGCACACGGCGCGCACCGCGTCGGAGTCGGCAGCGCGCATCTTCCGGTC 60  XBarc62 - GATCCAGATGGAGAGGCAGCAGAACGGCCCGTGAccgagcgaccgcatgcggtgcttggc 120 1AL_TaELF3 GATCCAGATGGAGAGGCAGCAGAACGGCCCGTGAccgagcgaccgcatgcagtggttggc 120 TuELF3 GATCCAGATGGAGAGGCAGCAGAACGGCCCGTGAccgagcgaccgcatgcggtggttggc 120 AtELF3 GATCCAGATGGAGAGGCAGCAGAACGGCCCGTGAcagagcgaccgcatgcggtggttggc 120 1DL_TaELF3 GATCCAGATGGAGAGGCAGCAGAACGGCCCGTGAcagagcgaccgcatgcggtggttggc 120 XBarc62 - codon (TGA). The sequence labelled Xbarc62 is the expressed sequence tag (EST) accession BV211449 used to design the XBarc 62 SSR marker (Song et al., 2005). The PCR primers are underlined and labelled primer 1, Primer 2 (designed to be specific to 1DL) while primer 3 and 4 were designed by Song et al., (2005) and amplify from both 1DL and 1AL (Fig. 1).
The difference between primer 2 and 3 is that primer 3 is shown by the single underline while primer 2 includes the whole of primer 3 and four additional bases (gaag) shown by double underlining which make primer 2 1DL specific (fig1). The D homeologue is 11bp longer than the A and B homeologues in the region between the two black downward arrows (Fig.   1). The start and end of the 'ATCT' SSR that is scored by the XBac62 marker is shown by the upward arrows and also by the dotted underline and the black horizontal bar flanked by the two upward facing arrows.