Chromosome Arm Locations of Barley Sucrose Transporter Gene in Transgenic Winter Wheat Lines

Abstract

Three transgenic HOSUT lines of winter wheat, HOSUT12, HOSUT20, and HOSUT24, each harbor a single copy of the cDNA for the barley sucrose transporter gene HvSUT1 (SUT), which was fused to the barley endosperm-specific Hordein B1 promoter (HO; the HOSUT transgene). Previously, flow cytometry combined with PCR analysis demonstrated that the HOSUT transgene had been integrated into different wheat chromosomes: 7A, 5D, and 4A in HOSUT12, HOSUT20, and HOSUT24, respectively. In order to confirm the chromosomal location of the HOSUT transgene by a cytological approach using wheat aneuploid stocks, we crossed corresponding nullisomic-tetrasomic lines with the three HOSUT lines, namely nullisomic 7A-tetrasomic 7B with HOSUT12, nullisomic 5D-tetrasomic 5B with HOSUT20, and nullisomic 4A-tetrasomic 4B with HOSUT24. We examined the resulting chromosomal constitutions and the presence of the HOSUT transgene in the F₂ progeny by means of chromosome banding and PCR. The chromosome banding patterns of the critical chromosomes in the original HOSUT lines showed no difference from those of the corresponding wild type chromosomes. The presence or absence of the critical chromosomes completely corresponded to the presence or absence of the HOSUT transgene in the F₂ plants. Investigating telocentric chromosomes occurred in the F₂ progeny, which were derived from the respective critical HOSUT chromosomes, we found that the HOSUT transgene was individually integrated on the long arms of chromosomes 4A, 7A, and 5D in the three HOSUT lines. Thus, in this study we verified the chromosomal locations of the transgene, which had previously been determined by flow cytometry, and moreover revealed the chromosome-arm locations of the HOSUT transgene in the HOSUT lines.

Keywords: HOSUT lines; barley sucrose transporter; chromosome banding; nullisomic-tetrasomics; telocentric chromosome; transgene; wheat.

FIGURE 1

Crossing scheme to verify the critical chromosomes carrying the HOSUT transgene (underlined). F₂ plants are expected to have various chromosome constitutions regarding the critical chromosomes. The transgene is expected to be present in the F₂ plants carrying the critical chromosome (HOSUT gene +).

FIGURE 2

Chromosome constitutions of a common wheat cultivar Certo and an F₁ hybrid between N7AT7B and HOSUT12. Chromosomes 4A, 7A, and 5D of Certo are similar to those of Chinese Spring wheat in terms of the C-banding pattern, and there is no wheat chromosome 1B in Certo (A). Sequential C-banding-FISH/GISH shows that Certo is disomic for 1BL.1RS translocation substituting for chromosome 1B. Probes for FISH/GISH are rye total genomic DNA (indicated with pink arrows), pSc 200 sequences (indicated with green arrows), and 18S.26S rDNA (indicated with green arrowheads at the secondary constrictions). (B). In the F₁ hybrid there was only one chromosome 7A from HOSUT12 and three doses of chromosome 7B. (C). Bar = 10 μm.

FIGURE 3

C-banding images (three for each) of chromosomes 7A, 4A, and 5D derived from Certo (upper row) and the HOSUT lines (lower row). Note that there was no obvious structural difference between the normal and transgene-carrying homologous chromosomes.

FIGURE 4

PCR analysis of part of the F₂ progeny from a cross between N7AT7B and HOSUT12. The HvSUT1 marker was not amplified in F₂ plants 3, 7, and 9. Neither of the 7A-specific markers (Owm186 and Owm 190) were amplified in F₂ plants 3 and 9, while Owm186 was amplified in F₂ plant 7, which was identified by C-banding to be monotelosomic 7AS (Supplementary Figure S2). This result suggested that the HvSUT1 marker was located on the long arm of chromosome 7A.

FIGURE 5

PCR analysis of part of the F₂ progeny from a cross between N5DT5B and HOSUT20. The HvSUT1 and two 5D-specific markers (Owm180 and Owm184) were not amplified in F₂ plants 2, 6, and 8. In F₂ plant 4, which was identified by C-banding to be monotelosomic 5DL (Supplementary Figure S4), the HvSUT1 and Owm180 markers were amplified, but Owm184 was not. This result suggested that the HvSUT1 marker was located on the long arm of chromosome 5D.

FIGURE 6

PCR analysis of part of the F₂ progeny from a cross between N4AT4B and HOSUT24. The HvSUT1 and two 4A-specific markers (Owm121 and Owm167) were not amplified in F₂ plants 7 and 9. All three markers were amplified in F₂ plant 3, which was identified by C-banding to be monotelosomic 4AL (Supplementary Figure S6). This result suggested that the HvSUT1 marker was located on the long arm of chromosome 4A.