Physical mapping of wheat-Aegilops longissimabreakpoints in mildew-resistant recombinant lines using FISH with highly repeated and low-copy DNA probes

Abstract

Fluorescence in situ hybridization (FISH) with multiple probes, consisting of highly repeated DNA sequences (pSc119.2 and pAs1) and of a low-copy, 3BS-specific RFLP sequence (PSR907), enabled determination of the physical position of the wheat-alien breakpoints (BPs) along the 3BS and 3DS arms of common wheat recombinant lines. These lines harbour 3SlS Aegilops longissima segments containing the powdery mildew resistance gene Pm13. In all 3B recombinants, the wheat-Aegilops longissima physical BPs lie within the interval separating the two most distal of the three pSc119.2 3BS sites. In all such recombinants a telomeric segment, containing the most distal of the pSc119.2 3BS sites, was in fact replaced by a homoeologous Ae. longissima segment, marked by characteristic pSc119.2 hybridization sites. Employment of the PSR907 RFLP probe as a FISH marker allowed to resolve further the critical region in the various 3B recombinant lines. Three of them, like the control common wheat, exhibited between the two most distal pSc119.2 sites a single PSR907 FISH site, which was missing in a fourth recombinant line. The amount of alien chromatin can thus be estimated to represent around 20% of the recombinant arm in the three former lines and a maximum of 27% in the latter. A similar physical length was calculated for the alien segment contained in three 3D recombinants, all characterized by the presence of the Ae. longissima pSc119.2 sites distal to the nearly telomeric pAs1 sites of normal 3DS. Comparison between the FISH-based maps and previously developed RFLP maps of the 3BS-3SlS and 3DS-3SlS arms revealed substantial differences between physical and genetic map positions of the wheat-alien BPs and of molecular markers associated with the critical chromosomal portions.Key words: wheat-alien recombinants, chromosome engineering, fluorescence in situ hybridization, highly repeated and low-copy DNA probes, physical versus genetic maps.

Wheat Genomics: Present Status and Future Prospects

“…FISH with some repeated sequences as probes was used for identification of individual chromosomes [ 107 – 110 ]. FISH was also utilized to physically map rRNA multigene family [ 111 , 112 ], RFLP markers [ 110 , 113 ], and unique sequences [ 114 – 116 ] and also for detecting and locating alien chromatin introgressed into wheat [ 117 – 119 ].…”

Wheat powdery mildew resistance gene Pm13 encodes a mixed lineage kinase domain-like protein

“…The donors of chromosomes 3S l #1 in R1B, 3S l #2 in TA3575, and 3S l #3 in TA7545 were derived accordingly from the Ae . longissima accessions TL01, TL20 and TA1910 (TAM4) 82 – 84 . The powdery mildew-susceptible wheat cultivar Fielder was used for the transformation of Pm13 .…”

Engineered Durum Wheat Germplasm with Multiple Alien Introgressions: Agronomic and Quality Performance

“…The Ae. longissima 3S l S segment contains Pm13, a highly effective resistance gene to powdery mildew, and replaces around 20% of the 3BS arm [26,52,53]. Finally, the 1DS chromosome segment derived from T. aestivum, harbours the Gli-D1/Glu-D3 storage protein genes, and replaces 17% of durum wheat 1AS arm, containing the Gli-A1/Glu-A3 homoeoloci [26,54,55].…”