The chromatin determinants and Ph1 gene effect at wheat sites with contrasting recombination frequency.

INTRODUCTION: Meiotic recombination is one of the most important processes of evolution and adaptation to environmental conditions. Even though there is substantial knowledge about proteins involved in the process, targeting specific DNA loci by the recombination machinery is not well understood. OBJECTIVES: This study aims to investigate a wheat recombination hotspot (H1) in comparison with a "regular" recombination site (Rec7) on the sequence and epigenetic level in conditions with functional and non-functional Ph1 locus. METHODS: The DNA sequence, methylation pattern, and recombination frequency were analyzed for the H1 and Rec7 in three mapping populations derived by crossing introgressive wheat line 8.1 with cv. Chinese Spring (with Ph1 and ph1 alleles) and cv. Tähti. RESULTS: The H1 and Rec7 loci are 1.586 kb and 2.538 kb long, respectively. High-density mapping allowed to delimit the Rec7 and H1 to 19 and 574 bp and 593 and 571 bp CO sites, respectively. A new method (ddPing) allowed screening recombination frequency in almost 66 thousand gametes. The screening revealed a 5.94-fold higher recombination frequency at the H1 compared to the Rec7. The H1 was also found out of the Ph1 control, similarly as gamete distortion. The recombination was strongly affected by larger genomic rearrangements but not by the SNP proximity. Moreover, chromatin markers for open chromatin and DNA hypomethylation were found associated with crossover occurrence except for the CHH methylation. CONCLUSION: Our results, for the first time, allowed study of wheat recombination directly on sequence, shed new light on chromatin landmarks associated with particular recombination sites, and deepened knowledge about role of the Ph1 locus in control of wheat recombination processes. The results are suggesting more than one recombination control pathway. Understanding this phenomenon may become a base for more efficient wheat genome manipulation, gene pool enrichment, breeding, and study processes of recombination itself.

Divergence between bread wheat and Triticum militinae in the powdery mildew resistance QPm.tut-4A locus and its implications for cloning of the resistance gene.

A segment of Triticum militinae chromosome 7G harbors a gene(s) conferring powdery mildew resistance which is effective at both the seedling and the adult plant stages when transferred into bread wheat (T. aestivum). The introgressed segment replaces a piece of wheat chromosome arm 4AL. An analysis of segregating materials generated to positionally clone the gene highlighted that in a plant heterozygous for the introgression segment, only limited recombination occurs between the introgressed region and bread wheat 4A. Nevertheless, 75 genetic markers were successfully placed within the region, thereby confining the gene to a 0.012 cM window along the 4AL arm. In a background lacking the Ph1 locus, the localized rate of recombination was raised 33-fold, enabling the reduction in the length of the region containing the resistance gene to a 480 kbp stretch harboring 12 predicted genes. The substituted segment in the reference sequence of bread wheat cv. Chinese Spring is longer (640 kbp) and harbors 16 genes. A comparison of the segments' sequences revealed a high degree of divergence with respect to both their gene content and nucleotide sequence. Of the 12 T. militinae genes, only four have a homolog in cv. Chinese Spring. Possible candidate genes for the resistance have been identified based on function predicted from their sequence.

The in silico identification and characterization of a bread wheat/Triticum militinae introgression line.

The capacity of the bread wheat (Triticum aestivum) genome to tolerate introgression from related genomes can be exploited for wheat improvement. A resistance to powdery mildew expressed by a derivative of the cross-bread wheat cv. Tähti × T. militinae (Tm) is known to be due to the incorporation of a Tm segment into the long arm of chromosome 4A. Here, a newly developed in silico method termed rearrangement identification and characterization (RICh) has been applied to characterize the introgression. A virtual gene order, assembled using the GenomeZipper approach, was obtained for the native copy of chromosome 4A; it incorporated 570 4A DArTseq markers to produce a zipper comprising 2132 loci. A comparison between the native and introgressed forms of the 4AL chromosome arm showed that the introgressed region is located at the distal part of the arm. The Tm segment, derived from chromosome 7G, harbours 131 homoeologs of the 357 genes present on the corresponding region of Chinese Spring 4AL. The estimated number of Tm genes transferred along with the disease resistance gene was 169. Characterizing the introgression's position, gene content and internal gene order should not only facilitate gene isolation, but may also be informative with respect to chromatin structure and behaviour studies.

Characterization of new allele influencing flowering time in bread wheat introgressed from Triticum militinae.

Flowering time variation was identified within a mapping population of doubled haploid lines developed from a cross between the introgressive line 8.1 and spring bread wheat cv. Tähti. The line 8.1 carried introgressions from tetraploid Triticum militinae in the cv. Tähti genetic background on chromosomes 1A, 2A, 4A, 5A, 7A, 1B and 5B. The most significant QTL for the flowering time variation was identified within the introgressed region on chromosome 5A and its largest effect was associated with the VRN-A1 locus, accounting for up to 70% of phenotypic variance. The allele of T. militinae origin was designated as VRN-A1f-like. The effect of the VRN-A1f-like allele was verified in two other mapping populations. QTL analysis identified that in cv. Tähti and cv. Mooni genetic background, VRN-A1f-like allele incurred a delay of 1.9-18.6 days in flowering time, depending on growing conditions. Sequence comparison of the VRN-A1f-like and VRN-A1a alleles from the parental lines of the mapping populations revealed major mutations in the promoter region as well as in the first intron, including insertion of a MITE element and a large deletion. The sequence variation allowed construction of specific diagnostic PCR markers for VRN-A1f-like allele determination. Identification and quantification of the effect of the VRN-A1f-like allele offers a useful tool for wheat breeding and for studying fine-scale regulation of flowering pathways in wheat.

Fine mapping, phenotypic characterization and validation of non-race-specific resistance to powdery mildew in a wheat-Triticum militinae introgression line.

Introgression of several genomic loci from tetraploid Triticum militinae into bread wheat cv. Tähti has increased resistance of introgression line 8.1 to powdery mildew in seedlings and adult plants. In our previous work, only a major quantitative trait locus (QTL) on chromosome 4AL of the line 8.1 contributed significantly to resistance, whereas QTL on chromosomes 1A, 1B, 2A, 5A and 5B were detected merely on a suggestive level. To verify and characterize all QTLs in the line 8.1, a mapping population of double haploid lines was established. Testing for seedling resistance to 16 different races/mixtures of Blumeria graminis f. sp. tritici revealed four highly significant non-race-specific resistance QTL including the main QTL on chromosome 4AL, and a race-specific QTL on chromosome 5B. The major QTL on chromosome 4AL (QPm.tut-4A) as well as QTL on chromosome 5AL and a newly detected QTL on 7AL were highly effective at the adult stage. The QPm.tut-4A QTL accounts on average for 33-49 % of the variation in resistance in the double haploid population. Interactions between the main QTL QPm.tut-4A and the minor QTL were evaluated and discussed. A population of 98 F(2) plants from a cross of susceptible cv. Chinese Spring and the line 8.1 was created that allowed mapping the QPm.tut-4A locus to the proximal 2.5-cM region of the introgressed segment on chromosome 4AL. The results obtained in this work make it feasible to use QPm.tut-4A in resistance breeding and provide a solid basis for positional cloning of the major QTL.

Adult plant and seedling resistance to powdery mildew in a Triticum aestivum x Triticum militinae hybrid line.

In the progeny of a cross between the common wheat cultivar Tähti and Triticum militinae, a member of the timopheevii group of tetraploid wheats, several hybrid lines were selected that are characterized by improved seedling and adult plant resistance (APR) to powdery mildew. An F2 single-seed descendant mapping population segregating for seedling resistance and APR to powdery mildew was analysed for the identification of quantitative trait loci (QTL). The main QTL responsible for APR was detected on the long arm of chromosome 4A tightly linked to the Xgwm160 locus on a T. militinae translocation explaining up to 54% of phenotypic variance. The same translocation influenced seedling resistance to powdery mildew upon inoculation of plants with a synthetic population of Blumeria graminis DC. f. sp. tritici, and explained 28-33% of the phenotypic variance.