Quantitative high resolution mapping of HvMLH3 foci in barley pachytene nuclei reveals a strong distal bias and weak interference

Abstract

In barley (Hordeum vulgare L.), chiasmata (the physical sites of genetic crossovers) are skewed towards the distal ends of chromosomes, effectively consigning a large proportion of genes to recombination coldspots. This has the effect of limiting potential genetic variability, and of reducing the efficiency of map-based cloning and breeding approaches for this crop. Shifting the sites of recombination to more proximal chromosome regions by forward and reverse genetic means may be profitable in terms of realizing the genetic potential of the species, but is predicated upon a better understanding of the mechanisms governing the sites of these events, and upon the ability to recognize real changes in recombination patterns. The barley MutL Homologue (HvMLH3), a marker for class I interfering crossovers, has been isolated and a specific antibody has been raised. Immunolocalization of HvMLH3 along with the synaptonemal complex transverse filament protein ZYP1, used in conjunction with fluorescence in situ hybridization (FISH) tagging of specific barley chromosomes, has enabled access to the physical recombination landscape of the barley cultivars Morex and Bowman. Consistent distal localization of HvMLH3 foci throughout the genome, and similar patterns of HvMLH3 foci within bivalents 2H and 3H have been observed. A difference in total numbers of HvMLH3 foci between these two cultivars has been quantified, which is interpreted as representing genotypic variation in class I crossover frequency. Discrepancies between the frequencies of HvMLH3 foci and crossover frequencies derived from linkage analysis point to the existence of at least two crossover pathways in barley. It is also shown that interference of HvMLH3 foci is relatively weak compared with other plant species.

The effect of temperature on the male and female recombination landscape of barley

“…Barley meiocytes at pachytene were embedded in acrylamide in order to preserve their three‐dimensional architecture, exactly as described in Phillips et al . (). The subsequent immunolocalisation and FISH procedure, including probe labelling conditions, were performed as described in Phillips et al .…”

“…Genetic background in barley has been shown to influence CO frequency in both cytological studies (Gale & Rees, ; Phillips et al ., ) and genetic mapping (Bengtsson & Säll, ; Säll, ). Differences in genetic background (together with environmental effects) potentially contribute to the differences in genetic map length found in more recent genome‐wide mapping studies though the general distal distribution of recombination is maintained (Close et al ., ; Mayer et al ., ).…”

The Synaptonemal Complex Protein ZYP1 Is Required for Imposition of Meiotic Crossovers in Barley

“…In barley, it marks late recombination nodules, the future sites of interference-dependent COs (Phillips et al, 2013). The number of MLH3 foci during prophase I in the empty vector control line (15 6 2.4, n = 10) was significantly greater (P < 0.001, 51 df) than the number of pooled MLH3 foci from the ZYP1 RNAi lines (2 6 0.2, n = 42) (Figures 7E and 7J; Supplemental Figure 10 and Supplemental Table 2).…”

“…Immunolocalization and other cytological techniques were performed according to Higgins (2013). Antibodies were used at a concentration of 1:200 including At-ASY1 rat, At-ZYP1 rat, gH2AX rabbit (Millipore), At-RAD51 rabbit, At-MSH4 rabbit, and Hv-MLH3 rabbit (Phillips et al, 2013). For the cytological time course, 10 mM BrdU (0.5 mL) was injected into barley influorescences, which were then fixed in 3:1 ethanol:acetic acid at various time points and detected with BrdU labeling and detection kit I (Roche Diagnostics), as in Higgins (2013).…”

Factors Underlying Restricted Crossover Localization in Barley Meiosis

“…Immunolocalization of MLH3 foci along barley chromosomes at pachytene has revealed that the mean inter-focus distances range from 29.2% to 44.35% of arm length for chromosomes 2H and 3H, with a minimum distance of 6.1% (83). Overall, nearly 40% of the MLH3 foci were separated by less than 20% of arm length.…”