Factors underlying restricted crossover localization in barley meiosis

Meiotic recombination results in the formation of cytological structures known as chiasmata at the sites of genetic crossovers (COs). The formation of at least one chiasma/CO between homologous chromosome pairs is essential for accurate chromosome segregation at the first meiotic division as well as for generating genetic variation. Although DNA double-strand breaks, which initiate recombination, are widely distributed along the chromosomes, this is not necessarily reflected in the chiasma distribution. In many species there is a tendency for chiasmata to be distributed in favored regions along the chromosomes, whereas in others, such as barley and some other grasses, chiasma localization is extremely pronounced. Localization of chiasma to the distal regions of barley chromosomes restricts the genetic variation available to breeders. Studies reviewed herein are beginning to provide an explanation for chiasma localization in barley. Moreover, they suggest a potential route to manipulating chiasma distribution that could be of value to plant breeders

Co-localization analysis between ASY1 and PRD3-HA in wild-type or ASY1 and SPO11-1-MYC foci in wild type and <i>prd3</i> at leptotene.

Co-localization between PRD3-HA and ASY1 in wild-type or ASY1 and SPO11-1-MYC foci in wild-type and prd3 were quantified and the percentage of foci co-localizing or not co-localizing are reported. A Mann-Whitney-Wilcoxon test was performed to test for significance. (DOCX)</p

Amino acid length and [S/T]Q sites of proteins involved in DSB formation.

(DOCX)</p

SPO11-1-MYC foci count at leptotene in DSB defective mutant lines.

ASY1 and SPO11-1-MYC were immunostained in DSB mutant lines. ASY1 staining was used to determine the meiotic stage and to count SPO11-1-MYC foci on nuclei at a comparable stage. (DOCX)</p

γH2AX and RAD51 foci count in Col and <i>asy3/+</i> male meiosis at leptotene.

ASY1 was immunostained with γH2AX or RAD51 in Col and asy3-1/+ male meiosis at letptotene stage. ASY1 staining was used to determine the meiotic stage and to count γH2AX or RAD51 foci on nuclei at a comparable stage. A Mann-Whitney-Wilcoxon test was performed to test for significance. (DOCX)</p

List of proteins identified in the ASY1 co-immunoprecipitation experiment with a known role in meiosis.

(DOCX)</p

Spatial and temporal localisation of PRD3 in early prophase I of meiosis.

(A) Localisation of ASY1 (red) and PRD3-HA (green) or SPO11-1-MYC (green) on wild type (Col), PRD3-HA prd3-3 and SPO11-1-MYC spo11-1-3 male meiocytes at leptotene and pachytene stages. White dashed lines represent the cross-sections used for the representative co-localisation analysis in Fig 2C. (B) Close-up representative images showing the differential co-localisation of PRD3-HA (green) or SPO11-1-MYC (green), in relation to ASY1 (red). (C) Representative co-localisation analysis of ASY1 and PRD3-HA or SPO11-1-MYC from the cross-sections of the cells represented with dashed lines in Fig 2A. (D) Localisation of SMC3 (green) and PRD3-HA (red) in asy3 at leptotene stage. (E) Plot showing the degree of overlap analysis of PRD3-HA foci with ASY1 or SPO11-1-MYC foci with ASY1 in wild type (Col) or prd3. To test for significance, images of PRD3-HA or SPO11-1-MYC were rotated 180 degrees and the degree of co-localisation with ASY1 was calculated (therein called “random of PRD3-HA” and “random of SPO11-1-MYC”). Black dots represent individual measurements, and red dots represent mean values. Mann-Whitney-Wilcoxon tests were performed to test for statistical difference. “*” indicates a statistically significant difference. (F) Plot showing the counts of PRD3-HA foci in wild type (Col) and asy3 in early prophase I. Black dots represent individual measurements, and red dots represent mean values. A Mann-Whitney-Wilcoxon test was used to test for statistical difference. “*” indicates a statistically significant difference. Scale bars = 10μM.</p

SPO11-1-MYC localisation in wild type and meiotic DSB defective mutants.

(A) Localisation of RAD51 or SPO11-1-MYC (red) with ASY1 (green) on wild type (Col) male meiocytes, from leptotene to pachytene stages. Scale bars = 10μM. (B) Localisation of SPO11-1-MYC (red) and ASY1 (green) in wild type (Col), prd1, prd2, prd3, spo11-2 and mtopVIb, during early prophase I. Scale bars = 10μM. (C) Plot showing RAD51 and SPO11-1-MYC foci counts in wild type (Col) at the leptotene (Lep), zygotene (Zyg) and pachytene (Pac) stages of meiosis. Black dots represent individual measurements, and red dots represent mean values. (D) Plot showing SPO11-1-MYC foci counts in wild type (Col) and DSB mutants in early prophase I. Black dots represent individual measurements, and red dots represent mean values. A Mann-Whitney-Wilcoxon test was used to compare SPO11-1-MYC foci count between wild type (Col) and mutants. “*” indicates a statistically significant difference. “n.s.” indicates a non-statistically significant difference.</p

Primers used to clone <i>ASY1</i>, <i>ASY3</i>, <i>PRD3</i> and <i>PCH2</i> into protoplast transient expression vectors.

(DOCX)</p

Co-localization analysis between ASY1 and randomised PRD3-HA or SPO11-1-MYC foci at leptotene.

Image of PRD3-HA and SPO11-1-MYC were rotated 180 degrees to test for random overlap between PRD3-HA and ASY1 or SPO11-1-MYC and ASY1. Co-localization between random PRD3-HA and ASY1 or random SPO11-1-MYC foci and ASY1 axis were quantified and the frequency of foci co-localizing or not colocalizing are reported. A Mann-Whitney-Wilcoxon test was performed to test for significance. (DOCX)</p