Redistribution of Meiotic Crossovers Along Wheat Chromosomes by Virus-Induced Gene Silencing.

Amir Raz,Cathy Melamed-Bessudo,Tal Dahan-Meir,Dena Leshkowitz,Avraham A Levy

doi:10.3389/fpls.2020.635139

Amir Raz, Cathy Melamed-Bessudo + Show 3 more

Open Access

https://doi.org/10.3389/fpls.2020.635139

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Abstract

Meiotic recombination is the main driver of genetic diversity in wheat breeding. The rate and location of crossover (CO) events are regulated by genetic and epigenetic factors. In wheat, most COs occur in subtelomeric regions but are rare in centromeric and pericentric areas. The aim of this work was to increase COs in both “hot” and “cold” chromosomal locations. We used Virus-Induced gene Silencing (VIGS) to downregulate the expression of recombination-suppressing genes XRCC2 and FANCM and of epigenetic maintenance genes MET1 and DDM1 during meiosis. VIGS suppresses genes in a dominant, transient and non-transgenic manner, which is convenient in wheat, a hard-to-transform polyploid. F1 hybrids of a cross between two tetraploid lines whose genome was fully sequenced (wild emmer and durum wheat), were infected with a VIGS vector ∼ 2 weeks before meiosis. Recombination was measured in F2 seedlings derived from F1-infected plants and non-infected controls. We found significant up and down-regulation of CO rates along subtelomeric regions as a result of silencing either MET1, DDM1 or XRCC2 during meiosis. In addition, we found up to 93% increase in COs in XRCC2-VIGS treatment in the pericentric regions of some chromosomes. Silencing FANCM showed no effect on CO. Overall, we show that CO distribution was affected by VIGS treatments rather than the total number of COs which did not change. We conclude that transient silencing of specific genes during meiosis can be used as a simple, fast and non-transgenic strategy to improve breeding abilities in specific chromosomal regions.

Highlights

During meiosis, homologous chromosomes pair and exchange DNA segments
Zavitan hybrid plants that underwent Virus-Induced gene Silencing (VIGS) and of negative controls that were treated with an empty vector, we developed a series of InDel markers, that are easy to screen for, through a whole genome comparison of the “Zavitan” and “Svevo” genomes
The transient nature of this method is advantageous over a stable gene silencing or knock out mutation for several reasons: it is nontransgenic and can be applied to any hybrid; it is transient so that if deleterious, the gene silencing effect is constrained in time; it is dominant and enables stacking of genes compared to the lengthy process of recessive mutations and double mutants production; when affecting meiotic recombination its effect is transmitted to the generation

Summary

Introduction

Homologous chromosomes pair and exchange DNA segments. This process, known as homologous recombination (HR), coupled with chromosome pairing, ensures proper segregation, and generates the genetic diversity among gametes. Type I COs are subject to CO interference, a process that regulates the distribution of COs along the chromosome, preventing the formation of multiple CO in close proximity (Copenhaver, 2005; Mercier et al, 2005) This is the most prominent CO pathway in plants (Higgins et al, 2004; Hodzic et al, 2004; Wan et al, 2004; Guillon et al, 2005; Mercier et al, 2005; Lhuissier et al, 2007; Falque et al, 2009). An increase in CO rate by a factor of up to 3.6 was reported in the fancm mutant (Crismani et al, 2012) and a 1.5 and 6.2 fold increase in the top3α and recq4a-recq4b mutants, respectively (Hartung et al, 2007; Séguéla-Arnaud et al, 2015) In these experiments most of the additional COs were of the type II CO pathway. Another anti-CO gene is the RAD51 paralog XRCC2. Serra et al (2013) found a 50% increase in recombination rate in the xrcc Arabidopsis mutant compared to wild type

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