A Cytological Analysis of Wheat Meiosis Targeted by Virus-Induced Gene Silencing (VIGS)

Desjardins, Stuart D.; Kanyuka, K.; Higgins, James D.

doi:10.1007/978-1-4939-9818-0_22

Cited by 9 publications

(13 citation statements)

References 12 publications

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“…The barley stripe mosaic virus, virus induced gene silencing system (BSMV:VIGS) 53 , 54 was used to target expression of TaFANCM in T. aestivum ‘Bobwhite’. Two non-overlapping regions with high predicted silencing efficiency were selected, following an in silico analysis of TaFANCM-A1 cds ( TraesCS4A02G217700 ) with si-Fi21 (siRNA Finder).…”

Section: Methodsmentioning

confidence: 99%

“…The amplicons were cloned into pCa-γbLIC to create a recombinant BSMV RNAγ. The recombinant pCa-γbLIC, as well pCaBS-α and pCaBS-β, were then transformed into electrocompetent Agrobacterium tumefaciens (GV3101), agroinfiltrated, and the virus accumulated in intermediate host Nicotiana benthamiana , prior to inoculation of wheat plants with infected sap at the 4–4.5 leaf stage (~28 days post-sowing)” 54 . For cytological analysis, anthers were dissected and fixed in 3:1 (v/v) ethanol: acetic acid at 14-days post inoculation.…”

Section: Methodsmentioning

confidence: 99%

“…Meiotic chromosome spreads were prepared from fixed anthers. Following digestion (30–60 min at 37 °C) in citrate buffer containing 0.33% cellulase ( Duchefa Biochemie) and 0.33% pectolyase ( MP Biomedicals ), the anthers were macerated in a drop of water, spread in 60–80% acetic acid at 45 °C, and refixed in 3:1 (v/v) ethanol: acetic acid 54 , 56 . Slides were mounted in VECTASHIELD ® Mounting Medium with DAPI (Vector Labaratories).…”

Section: Methodsmentioning

confidence: 99%

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FANCM promotes class I interfering crossovers and suppresses class II non-interfering crossovers in wheat meiosis

et al. 2022

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FANCM suppresses crossovers in plants by unwinding recombination intermediates. In wheat, crossovers are skewed toward the chromosome ends, thus limiting generation of novel allelic combinations. Here, we observe that FANCM maintains the obligate crossover in tetraploid and hexaploid wheat, thus ensuring that every chromosome pair exhibits at least one crossover, by localizing class I crossover protein HEI10 at pachytene. FANCM also suppresses class II crossovers that increased 2.6-fold in fancm msh5 quadruple mutants. These data are consistent with a role for FANCM in second-end capture of class I designated crossover sites, whilst FANCM is also required to promote formation of non-crossovers. In hexaploid wheat, genetic mapping reveals that crossovers increase by 31% in fancm compared to wild type, indicating that fancm could be an effective tool to accelerate breeding. Crossover rate differences in fancm correlate with wild type crossover distributions, suggesting that chromatin may influence the recombination landscape in similar ways in both wild type and fancm.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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FANCM promotes class I interfering crossovers and suppresses class II non-interfering crossovers in wheat meiosis

et al. 2022

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“…Chromosome spreads were performed as described previously (Higgins, 2013;Desjardins et al, 2020). Nikon Ni-E and Eclipse Ci fluorescence microscopes equipped with NIS elements software were used to image chromosomes.…”

Section: Cytological Proceduresmentioning

confidence: 99%

MutS homologue 4 and MutS homologue 5 Maintain the Obligate Crossover in Wheat Despite Stepwise Gene Loss following Polyploidization

et al. 2020

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Crossovers (COs) ensure accurate chromosome segregation during meiosis while creating novel allelic combinations. Here, we show that allotetraploid (AABB) durum wheat (Triticum turgidum ssp. durum) utilizes two pathways of meiotic recombination. The class I pathway requires MSH4 and MSH5 (MutSg) to maintain the obligate CO/chiasma and accounts for ;85% of meiotic COs, whereas the residual ;15% are consistent with the class II CO pathway. Class I and class II chiasmata are skewed toward the chromosome ends, but class II chiasmata are significantly more distal than class I chiasmata. Chiasma distribution does not reflect the abundance of double-strand breaks, detected by proxy as RAD51 foci at leptotene, but only ;2.3% of these sites mature into chiasmata. MutSg maintains the obligate chiasma despite a 5.4-kb deletion in MSH5B rendering it nonfunctional, which occurred early in the evolution of tetraploid wheat and was then domesticated into hexaploid (AABBDD) common wheat (Triticum aestivum), as well as an 8-kb deletion in MSH4D in hexaploid wheat, predicted to create a nonfunctional pseudogene. Stepwise loss of MSH5B and MSH4D following hybridization and whole-genome duplication may have occurred due to gene redundancy (as functional copies of MSH5A, MSH4A, and MSH4B are still present in the tetraploid and MSH5A, MSH5D, MSH4A, and MSH4B are present in the hexaploid) or as an adaptation to modulate recombination in allopolyploid wheat.

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“…This was successfully used in wheat for both basic and applicative researches ( Bennypaul et al, 2012 ; Lee et al, 2015 ). Moreover, VIGS treatment were successfully applied to manipulate meiotic-specific processes in wheat and Arabidopsis ( Bhullar et al, 2014 ; Calvo-Baltanás et al, 2020 ; Desjardins et al, 2020b ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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A Cytological Analysis of Wheat Meiosis Targeted by Virus-Induced Gene Silencing (VIGS)

Abstract: The output can be accessed at: https://repository.rothamsted.ac.uk/item/96x11/acytological-analysis-of-wheat-meiosis-targeted-by-virus-induced-gene-silencing-vigs.

Cited by 9 publications

References 12 publications

FANCM promotes class I interfering crossovers and suppresses class II non-interfering crossovers in wheat meiosis

FANCM promotes class I interfering crossovers and suppresses class II non-interfering crossovers in wheat meiosis

MutS homologue 4 and MutS homologue 5 Maintain the Obligate Crossover in Wheat Despite Stepwise Gene Loss following Polyploidization

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

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