Periodic Reporting for period 2 - MEIOBARMIX (Meiosis in barley: Mixing it up)
Período documentado: 2022-08-01 hasta 2024-01-31
To assure Food Security in the future, rapid crop improvements are required. Traditional plant breeding relying on natural genetic variation that arises during meiosis together with genetic engineering will play key roles.
Natural genetic variation harnessed during breeding is primarily achieved during meiosis through homologous recombination (HR). During HR, programmed meiotic DNA double strand breaks (DSBs) are either repaired on the sister chromatid as a non-crossover (NCO), leaving no genetic trace, or they are repaired on the homologous chromosome, that can result in the formation of a crossover (CO) leading to reciprocal genetic exchange between parental homologous chromosomes and thus new allelic combinations. In plants including barley (Hordeum vulgare) CO-repair results in a limited number of CO that are skewed towards chromosome ends. Hence, large portions of the genome (in particular interstitial chromosome regions) are kept untapped during breeding. Therefore, new strategies and tools are needed to modify meiotic HR outcome. Notably, non-CO repair on the homologous chromosome can result in a so-called NCO gene conversion (NCO-GC). In this case, a non-reciprocal exchange of short DNA patches between alleles occurs leading to novel alleles. Interestingly, most meiotic DSBs (>90%) are repaired as a NCO and NCO-GCs are typically not considered in breeding practices as little is known about their tract length or frequency.
MEIOBARMIX aims to identify new strategies and to establish novel tools to modify meiotic HR outcome in plants in order to accelerate plant breeding. To determine NCO-GC frequency and tract length as potential natural source of genetic variation and to isolate components regulating their formation, genetic and genotyping tools are employed (aim 1). Targeted meiotic recombination approaches to trigger locally HR at pre-defined genomic sites are explored in Arabidopsis thaliana and subsequently tested in barley (aim 2). Novel methodologies are developed in barley that enable modifying (virus-based knockdown or knockout of meiotic candidates and application of ‘stresses’ during reproduction) and assessing (single pollen nucleus genotyping) meiotic recombination outcome and to transfer the knowledge acquired in Arabidopsis (aim 3).
In a nutshell, deciphering strategies to target, manipulate and harness meiotic HR outcome will benefit plant breeding progress to assure food security in the near future.
Natural genetic variation harnessed during breeding is primarily achieved during meiosis through homologous recombination (HR). During HR, programmed meiotic DNA double strand breaks (DSBs) are either repaired on the sister chromatid as a non-crossover (NCO), leaving no genetic trace, or they are repaired on the homologous chromosome, that can result in the formation of a crossover (CO) leading to reciprocal genetic exchange between parental homologous chromosomes and thus new allelic combinations. In plants including barley (Hordeum vulgare) CO-repair results in a limited number of CO that are skewed towards chromosome ends. Hence, large portions of the genome (in particular interstitial chromosome regions) are kept untapped during breeding. Therefore, new strategies and tools are needed to modify meiotic HR outcome. Notably, non-CO repair on the homologous chromosome can result in a so-called NCO gene conversion (NCO-GC). In this case, a non-reciprocal exchange of short DNA patches between alleles occurs leading to novel alleles. Interestingly, most meiotic DSBs (>90%) are repaired as a NCO and NCO-GCs are typically not considered in breeding practices as little is known about their tract length or frequency.
MEIOBARMIX aims to identify new strategies and to establish novel tools to modify meiotic HR outcome in plants in order to accelerate plant breeding. To determine NCO-GC frequency and tract length as potential natural source of genetic variation and to isolate components regulating their formation, genetic and genotyping tools are employed (aim 1). Targeted meiotic recombination approaches to trigger locally HR at pre-defined genomic sites are explored in Arabidopsis thaliana and subsequently tested in barley (aim 2). Novel methodologies are developed in barley that enable modifying (virus-based knockdown or knockout of meiotic candidates and application of ‘stresses’ during reproduction) and assessing (single pollen nucleus genotyping) meiotic recombination outcome and to transfer the knowledge acquired in Arabidopsis (aim 3).
In a nutshell, deciphering strategies to target, manipulate and harness meiotic HR outcome will benefit plant breeding progress to assure food security in the near future.
Aim 1: Dissection of local HR frequency/outcome and identification of players involved.
Approaches to dissect local outcome and frequency of HR events (CO vs NCO-GC) including the tract length of NCO-GCs were established. In Arabidopsis a GC tract length of a few bp up to the kbp range was found. At a given locus the frequency of NCO-GCs by far exceeds CO numbers, suggesting a large number of meiotic DSBs being repaired as NCO-GC representing a yet untapped source of natural genetic variation. Further systems to dissect local HR outcome both in an inbred and hybrid background (local sequence diversity) are being explored. The impact of genetic factors on local HR outcome is being dissected and efforts are made to dissect local HR outcome/frequency based on sequencing approaches.
Aim 2 (WP 2): Dissection of the repair outcome of targeted site-specific meiotic DSBs.
Different strategies based on genome editing tools are being explored to test whether targeted site-specific meiotic DSBs can serve as template for HR and if they are repaired as CO or as NCO-GC. Mutant complementation and no negative impact in a WT background were found, suggesting all targeted recombination tools being functional. Moreover, guide (g)RNAs target sites addressing different genomic environments were selected. Finally, nearly 40 constructs were transformed in materials generated within the first aim and are being analyzed for repair outcome at target sites.
Aim 3 (WP 3): Impact of ‘stresses’ and genetic factors on the barley recombination landscape.
To measure meiotic recombination rates in pollen without the need of growing segregating populations, single pollen nucleus genotyping has been extended to multiple linked genetic intervals. Virus-induced genome editing in Cas9-expressing plants (BSMVIGE) was developed for barley, i.e. mutants can be rapidly isolated without the need of stable genetic transformation. A workflow based on virus-induced gene silencing (VIGS) or application of ‘stresses’ together with single pollen nucleus genotyping was established in barley to identify genotype- and GM-independent methods for breeding practices that can alter meiotic recombination rates. Initial results revealed an impact of selected factors on the barley recombination landscape. BSMVIGE is used to rapidly generate edited barley plants.
Approaches to dissect local outcome and frequency of HR events (CO vs NCO-GC) including the tract length of NCO-GCs were established. In Arabidopsis a GC tract length of a few bp up to the kbp range was found. At a given locus the frequency of NCO-GCs by far exceeds CO numbers, suggesting a large number of meiotic DSBs being repaired as NCO-GC representing a yet untapped source of natural genetic variation. Further systems to dissect local HR outcome both in an inbred and hybrid background (local sequence diversity) are being explored. The impact of genetic factors on local HR outcome is being dissected and efforts are made to dissect local HR outcome/frequency based on sequencing approaches.
Aim 2 (WP 2): Dissection of the repair outcome of targeted site-specific meiotic DSBs.
Different strategies based on genome editing tools are being explored to test whether targeted site-specific meiotic DSBs can serve as template for HR and if they are repaired as CO or as NCO-GC. Mutant complementation and no negative impact in a WT background were found, suggesting all targeted recombination tools being functional. Moreover, guide (g)RNAs target sites addressing different genomic environments were selected. Finally, nearly 40 constructs were transformed in materials generated within the first aim and are being analyzed for repair outcome at target sites.
Aim 3 (WP 3): Impact of ‘stresses’ and genetic factors on the barley recombination landscape.
To measure meiotic recombination rates in pollen without the need of growing segregating populations, single pollen nucleus genotyping has been extended to multiple linked genetic intervals. Virus-induced genome editing in Cas9-expressing plants (BSMVIGE) was developed for barley, i.e. mutants can be rapidly isolated without the need of stable genetic transformation. A workflow based on virus-induced gene silencing (VIGS) or application of ‘stresses’ together with single pollen nucleus genotyping was established in barley to identify genotype- and GM-independent methods for breeding practices that can alter meiotic recombination rates. Initial results revealed an impact of selected factors on the barley recombination landscape. BSMVIGE is used to rapidly generate edited barley plants.
To develop new and superior varieties, plant breeders are dependent on novel genetic variations. These arise through homologous recombination during meiosis in breeding materials. However, in plants including cereal crops such as barley limited and skewed meiotic recombination events represent a bottleneck in breeding programs. MEIOBARMIX aims to deliver novel insights on local homologous recombination frequency and outcome combined with the application of novel approaches and tools to modify and assess meiotic recombination outcome. Therefore, they key impact of MEIOBARMIX will be:
Detection methods to decipher locally meiotic recombination outcome including frequency and tract lengths of gene conversions and identification of players involved in their formation as well as strategies for targeted meiotic recombination will allow to break into uncharted territory with direct implications for plant breeding. In addition, new virus-based tools, ‘stresses’ and methods to readily detect HR frequency and outcome in pollen will offer new ways to be implemented in barley breeding programs as well as they will enable to modify naturally limited and skewed distribution of meiotic recombination in barley. Deciphering methodologies and approaches to modify the recombination landscape in barley, may ultimately pave the way to enable similar modifications of meiotic recombination in (closely) related crops such as wheat, rye, oat, maize or even rice with a similarly skewed heterogeneous distribution of CO events due to high conservation of the underlying meiotic program.
Detection methods to decipher locally meiotic recombination outcome including frequency and tract lengths of gene conversions and identification of players involved in their formation as well as strategies for targeted meiotic recombination will allow to break into uncharted territory with direct implications for plant breeding. In addition, new virus-based tools, ‘stresses’ and methods to readily detect HR frequency and outcome in pollen will offer new ways to be implemented in barley breeding programs as well as they will enable to modify naturally limited and skewed distribution of meiotic recombination in barley. Deciphering methodologies and approaches to modify the recombination landscape in barley, may ultimately pave the way to enable similar modifications of meiotic recombination in (closely) related crops such as wheat, rye, oat, maize or even rice with a similarly skewed heterogeneous distribution of CO events due to high conservation of the underlying meiotic program.