Universität zu Köln

Mohd Zaidan, Mohd Waznul Adly (2024). Generation And Analysis Of Pro- And Anti-Crossover Meiotic Mutants In Tomato. PhD thesis, Universität zu Köln.

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Abstract

One of the unique characteristics of meiosis is the formation of meiotic crossovers (CO) between homologous chromosomes that leads to the reciprocal exchange of DNA and eventually diverse haploid gametes. Meiotic CO leads to generation of genetic variation and therefore creates diversity at the level of the individual organism and population. Research and knowledge on the fundamental mechanisms of meiotic CO could ultimately expedite the improvement of crop plant varieties and animal breeds. As has been reported previously in many types of organisms, the class I CO pathway is usually responsible for the majority of CO events, while the class II CO pathway is the minor CO pathway. The genetic knock-out of some factors that facilitate non-crossover (NCO) repair have been found to lead to increased activity of the class II CO pathway. Information on the regulation of meiotic CO for both class I CO and NCO are previously extensively described in the model plant, A. thaliana. However, there is a lack of information available in other dicot plants especially those used as a food crops. Therefore, this thesis described the generation of mutants in the class I CO (SlMLH1 and SlZIP4) and NCO repair (SlRECQ4) genes, the role of those genes and their products in promoting and regulating meiotic CO, and also the importance of those genes for the fertility in tomato. CRISPR/Cas9 mutagenesis was applied to produce three different mutants in the dwarf tomato variety, Micro-Tom. Slmlh1 and Slzip4 mutants are associated with reduced activity of the class I CO pathway, while the Slrecq4 mutant is a NCO repair gene. All three mutants exhibited a significant reduction in plant fertility. The average percentage of viable pollen is 1.49%, 13.4% and 37.3% for Slzip4, Slmlh1 and Slrecq4 mutants respectively, when compared to wildtype Micro-Tom (97.6%). This showed that both class I CO genes and NCO genes are essential to ensure tomato fertility probably due to their important functions in DNA repair during meiosis. A lack of meiotic CO between homologous chromosomes leads to univalent formation and unequal segregation at the end of the first meiotic division. Cytological analysis of meiotic chromosomes spreads indicated that reduced fertility in all three mutants was associated with univalent formation, and other meiotic defects. A higher percentage of univalent were observed in the most infertile Slzip4 mutant, whilst the Slrecq4 had the least number of univalent. However, Slrecq4 mutants also exhibit DNA fragmentation during meiosis leading to a further reduction in fertility rate. This fragmentation in Slrecq4 suggests that either too much CO has occurred or the intermediate for Holiday Junction (HJ) is not being fully repaired. Despite the reduced fertility rate of Slrecq4, it can still partially supress class I CO infertility as double mutant of Slmlh1 Slrecq4 and Slzip4 Slrecq4 had a significant improvement on the percentage of viable pollen, fruit number, fruit size and seed set. The number of univalent during diakinesis stage was also reduced significantly in the Slmlh1 Slrecq4 and Slzip4 Slrecq4 double mutants. As similarly observed in A. thaliana recq4 mutant, the Slrecq4 mutant can supress both tomato class I CO mutants suggesting that the elevated CO in Slrecq4 occurs via the class II CO pathway. Another interesting observation is the introgression of the mlh1 mutation in the S. lycopersicum background into the wild tomato species, Solanum pimpinellifolium can significantly increase meiotic CO number and also the percentage of viable pollen. This suggests that there is a potential modifier of meiotic CO rate between the two accessions studied. Overall, it is suggested that meiotic CO and NCO mutants can have different phenotypes depending on the context of other genomic factors. For example, the SlRECQ4 gene was found to be very important not only to regulate meiotic CO but also to ensure normal DNA repair, correct chromosome segregation and also to prevent abnormal gametes formation. In the future, it will be of interest to identify the parity value between CO and NCO regulation in tomato which could be used to stimulate more CO without have negative side effects on the fertility rate. Another interesting point of view is to look further into the genetic control of meiotic CO in wild tomato species which might have interesting genetic modifiers to stimulate higher meiotic CO in tomato breeding.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Mohd Zaidan, Mohd Waznul Adly waznuladly@gmail.com UNSPECIFIED UNSPECIFIED
Corporate Creators:	Max Planck Institute For Plant Breeding Research, Universität zu Köln
URN:	urn:nbn:de:hbz:38-719453
Date:	2024
Publisher:	University of Cologne
Place of Publication:	Cologne
Language:	English
Faculty:	External institution
Divisions:	Außeruniversitäre Forschungseinrichtungen > MPI for Plant Breeding Research
Subjects:	Life sciences Agriculture
Uncontrolled Keywords:	Keywords Language Tomato English Solanum lycopersicum English Meiosis English Meiotic Crossover English Class I Crossover English Cytology English Anti-Crossover English Solanum pimpinellifolium English Fertility English
Date of oral exam:	20 September 2023
Referee:	Name Academic Title Mercier, Raphaël Prof. Underwood, Charles J. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/71945