Generation And Analysis Of Pro- And Anti-Crossover Meiotic Mutants In Tomato

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

Microbiology and outcomes of polymicrobial peritonitis associated with peritoneal dialysis: a register-based cohort study from the French Language Peritoneal Dialysis Registry

International audienceBACKGROUND: Previous studies have reported that polymicrobial peritonitis in peritoneal dialysis (PD) is associated with poor outcomes, but recent data from European cohorts are scarce. METHODS: We included from the French Language Peritoneal Dialysis Registry all patients ≥18 years of age who started PD between January 2014 and November 2020. We compared microbiology and patient characteristics associated with mono- and polymicrobial peritonitis. We assessed patient outcomes after a first polymicrobial peritonitis using survival analysis with competing events. We differentiated microorganisms isolated from dialysis effluent as enteric or non-enteric pathogens. RESULTS: A total of 8848 patients contributed 13 023 patient-years of follow-up and 3348 culture-positive peritonitis episodes, including 251 polymicrobial ones. This corresponded to rates of 0.32 and 0.02 episodes/patient-year, respectively. For most patients (72%) who experienced polymicrobial peritonitis, this was their first peritonitis episode. Enteric pathogens were more frequently isolated in polymicrobial than in monomicrobial peritonitis (57 versus 44%; P < .001). In both cases of peritonitis with and without enteric pathogens, the polymicrobial versus monomicrobial character of the peritonitis was not associated with mortality in patients who did not switch to haemodialysis {adjusted cause-specific hazard ratio [acsHR] 1.2 [95% confidence interval (CI) 0.3-5.0], P = .78 and 1.1 [95% CI 0.7-1.8], P = .73, respectively}. However, the risks of death and switch to haemodialysis were higher for monomicrobial peritonitis with enteric pathogens compared with those without [acsHR 1.3 (95% CI 1.1-1.7), P = .02 and 1.9 (95% CI 1.5-2.4), P < .0001, respectively]. CONCLUSION: Isolation of enteric pathogens, rather than the polymicrobial character of the peritonitis, is associated with poorer outcomes

Reconstruction of Curcuma aeruginosa secondary metabolite biosynthetic pathway using omics data

Curcuma aeruginosa or temu hitam is herbaceous plant with high therapeutic values in its rhizome that is widely used in traditional medicine. However, molecular studies on the secondary metabolite biosynthetic pathway of C. aeruginosa is still limited. Hence, the aim of this study was to explore and reconstruct the secondary metabolite biosynthetic pathway of C. aeruginosa rhizome by integrating the metabolite profiling and transcriptomic data. A total of 81 metabolites were identified in the rhizome of C. aeruginosa; amongst others are curzerene and β-Cubebene which are potent antioxidants. A total of 28,225 unigene were obtained from the transcriptomic sequencing of C. aeruginosa rhizome and analysed to identify potential genes associated with the biosynthesis of its metabolites. Of these, 43 unigenes were identified and mapped onto five sub-pathways; i.e. carotenoid biosynthetic pathway, diterpenoid biosynthetic pathway, monoterpenoid biosynthetic pathway, terpenoid and steroid biosynthetic pathway, and sesquiterpenoid and triterpenoid biosynthetic pathway. This study demonstrated a systematic bioinformatic approach to reconstruct a metabolic pathway in the rhizome of C. aeruginosa using bioinformatic approach

A chromosome scale tomato genome built from complementary PacBio and Nanopore sequences alone reveals extensive linkage drag during breeding

The assembly and scaffolding of plant crop genomes facilitate the characterization of genetically diverse cultivated and wild germplasm. The cultivated tomato (Solanum lycopersicum) has been improved through the introgression of genetic material from related wild species, including resistance to pandemic strains of tobacco mosaic virus (TMV) from Solanum peruvianum. Here we applied PacBio HiFi and ONT Nanopore sequencing to develop independent, highly contiguous and complementary assemblies of an inbred TMV-resistant tomato variety. We show specific examples of how HiFi and ONT datasets can complement one another to improve assembly contiguity. We merged the HiFi and ONT assemblies to generate a long-read-only assembly where all 12 chromosomes were represented as 12 contiguous sequences (N50 = 68.5 Mbp). This chromosome scale assembly did not require scaffolding using an orthogonal data type. The merged assembly was validated by chromosome conformation capture data and is highly consistent with previous tomato genome assemblies that made use of genetic maps and Hi-C for scaffolding. Our long-read-only assembly reveals that a complex series of structural variants linked to the TMV resistance gene likely contributed to linkage drag of a 64.1-Mbp region of the S. peruvianum genome during tomato breeding. Through marker studies and ONT-based comprehensive haplotyping we show that this minimal introgression region is present in six cultivated tomato hybrid varieties developed in three commercial breeding programs. Our results suggest that complementary long read technologies can facilitate the rapid generation of near-complete genome sequences