Biology subjects

Cobo, N.

Publications and source records attributed to Cobo, N..

2 recordsLinked to original sources

bioRxiv2019

Identification of a dominant chlorosis phenotype through a forward screen of the Triticum turgidum cv. Kronos TILLING population

Durum wheat (Triticum turgidum) derives from a hybridization event approximately 400,000 years ago which led to the creation of an allotetraploid genome. Unlike with more ancient whole genome duplications, the evolutionary recent origin of durum wheat means that its genome has not yet been fully diploidised. As a result, many of the genes present in the durum genome act in a redundant fashion, meaning that, in many cases, loss-of-function mutations must be present in both gene copies to observe a phenotypic effect. This redundancy has hindered the use of forward genetic screens in durum wheat. Here we use a novel set of induced variation within the cv. Kronos TILLING population to identify a locus controlling a dominant, environmentally-dependent chlorosis phenotype. We carried out a forward screen of the sequenced cv. Kronos TILLING lines for senescence phenotypes and identified a single line with a dominant early senescence and chlorosis phenotype. Mutant plants contained overall less chlorophyll throughout their development and displayed premature flag leaf senescence. A segregating population was classified into discrete phenotypic groups and subjected to bulked-segregant analysis using exome capture followed by next-generation sequencing. This allowed the identification of a single region on chromosome 3A, Yellow Early Senescence 1 (YES-1), which was associated with the mutant phenotype. To obtain further SNPs for fine-mapping, we isolated chromosome 3A using flow sorting and sequenced the entire chromosome. By mapping these reads against both the cv. Chinese Spring reference sequence and the cv. Kronos assembly, we could identify high-quality, novel EMS-induced SNPs in non-coding regions within YES-1 that were previously missed in the exome capture data. This allowed us to fine-map YES-1 to 4.3 Mb, containing 59 genes. Our study shows that populations containing induced variation can be sources of novel dominant variation in polyploid crop species, highlighting their importance in future genetic screens. We also demonstrate the value of using cultivar-specific genome assemblies alongside the gold-standard reference genomes particularly when working with non-coding regions of the genome. Further fine-mapping of the YES-1 locus will be needed to identify the causal SNP underpinning this dominant, environmentally dependent phenotype.

plant biology↗

bioRxiv2019

Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence

BackgroundNAC transcription factors contain five highly conserved subdomains which are required for protein dimerisation and DNA binding. Few residues within these subdomains have been identified as essential for protein function, and fewer still have been shown to be of biological relevance in planta. Here we use a positive regulator of senescence in wheat, NAM-A1, to test the impact of missense mutations at specific, highly conserved residues of the NAC domain on protein function.\n\nResultsWe identified missense mutations in five highly conserved residues of the NAC domain of NAM-A1 in a tetraploid TILLING population. TILLING lines containing these mutations, alongside synonymous and non-conserved mutation controls, were grown under glasshouse conditions and scored for senescence. Four of the five mutations showed a significant and consistent delay in peduncle senescence but had no consistent effects on flag leaf senescence. All four mutant alleles with the delayed senescence phenotype also lost the ability to interact with the homoeolog NAM-B1 in a yeast two-hybrid assay. Two of these residues were previously shown to be involved in NAC domain function in Arabidopsis, suggesting conservation of residue function between species. Three of these four alleles led to an attenuated cell death response compared to wild-type NAM-A1 when transiently over-expressed in Nicotiana benthamiana. One of these mutations was further tested under field conditions, in which there was a significant and consistent delay in both peduncle and leaf senescence.\n\nConclusionsWe combined field and glasshouse studies of a series of mutant alleles with biochemical analyses to identify four residues of the NAC domain which are required for NAM-A1 function and protein interaction. We show that mutations in these residues lead to a gradient of phenotypes, raising the possibility of developing allelic series of mutations for traits of agronomic importance. We also show that mutations in NAM-A1 more severely impact peduncle senescence, compared to the more commonly studied flag leaf senescence, highlighting this as an area deserving of further study. The results from this integrated approach provide strong evidence that conserved residues within the functional domains of NAC transcription factors have biological significance in planta.

plant biology↗