The Evolution Of Resistance Against Tobacco Mosaic Virus In Nicotiana Species And Fine-Mapping Of A Major Gene Controlling Leaf Shape In Lettuce

The interaction between tobacco mosaic virus（TMV）and Nicotiana has been a model system to study plant-pathogen interactions.The N gene in cultivated tobacco was introgressed from Nicotiana glutinosa.The N gene is alternatively spliced,producing two transcripts named N_S and N_L.The alternative exon in N_L transcript is derived from a transposable element in intron III.The TMV resistance genes in N.paniculata and N.undulata were mapped to the N locus,and were likely homologues of the N gene.However,the reactions of most Nicotiana species against TMV remain unclear and the origin and evolution of TMV resistance in Nicotiana has not been studied.In this study,molecular approaches were applied to clone TMV resistance genes in Nicotiana and to study their evolution.Screening 40 Nicotiana species identified 10 TMV resistant ones which scattered all over the phylogenetic tree for Nicotiana species.Unlike the N-encoded TMV resistance,TMV resistance in some species such as N.goodspeedii is temperature insensitive,and it provides novel breeding resources for disease resistance.The TMV resistance genes in these 10 species recognize two different avirulence proteins,helicase domain（p50）of TMV replicase or coat protein（CP）.Transient expression assay showed that the N orthologue Np from N.paniculata was the TMV resistance gene,recognizing helicase p50 as N gene does.However,the Np and N genes have only 93.9%nucleotide identity and exhibit diversifying selection at the hypersensitive sites in LRR region.The sequences of the N orthologues showed that none of the N orthologues investigated in this study have the functionally indispensible alternative exon of the N gene.Therefore,the alternative exon of the N gene,and its consequent TMV resistance function was originated after the speciation of N.glutinosa.The gene Nw302471-N5 in N.wigandioides is the N orthologue most similar to the N gene（96%）,but it does not encode TMV resistance.The Ka/Ks ratios between the hypersensitive sites of N gene and eight N orthologues were higher than one,showing diversifying selection after speciation.TMV resistance in at least four Nicotiana species is conferred by the N’gene family,encoding CC-NBS-LRR domains and recognizing TMV coat protein.The resistance against TMV-Cg in some Nicotiana species is also conferred by N’orthologues.The N’orthologues encoding resistance against TMV-U1 also encode resistance against TMV-Cg,but not vice versa.The N’orthologues in some species（e.g.,N.amplexicaulis）confer resistance to TMV-Cg only.The results from N’orthologues with single nucleotide subsitution and chimeras between different N’orthologues indicate that the differential resistance of N’orthologues was caused by accumulated point mutations in different sites of LRR region rather than single point mutation.There are several studies on leaf-shape related genes in model plant Arabidopsis.The ratio between leaf length and width is controlled by the two-dimensional cell division and expansion.Most of the leaf-shape related genes were cloned by sreening mutants or map-based cloing from segregating population of mutants.Similarly,most of the leaf-shape related genes in rice were cloned by map-based cloing from segregating population of mutants.Studies on the QTL genes controlling leaf shape are still at early stage such as prilimary genetic mapping.Lactuca sativa var.ramosa Hort.and L.sativa var.longifoliaf.Lam are two popular cultivars of Lactuca sativa,with considerable leaf shape difference.In our study,a loose leaf cultivar S1（with obovate leaves）was crossed with a var.longifoliaf cultivar Y37（with acuminate tips）,and the F₁ was selfed to generate a F₂ segregating population.RNA-seq was performed for the extreme round-leaf pool and extreme acuminate-leaf pool from the F₂population.The RNA-seq data suggest5 loci controlling leaf shape,a major one on chromosomes 5,and four minor ones on chromosomes 1,4,7 and 9.Individuals,which are heterozygous at the detected locus on chromosome 5 were chosen using molecular markers and selfed to find a single gene segregating populations.The major gene was mapped to a 1.2 Mb interval.Expression construct was prepared for one candidate gene Ls RTFL12 and its transformation and function analysis are underway.