Defining the full tomato NB-LRR resistance gene repertoire using genomic and cDNA RenSeq

Abstract

BackgroundThe availability of draft crop plant genomes allows the prediction of the full complement of genes that encode NB-LRR resistance gene homologs, enabling a more targeted breeding for disease resistance. Recently, we developed the RenSeq method to reannotate the full NB-LRR gene complement in potato and to identify novel sequences that were not picked up by the automated gene prediction software. Here, we established RenSeq on the reference genome of tomato (Solanum lycopersicum) Heinz 1706, using 260 previously identified NB-LRR genes in an updated Solanaceae RenSeq bait library.ResultUsing 250-bp MiSeq reads after RenSeq on genomic DNA of Heinz 1706, we identified 105 novel NB-LRR sequences. Reannotation included the splitting of gene models, combination of partial genes to a longer sequence and closing of assembly gaps. Within the draft S. pimpinellifolium LA1589 genome, RenSeq enabled the annotation of 355 NB-LRR genes. The majority of these are however fragmented, with 5′- and 3′-end located on the edges of separate contigs. Phylogenetic analyses show a high conservation of all NB-LRR classes between Heinz 1706, LA1589 and the potato clone DM, suggesting that all sub-families were already present in the last common ancestor. A phylogenetic comparison to the Arabidopsis thaliana NB-LRR complement verifies the high conservation of the more ancient CCRPW8-type NB-LRRs. Use of RenSeq on cDNA from uninfected and late blight-infected tomato leaves allows the avoidance of sequence analysis of non-expressed paralogues.ConclusionRenSeq is a promising method to facilitate analysis of plant resistance gene complements. The reannotated tomato NB-LRR complements, phylogenetic relationships and chromosomal locations provided in this paper will provide breeders and scientists with a useful tool to identify novel disease resistance traits. cDNA RenSeq enables for the first time next-gen sequencing approaches targeted to this very low-expressed gene family without the need for normalization.

Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing

“…We also observed an expansion of certain clades (e.g. CNL-10) and emergence of a novel clade CNL-17, compared to tomato and potato NLR phylogenetic trees 8,10 .…”

“…RenSeq on genomic DNA from previously unsequenced parents, and from bulked resistant and susceptible genotypes, followed by short-read sequencing, assembly and comparison, enables genetic mapping of NLR-type R genes. It also enables (re)annotation of NLR complements, and identification of transcribed NLRs (cDNA RenSeq 4,8 ). However, large copy numbers and highly repetitive coding sequences impair accurate de novo assembly of complete NLR genes using short reads 4,8 Many Solanum species have been assessed for genetic variation in resistance to P. infestans, but resistance is rare 3 .…”

“…It also enables (re)annotation of NLR complements, and identification of transcribed NLRs (cDNA RenSeq 4,8 ). However, large copy numbers and highly repetitive coding sequences impair accurate de novo assembly of complete NLR genes using short reads 4,8 Many Solanum species have been assessed for genetic variation in resistance to P. infestans, but resistance is rare 3 . The hexaploid S. nigrum is reported as resistant, and S. americanum is a likely diploid ancestor of S. nigrum 9 .…”

“…We identified 14 complete (single ORF, full-length cds; Supplementary Figure 1d) and 10 pseudogenised NLRs (>80% identity >1kb). Mapping of cDNA RenSeq short-read data to these sequences 8,21 provided evidence for the expression of six candidates with a uniform cDNA coverage over the whole sequence (Supplementary Table 5). …”

Fine mapping of the Rpi-rzc1 gene conferring broad-spectrum resistance to potato late blight

“…So far, 14 of them have been shown to belong to the same Coiled Coil -Nucleotide Binding -Leucine -Rich Repeat (CC-NB-LRR) gene family and further nine -to contain NB and LRR domains (Rodewald and Trognitz 2013). More late blight resistance genes are to be discovered since in a single doubled haploid genome DM (S. tuberosum) there are 755 NB-LRR genes annotated (Jupe et al 2013) while in tomato (S. lycopersicum) 326 are found (Andolfo et al 2014). How many of them are involved in interactions with P. infestans remains unknown but the above numbers of potential R genes grow when wild Solanum diversity, frequent polyploidy and heterozygosity are taken into account.…”