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Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, …


Biology Articles » Botany » Plant Pathology » A physical map of the heterozygous grapevine 'Cabernet Sauvignon' allows mapping candidate genes for disease resistance » Background

Background
- A physical map of the heterozygous grapevine 'Cabernet Sauvignon' allows mapping candidate genes for disease resistance

Physical maps built from large-insert BAC clones and anchored to linkage maps assist sequence assembly in whole genome shotgun (WGS) sequencing projects [1], enable positional cloning of genes/QTLs and structural studies on gene families [2,3], and facilitate the isolation of homologous genes from model plants in heterozygous or polyploid species [4].

In plants, physical maps have been constructed for Arabidopsis thaliana [5], sorghum [6], rice [7], soybean [8], apple [9], black cottonwood [10], and grapevine [11]. Strategies based on BAC fingerprints detect overlaps among BAC clones for the development of physical maps. Briefly, BAC clones are digested with restriction enzymes and the fragments are separated by electrophoresis, producing a pattern of bands. The overlap between adjacent clones is identified by pairwise comparison of band profiles and calculation of the proportion of shared bands [12]. Technologies for producing BAC fingerprints have evolved rapidly, from using one to five restriction enzymes, and moving from agarose-based gel to sequencer-based electrophoresis to generate the profiles [13]. The methods that combine the use of several restriction enzymes and acrylamide gels or sequencers are usually referred to as High Information Content Fingerprinting (HICF), because they have allowed a dramatic increase in the sensitivity of the process. Since all of these methodologies were not applied to the same biological materials, the performances are not homogeneously comparable, and the debate on the advantages and disadvantages of the different protocols still persists [14]. For instance, it appears that the five-enzyme restriction protocol developed by [13] leads to a higher error rate per fingerprint, but provides the highest sensitivity compared with alternative techniques based on two or three-enzyme restrictions [15]. In the present work, the five-enzyme restriction protocol was adopted because of its higher sensitivity and throughput.

The grapevine genome has recently disclosed two peculiarities: grapes are highly heterozygous and they have descended from an ancient hexaploid ancestor [1,16,17]. The polyploid origin of the grapevine genome was revealed by whole proteome comparison [1] but was undetectable using STS markers or nucleotide alignments. Thus homeologous regions are not expected to hamper the construction of a physical map of the grapevine genome, as their respective fingerprints are substantially different. In turn, heterozygosity is likely to affect the correct assembly of BAC fingerprints, as it did in the DNA assembly of Ciona savignyi [18]. This aspect was addressed in poplar by [10], but was somewhat neglected in grapevine [11] and in apple [9]. Here, a thorough analysis of the effects of heterozygosity on physical map construction is presented that unveils contig features that were not previously described in poplar. It is also shown that the final map is an effective tool for mapping candidate genes for agronomic traits like disease resistance, as well as for developing new genetic markers.

Strengthening the resistance to diseases is one of the major objectives in grapevine breeding [19]. Two types of defence can be categorized in plants, based on the width of the host range. Non-host resistance is effective across an entire plant taxon against all isolates of a pathogen. Host resistance, the second type of resistance, is exerted at a genotype-to-genotype level: only some of the genotypes of a plant taxon to which a pathogen has adapted are resistant to any or all pathogenic strains. This classification agrees well with that based on the type of mechanisms and genes involved. Pre-invasion barriers and reactions triggered by pathogen-associated molecular patterns (PAMPs), called PAMP-triggered immunity (PTI), disrupt the potential ability of a pathogen to attack a plant. Overall, the concepts of non-host resistance and PTI overlap. Host resistance is a second line of defence towards pathogens that have gained the capability of suppressing basal or PAMP-triggered resistance. Host resistance can be either complete or partial. The mechanisms underlying complete resistance are similar across many types of plants. Complete host resistance is conferred by R proteins that recognise pathogen effectors/suppressors or modifications of their cellular targets (effector triggered immunity, ETI). R genes are mostly arrayed in clusters, a physical organisation that generates new variants at a rate higher than in any other class of genes [20,21]. The links between ETI, PTI, complete host, and non-host resistance were modelled by [22]. Both PTI and ETI rely first on pathogen recognition carried out by receptors, consisting of transmembrane proteins for PTI [23,24], and cytoplasmic proteins with a nucleotide-binding site and a leucine-rich repeat domain (NBS-LRR) or receptor-like kinases (RLK) for ETI [25]. These two sides of the immune system are connected by proteins of the downstream signalling pathways, such as SGT1 and RAR1 [26-28], and by gene products of the salicilic acid (SA), jasmonic acid (JA), ethylene (ET), and MAPK cascade pathways [29].

Non-host resistance is the outcome of a heterogeneous set of genes, and the proteins they specify, that are implicated in pathogen accessibility/inaccessibility (i.e. lipase-like EDS1, synthaxin-like PEN1, etc.), cytoskeletal rearrangements and protein turnover (i.e. SGT1), PAMP-triggered responses, and synthesis of toxic metabolites [30-32]. The location of their homologues in the grapevine genome was recently established using information from the grapevine genome sequence [1,17], which was unknown when this study began. In contrast, the genes triggering complete host resistance all conform to a few classes of receptor-coding genes that are functionally similar. Clues on the size and the genomic organisation of R gene families in grape were given by genetic map data [2,33,34] and by a survey in the draft genome sequence [17].

In this paper, we present (1) the assembly of a 'Cabernet Sauvignon' physical map based on restriction enzyme BAC fingerprinting, (2) the anchorage of the physical contigs on the meiotic linkage maps, (3) the use of this map for the placement of candidate genes for disease resistance, and (4) the alignment between the physical location of resistance gene analogues and phenotypic loci for pathogen resistance based on bridging markers. How the high heterozygosity in the 'Cabernet Sauvignon' genome has shaped some features of the physical map is also studied in detail and discussed.


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