Genetic and biological analysis of root lesion nematode (Pratylenchus thornei) resistance loci in wheat

Abstract

The root lesion nematode Pratylenchus thornei feeds on roots of wheat (Triticum aestivum) plants, causing significant damage to the roots at the cellular level, resulting in yield reduction. In a previous study, P. thornei resistance QTL, QRlnt.sk-6D and QRlnt.sk-2B were identified in a Sokoll/Krichauff wheat DH population. The current project was undertaken with the aim to dissect the genetic and biological basis of this resistance. To better define the genetic basis of resistance, both resistance loci were fine mapped using the Sokoll/Krichauff DH population and six newly developed RIL populations. Bulked segregation analysis with the 90K Wheat SNP array identified linked SNPs, which were subsequently converted to KASP assays for mapping in the DH and RIL populations. QRlnt.sk-6D was delimited to a 3.5 cM interval, representing 1.77 Mbp in the bread wheat cv. Chinese Spring reference genome sequence and 2.29 Mbp in the Ae. tauschii genome sequence. These intervals contained 42 and 43 gene models in the respective annotated genome sequences. QRlnt.sk-2B was delimited to 1.4 cM, corresponding 3.14 Mbp in the durum wheat cv. Svevo reference sequence and 2.19 Mbp in Chinese Spring. The interval in Chinese Spring contained 56 high confidence gene models. Intervals for both QTL contained genes with similarity to those previously reported to be involved in disease resistance, namely genes for phenylpropanoid-biosynthetic-pathway-related enzymes, NBS-LRR proteins and protein kinases. The potential roles of these candidate genes in P. thornei resistance are discussed. The KASP markers reported in this study could potentially be used for marker assisted breeding of P. thornei resistant wheat cultivars. To quantify P. thornei from wheat root, a qPCR-based assay was developed. A standard curve was produced to quantify P. thornei from wheat root samples. The standard curve was validated by estimating P. thornei from sixteen wheat lines with known levels of resistance. Overall, the assay was 2.4-fold less expensive compared to the commercial service (PreDicta B test, SARDI). The DNA extraction protocol was inexpensive as it works without using a commercial DNA extraction kit. In order to identify metabolites associated with resistance loci, the GC-MS based metabolic profiles of root exudates and root tissues from the resistant lines were compared with the susceptible lines. In root exudates, 21 metabolites were found to be associated with resistance QTL. Likewise, from root tissue, 15 metabolites were found to be associated with the resistance QTL. These metabolites were derived from diverse biochemical groups, including amino acids and amines, organic acids, sugars, sugar alcohols and sugar phosphates. The possible roles of these resistance compounds in P. thornei resistance is largely unknown. However, their nematotoxic properties against other plant parasitic nematodes were discussed. In response to P. thornei infection, the histological and histochemical responses of wheat roots were investigated. The use of the fluorescent dye PKH26 (for P. thornei labelling) and confocal microscopy enabled visualisation of live P. thornei both out and inside wheat root tissue. In response to P. thornei infection, secondary cell wall thickening (deposition of cellulose, callose, lignin and suberin) was observed in the P. thornei resistant cultivar, Sokoll. Secondary cell wall thickening might result in physical reinforcement of the cell wall restricting P. thornei migration in the resistant root tissues.