Assessment and identification of molecular markers underpinning for Helicoverpa armigera Hübner resistance in Australian wild Cajanus species

Abstract

The wild relatives of domesticated crops possess enormous genetic variation that is useful to produce resilient crop varieties. Pigeonpea (Cajanus cajan (L) Millsp.) is one such leguminous crop, where potential traits of agronomic importance from wild species have already been used in crop improvement. However, production of this semi-arid crop is greatly hindered by pod borer (Helicoverpa armigera Hubner), causing losses of approximately $3.6 billion/year.Australia represents the second most significant centre of diversity for Cajanus species possessing 15 out of 32 species. The current study included seven wild species, viz; C. acutifolius, C. lanceolatus, C. latisepalus, C. reticulatus, C. scarabaeoides from the secondary genepool and C. lanuginosus and C. pubescens, from the tertiary genepool along with the interspecific hybrids (ISH) of C. acutifolius to investigate insect resistance and to determine the biochemical and molecular basis of the resistance.The first objective of this study was to identify the phenotypic variation in the selected subset of species focusing on 17 qualitative and six quantitative traits. A total of 74 genotypes were characterised, and significant variation including four plant types, seven leaf shapes, three base flower colours, four patterns of streaks, three flowering patterns were observed. Significant variation in stem thickness, days to 50% flowering (dff) (P < 0.001), and raceme number (P < 0.021) within group, plant height, stem thickness, leaf size and dff (P < 0.001) between groups were found.The second objective was to screen the selected species and ISH to H. armigera resistance and identify the specific phenolic compounds responsible for the resistant component. Initially, a subset of 24 genotypes, including only C. acutifolius accessions and its inter specific hybrids (ISH), were screened using detached leaf bioassay. All the wild accessions and the backcross population with wild accession were highly resistant compared to the other genotypes. Though the UPLC analysis of phenolic compounds could not reveal the resistance related component, estimation of total phenolic content (TPC) showed a strong negative correlation (r2= 0.64) between TPCs and the insect survival that was highly significant (P < 0.001). The outcome of higher TPC contributing to host plant resistance (HPR) was reconfirmed by using 17 genotypes comprised of 12 wild accessions and seven domesticated accessions. However, two wild genotypes were identified as susceptible despite having higher TPC. We have also identified an entirely new flavonoid isoorientin as the major compound present in Australian wild species, irrespective of their resistance and susceptibility.The third objective was to identify the specific genetic markers linked to the resistance and high TPC concentrations in wild species. For this, 19 genotypes were subjected to DNA extraction and whole-genome sequencing (Illumina). The extreme phenotype genome wide association (XP-GWAS) method was used to associate polymorphisms with traits (Resistance, TPC). The non-synonymous SNPs between two extreme phenotype bulks for both the traits were subjected to functional analysis. KEGG pathway analysis revealed the presence of SNPs in five candidate pathways, including “Cutin, suberine and wax synthesis”, contributing towards structural defence. Jasmonic acid (JA) synthesis pathway was identified as the pathway separating the resistant from the susceptible group with SNPs at gene loci LOC109800624, LOC109807875, LOC109802084, ACX and MFP2, indicating active JA synthesis and further defence response. Other polymorphic genes include lanosterol synthase at gene loci LOC109808037 (Chr11: 166,722 - 175,493) and “aldehyde dehydrogenase enzyme at gene loci LOC109788000 (Un|NW_017984482.1: 183,541 - 193,224). These genes are involved in the synthesis of lanosterol and juvenile hormone, acting antagonistically with insect survival. Analysis of SNPs associated with TPC led to the identification of a divergent pathway of secondary metabolite synthesis in wild Cajanus species with by “L-rhamnosyltransferase” or UDP-glycosyltransferase” (Un|NW_017990452.1: 36 – 482) enzymes. Our results demonstrated that TPCs could be used as markers for HPR for H. armigera in pigeonpea. Australian native species C. pubescens belonging to tertiary genepool and C. lanceolatus (AGG300129WCAJ1), C. reticulatus (AGG300162WCAJ1), and C. latisepalus (AGG309208WCAJ1) of secondary genepool, demonstrated high to moderate levels of resistance. C. lanceolatus (AGG300129WCAJ1) was highly resistant to H. armigera compared to C. acutifolius. The genotypes backcrossed with C. acutifolius also exhibited high resistance, suggesting that the resistance trait is transferable from the secondary genepool without altering functionality. These findings support the hypothesis that the Australian wild species exhibit enormous variation in phenotype, biochemical and molecular aspects. Hence they are a great source of pod borer resistance in crop improvement programs for producing superior verities.