Microsatellites reveal genetic diversity in Rotylenchulus reniformis populations.
ABSTRACT: Rotylenchulus reniformis is the predominant parasitic nematode of cotton in the Mid South area of the United States. Although variable levels of infection and morphological differences have been reported for this nematode, genetic variability has been more elusive. We developed microsatellite-enriched libraries for R. reniformis, produced 1152 clones, assembled 694 contigs, detected 783 simple sequence repeats (SSR) and designed 192 SSR-markers. The markers were tested on six R. reniformis cultures from four states, Texas, Louisiana, Mississippi and Georgia, in the USA. Based on performance we selected 156 SSR markers for R. reniformis from which 88 were polymorphic across the six reniform nematode populations, showing as the most frequent motif the dinucleotide AG. The polymorphic information content of the markers ranged from 0.00 to 0.82, and the percentage of multiallelic loci of the isolates was between 40.9 and 45.1%. An interesting finding in this study was the genetic variability detected among the three Mississippi isolates, for which 22 SSR markers were polymorphic. We also tested the level of infection of these isolates on six cotton genotypes, where significant differences were found between the Texas and Georgia isolates. Coincidentally, 62 polymorphic markers were able to distinguish these two populations. Further studies will be necessary to establish possible connections, if any, between markers and level of pathogenicity of the nematode. The SSR markers developed here will be useful in the assessment of the genetic diversity of this nematode, could assist in management practices for control of reniform nematode, be used in breeding programs for crop resistance, and help in detecting the origin and spread of this nematode in the United States.
Project description:<h4>Background</h4>Reniform nematode (Rotylenchulus reniformis) has emerged as one of the most destructive root pathogens of upland cotton (Gossypium hirsutum) in the United States. Management of R. reniformis has been hindered by the lack of resistant G. hirsutum cultivars; however, resistance has been frequently identified in germplasm accessions from the G. arboreum collection. To determine the genetic basis of reniform nematode resistance, a genome-wide association study (GWAS) was performed using 246 G. arboreum germplasm accessions that were genotyped with 7220 single nucleotide polymorphic (SNP) sequence markers generated from genotyping-by-sequencing.<h4>Results</h4>Fifteen SNPs representing 12 genomic loci distributed over eight chromosomes showed association with reniform nematode resistance. For 14 SNPs, major alleles were shown to be associated with resistance. From the 15 significantly associated SNPs, 146 genes containing or physically close to these loci were identified as putative reniform nematode resistance candidate genes. These genes are involved in a broad range of biological pathways, including plant innate immunity, transcriptional regulation, and redox reaction that may have a role in the expression of resistance. Eighteen of these genes corresponded to differentially expressed genes identified from G. hirsutum in response to reniform nematode infection.<h4>Conclusions</h4>The identification of multiple genomic loci associated with reniform nematode resistance would indicate that the G. arboreum collection is a significant resource of novel resistance genes. The significantly associated markers identified from this GWAS can be used for the development of molecular tools for breeding improved reniform nematode resistant upland cotton with resistance introgressed from G. arboreum. Additionally, a greater understanding of the molecular mechanisms of reniform nematode resistance can be determined through genetic structure and functional analyses of candidate genes, which will aid in the pyramiding of multiple resistance genes.
Project description:The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.
Project description:The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) is a semi-endoparasitic nematode that is a pathogen of numerous major crops such as cotton and soybean. Here, the authors present transcriptome assemblies of the egg, second-stage juvenile (J2), J3, vermiform adult, and sedentary female life stages of this important plant pathogen.The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) is a semi-endoparasitic nematode that is a pathogen of numerous major crops such as cotton and soybean. Here, the authors present transcriptome assemblies of the egg, second-stage juvenile (J2), J3, vermiform adult, and sedentary female life stages of this important plant pathogen.
Project description:Reniform nematode (Rotylenchulus reniformis) is a major pest of cotton in the southeastern United States. The objective of this study was to examine the variation of reniform nematode populations from cotton-growing locations in the United States where it is prevalent. Multivariate analysis of variance and discriminant analysis were used to determine the variability of morphology in males and immature females. Reproduction indices of populations were measured on selected soybean and cotton genotypes in the greenhouse. High variability in morphometrics and reproduction was observed within all the populations, and several differences were found among populations. DNA sequences of the nuclear ribosomal first internal transcribed spacer region (ITS1) were compared among populations from the United States and to sequences of populations from Brazil, Colombia, Honduras, and Japan. No polymorphic nucleotide sites were observed among the amphimictic populations. Only a parthenogenic population from Japan was distinct. The phenotypic polymorphism of the species in the United States could impact the effectiveness of management strategies based on host plant resistance.
Project description:The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.
Project description:Genetic and physical framework mapping in cotton (Gossypium spp.) were used to discover putative gene sequences involved in resistance to common soil-borne pathogens. Chromosome (Chr) 11 and its homoeologous Chr 21 of Upland cotton (G. hirsutum) are foci for discovery of resistance (R) or pathogen-induced R (PR) genes underlying QTLs involved in response to root-knot nematode (Meloidogyne incognita), reniform nematode (Rotylenchulus reniformis), Fusarium wilt (Fusarium oxysporum f.sp. vasinfectum), Verticillium wilt (Verticillium dahliae), and black root rot (Thielaviopsis basicola). Simple sequence repeat (SSR) markers and bacterial artificial chromosome (BAC) clones from a BAC library developed from the Upland cotton Acala Maxxa were mapped on Chr 11 and Chr 21. DNA sequence through Gene Ontology (GO) of 99 of 256 Chr 11 and 109 of 239 Chr 21 previously mapped SSRs revealed response elements to internal and external stimulus, stress, signaling process, and cell death. The reconciliation between genetic and physical mapping of gene annotations from new DNA sequences of 20 BAC clones revealed 467 (Chr 11) and 285 (Chr 21) G. hirsutum putative coding sequences, plus 146 (Chr 11) and 98 (Chr 21) predicted genes. GO functional profiling of Unigenes uncovered genes involved in different metabolic functions and stress response elements (SRE). Our results revealed that Chrs 11 and 21 harbor resistance gene rich genomic regions. Sequence comparisons with the ancestral diploid D5 (G. raimondii), A2 (G. arboreum) and domesticated tetraploid TM-1 AD1 (G. hirsutum) genomes revealed abundance of transposable elements and confirmed the richness of resistance gene motifs in these chromosomes. The sequence information of SSR markers and BAC clones and the genetic mapping of BAC clones provide enhanced genetic and physical frameworks of resistance gene-rich regions of the cotton genome, thereby aiding discovery of R and PR genes and breeding for resistance to cotton diseases.
Project description:U.S. cotton production is suffering from the yield loss caused by the reniform nematode (RN), Rotylenchulus reniformis. Management of this devastating pest is of utmost importance because, no upland cotton cultivar exhibits adequate resistance to RN. Nine populations of RN from distinct regions in Alabama and one population from Mississippi were studied and thirteen morphometric features were measured on 20 male and 20 female nematodes from each population. Highly correlated variables (positive) in female and male RN morphometric parameters were observed for body length (L) and distance of vulva from the lip region (V) (r = 0.7) and tail length (TL) and c' (r = 0.8), respectively. The first and second principal components for the female and male populations showed distinct clustering into three groups. These results show pattern of sub-groups within the RN populations in Alabama. A one-way ANOVA on female and male RN populations showed significant differences (p ≤ 0.05) among the variables. Multiple sequence alignment (MSA) of 18S rRNA sequences (421) showed lengths of 653 bp. Sites within the aligned sequences were conserved (53%), parsimony-informative (17%), singletons (28%), and indels (2%), respectively. Neighbor-Joining analysis showed intra and inter-nematodal variations within the populations as clone sequences from different nematodes irrespective of the sex of nematode isolate clustered together. Morphologically, the three groups (I, II and III) could not be distinctly associated with the molecular data from the 18S rRNA sequences. The three groups may be identified as being non-geographically contiguous.
Project description:Soybean cyst nematode (SCN, Heterodera glycine Ichinohe), southern root-knot nematode [SRKN, Meloidogyne incognita (Kofoid and White) Chitwood] and reniform nematode (RN, Rotylenchulus reniformis Linford and Oliveira) are three important plant-parasitic pests in soybean. Previous study showed that plant introduction (PI) 567516C harbored novel quantitative trait loci (QTL) conferring SCN resistance to soybean. However, QTL underlying resistance to SRKN and RN in PI 567516C remain unknown. The objectives of this study were to identify QTL for resistance to SRKN and RN in PI 567516C. Two hundred and forty-seven F6:9 recombinant inbred lines, derived from a cross between cultivar Magellan and PI 567516C, were evaluated for resistance to SRKN and RN. Two hundred and thirty-eight simple sequence repeats and 687 single nucleotide polymorphism markers were used to construct a genetic linkage map. Three significant QTL associated with resistance to SRKN were mapped on chromosomes (Chrs.) 10, 13 and 17. Two significant QTL associated with resistance to RN were detected on Chrs. 11 and 18. Whole-genome resequencing revealed that there might be Peking-type Rhg1 in PI 567516C. Our study provides useful information to employ PI 567516C in soybean breeding in order to develop new cultivars with resistance to multiple nematodes.
Project description:The biotrophic parasitic fungus Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, a devastating disease of wheat, endangering global food security. Because the Pst population is highly dynamic, it is difficult to develop wheat cultivars with durable and highly effective resistance. Simple sequence repeats (SSRs) are widely used as molecular markers in genetic studies to determine population structure in many organisms. However, only a small number of SSR markers have been developed for Pst. In this study, a total of 4,792 SSR loci were identified using the whole genome sequences of six isolates from different regions of the world, with a marker density of one SSR per 22.95 kb. The majority of the SSRs were di- and tri-nucleotide repeats. A database containing 1,113 SSR markers were established. Through in silico comparison, the previously reported SSR markers were found mainly in exons, whereas the SSR markers in the database were mostly in intergenic regions. Furthermore, 105 polymorphic SSR markers were confirmed in silico by their identical positions and nucleotide variations with INDELs identified among the six isolates. When 104 in silico polymorphic SSR markers were used to genotype 21 Pst isolates, 84 produced the target bands, and 82 of them were polymorphic and revealed the genetic relationships among the isolates. The results show that whole genome re-sequencing of multiple isolates provides an ideal resource for developing SSR markers, and the newly developed SSR markers are useful for genetic and population studies of the wheat stripe rust fungus.
Project description:The reniform nematode, Rotylenchulus reniformis, is a sedentary semi-endoparasitic species with a host range that encompasses more than 77 plant families. Nematode effector proteins containing plant-ligand motifs similar to CLAVATA3/ESR (CLE) peptides have been identified in the Heterodera, Globodera, and Meloidogyne genera of sedentary endoparasites. Here, we describe the isolation, sequence analysis, and spatiotemporal expression of three R. reniformis genes encoding putative CLE motifs named Rr-cle-1, Rr-cle-2, and Rr-cle-3. The Rr-cle cDNAs showed >98% identity with each other and the predicted peptides were identical with the exception of a short stretch of residues at the carboxy(C)-terminus of the variable domain (VD). Each RrCLE peptide possessed an amino-terminal signal peptide for secretion and a single C-terminal CLE motif that was most similar to Heterodera CLE motifs. Aligning the Rr-cle cDNAs with their corresponding genomic sequences showed three exons with an intron separating the signal peptide from the VD and a second intron separating the VD from the CLE motif. An alignment of the RrCLE1 peptide with Heterodera glycines and Heterodera schachtii CLE proteins revealed a high level of homology within the VD region associated with regulating in planta trafficking of the processed CLE peptide. Quantitative RT-PCR (qRT-PCR) showed similar expression profiles for each Rr-cle transcript across the R. reniformis life-cycle with the greatest transcript abundance being in sedentary parasitic female nematodes. In situ hybridization showed specific Rr-cle expression within the dorsal esophageal gland cell of sedentary parasitic females.