Acala NemX Research Articles

Root-Knot Nematode Resistance in Gossypium hirsutum Determined by a Constitutive Defense-Response Transcriptional Program Avoiding a Fitness Penalty.

Cotton (Gossypium spp.) is the most important renewable source of natural textile fiber and one of the most cultivated crops around the world. Plant-parasitic nematode infestations, such as the southern Root-Knot Nematode (RKN) Meloidogyne incognita, represent a threat to cotton production worldwide. Host-plant resistance is a highly effective strategy to manage RKN; however, the underlying molecular mechanisms of RKN-resistance remain largely unknown. In this study, we harness the differences in RKN-resistance between a susceptible (Acala SJ-2, SJ2), a moderately resistant (Upland Wild Mexico Jack Jones, WMJJ), and a resistant (Acala NemX) cotton entries, to perform genome-wide comparative analysis of the root transcriptional response to M. incognita infection. RNA-seq data suggest that RKN-resistance is determined by a constitutive state of defense transcriptional behavior that prevails in the roots of the NemX cultivar. Gene ontology and protein homology analyses indicate that the root transcriptional landscape in response to RKN-infection is enriched for responses related to jasmonic and salicylic acid, two key phytohormones in plant defense responses. These responses are constitutively activated in NemX and correlate with elevated levels of these two hormones while avoiding a fitness penalty. We show that the expression of cotton genes coding for disease resistance and receptor proteins linked to RKN-resistance and perception in plants, is enhanced in the roots of RKN-resistant NemX. Members of the later gene families, located in the confidence interval of a previously identified QTL associated with RKN resistance, represent promising candidates that might facilitate introduction of RKN-resistance into valuable commercial varieties of cotton. Our study provides novel insights into the molecular mechanisms that underlie RKN resistance in cotton.

Sequence Composition of Bacterial Chromosome Clones in a Transgressive Root-Knot Nematode Resistance Chromosome Region in Tetraploid Cotton.

Plants evolve innate immunity including resistance genes to defend against pest and pathogen attack. Our previous studies in cotton (Gossypium spp.) revealed that one telomeric segment on chromosome (Chr) 11 in G. hirsutum cv. Acala NemX (rkn1 locus) contributed to transgressive resistance to the plant parasitic nematode Meloidogyne incognita, but the highly homologous segment on homoeologous Chr 21 had no resistance contribution. To better understand the resistance mechanism, a bacterial chromosome (BAC) library of Acala N901 (Acala NemX resistance source) was used to select, sequence, and analyze BAC clones associated with SSR markers in the complex rkn1 resistance region. Sequence alignment with the susceptible G. hirsutum cv. TM-1 genome indicated that 23 BACs mapped to TM-1-Chr11 and 18 BACs mapped to TM-1-Chr 21. Genetic and physical mapping confirmed less BAC sequence (53–84%) mapped with the TM-1 genome in the rkn1 region on Chr 11 than to the homologous region (>89%) on Chr 21. A 3.1-cM genetic distance between the rkn1 flanking markers CIR316 and CIR069 was mapped in a Pima S-7 × Acala NemX RIL population with a physical distance ∼1 Mbp in TM-1. NCBI Blast and Gene annotation indicated that both Chr 11 and Chr 21 harbor resistance gene-rich cluster regions, but more multiple homologous copies of Resistance (R) proteins and of adjacent transposable elements (TE) are present within Chr 11 than within Chr 21. (CC)-NB-LRR type R proteins were found in the rkn1 region close to CIR316, and (TIR)-NB-LRR type R proteins were identified in another resistance rich region 10 cM from CIR 316 (∼3.1 Mbp in the TM-1 genome). The identified unique insertion/deletion in NB-ARC domain, different copies of LRR domain, multiple copies or duplication of R proteins, adjacent protein kinases, or TE in the rkn1 region on Chr 11 might be major factors contributing to complex recombination and transgressive resistance.

Registration of BARBREN-713 Germplasm Line of Upland Cotton Resistant to Reniform and Root-Knot Nematodes

Cotton (Gossypium hirsutum L.) germplasm line BARBREN-713 (Reg. No. GP-987, PI 671965) was developed and released by the USDA–ARS, Mississippi Agricultural and Forestry Experiment Station, Texas A&M AgriLife Research, and Cotton Incorporated in 2012. The objective of the release was to provide public and private breeders with an agronomically desirable germplasm that is resistant to both the reniform nematode (Rotylenchulus reniformis Linford and Oliveira) and the root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood]. The line also has excellent seedling vigor in fields infested with nematodes and fungal root rot pathogens, such as Thielaviopsis basicola, Rhizoctonia solani and Fusarium spp. Resistance to reniform nematode was transferred from G. barbadense GB713 (PI 608139) and is associated primarily with the Ren2GB713 gene on chromosome 21. Resistance to root-knot nematode was transferred from the germplasm line M-315 RNR (PI 592514), ‘LA 887’, or ‘Acala Nem-X’ and is associated primarily with the Mi-1 gene on chromosome 11. The codominant simple sequence repeat markers, BNL 3279_105 and CIR 316_202, respectively, were closely linked to the resistance genes. A single nucleotide polymorphism marker, Gl-187401, also was developed to detect the Ren2GB713 gene. In controlled environment assays, BARBREN-713 suppressed reproduction of both nematodes by 90% or more. It also reduced reniform nematode populations in the field at eight locations in four states. The line has fiber quality similar to M-315 RNR but yielded more than M-315 RNR in reniform nematode-infested fields.

Mapping Fusarium wilt race 1 resistance genes in cotton by inheritance, QTL and sequencing composition

Knowledge of the inheritance of disease resistance and genomic regions housing resistance (R) genes is essential to prevent expanding pathogen threats such as Fusarium wilt [Fusarium oxysporum f.sp. vasinfectum (FOV) Atk. Sny & Hans] in cotton (Gossypium spp.). We conducted a comprehensive study combining conventional inheritance, genetic and quantitative trait loci (QTL) mapping, QTL marker-sequence composition, and genome sequencing to examine the distribution, structure and organization of disease R genes to race 1 of FOV in the cotton genome. Molecular markers were applied to F(2) and recombinant inbred line (RIL) interspecific mapping populations from the crosses Pima-S7 (G. barbadense L.)×'Acala NemX' (G. hirsutum L.) and Upland TM-1 (G. hirsutum)×Pima 3-79 (G. barbadense), respectively. Three greenhouse tests and one field test were used to obtain sequential estimates of severity index (DSI) of leaves, and vascular stem and root staining (VRS). A single resistance gene model was observed for the F(2) population based on inheritance of phenotypes. However, additional inheritance analyses and QTL mapping indicated gene interactions and inheritance from nine cotton chromosomes, with major QTLs detected on five chromosomes [Fov1-C06, Fov1-C08, (Fov1-C11 ( 1 ) and Fov1-C11 ( 2)) , Fov1-C16 and Fov1-C19 loci], explaining 8-31% of the DSI or VRS variation. The Fov1-C16 QTL locus identified in the F(2) and in the RIL populations had a significant role in conferring FOV race 1 resistance in different cotton backgrounds. Identified molecular markers may have important potential for breeding effective FOV race 1 resistance into elite cultivars by marker-assisted selection. Reconciliation between genetic and physical mapping of gene annotations from marker-DNA and new DNA sequences of BAC clones tagged with the resistance-associated QTLs revealed defenses genes induced upon pathogen infection and gene regions rich in disease-response elements, respectively. These offer candidate gene targets for Fusarium wilt resistance response in cotton and other host plants.

Identification of QTL regions and SSR markers associated with resistance to reniform nematode in Gossypium barbadense L. accession GB713

The identification of molecular markers that are closely linked to gene(s) in Gossypium barbadense L. accession GB713 that confer a high level of resistance to reniform nematode (RN), Rotylenchulus reniformis Linford & Oliveira, would be very useful in cotton breeding programs. Our objectives were to determine the inheritance of RN resistance in the accession GB713, to identify SSR markers linked with RN resistance QTLs, and to map these linked markers to specific chromosomes. We grew and scored plants for RN reproduction in the P(1), P(2), F(1), F(2), BC(1)P(1), and BC(1)P(2) generations from the cross of GB713 × Acala Nem-X. The generation means analysis using the six generations indicated that one or more genes were involved in the RN resistance of GB713. The interspecific F(2) population of 300 plants was genotyped with SSR molecular markers that covered most of the chromosomes of Upland cotton (G. hirsutum L.). Results showed two QTLs on chromosome 21 and one QTL on chromosome 18. One QTL on chromosome 21 was at map position 168.6 (LOD 28.0) flanked by SSR markers, BNL 1551_162 and GH 132_199 at positions 154.2 and 177.3, respectively. A second QTL on chromosome 21 was at map position 182.7 (LOD 24.6) flanked by SSR markers BNL 4011_155 and BNL 3279_106 at positions 180.6 and 184.5, respectively. Our chromosome 21 map had 61 SSR markers covering 219 cM. One QTL with smaller genetic effects was localized to chromosome 18 at map position 39.6 (LOD 4.0) and flanked by SSR markers BNL 1721_178 and BNL 569_131 at positions 27.6 and 42.9, respectively. The two QTLs on chromosome 21 had significant additive and dominance effects, which were about equal for each QTL. The QTL on chromosome 18 showed larger additive than dominance effects. Following the precedent set by the naming of the G. longicalyx Hutchinson & Lee and G. aridum [(Rose & Standley) Skovsted] sources of resistance, we suggest the usage of Ren (barb1) and Ren (barb2) to designate these QTLs on chromosome 21 and Ren (barb3) on chromosome 18.

Introgression of Root‐Knot Nematode Resistance into Tetraploid Cottons

Root‐knot nematode (RKN) resistance introgression into tetraploid cotton (Gossypium spp.) and its ancestral genome origin were examined. Resistance sources (‘Acala NemX’, ‘Clevewilt 6’, Auburn 623 RNR) were compared with diverse germplasm using simple sequence repeat (SSR) markers from chromosomes 7, 11, and 14 and DNA sequence information. Differences (P < 0.05) were observed for mean root galling index (GI; scale 0–10) between the 56 resistant and susceptible entries. In resistance sources, GI ranged from 0.3 to 2.9, but no consistent differences were observed at allele‐marker or DNA sequence level. Except for CIR316 allele (206–207 bp) on chromosome 11, no alleles from other SSRs were observed on resistant entries (GI < 3). Allotetraploid G. hirsutum L. (AD1) and G. barbadense L. (AD2) showing the same SSR marker alleles as G. arboreum L. (A2) might suggest resistance introduction from the diploid cotton A2 genome (genetic distance 0.19 to 0.27). However, percent sequence identity from MUCS088 and CIR316 revealed G. barbadense and resistant G. hirsutum (206–207 bp) also were close to diploids G. herbaceum L. (A1), G. thurberi Tod. (D1), and G. trilobum (DC.) Skovst. (D8). Other G. hirsutum DNA sequences were closer to G. raimondii Ulbr. (D5). These analyses indicated resistance introgression into G. hirsutum allotetraploid cottons occurred by artificial hybridization with ancestral genome origin from G. arboreum as well as G. thurberi and not during cotton genome evolution.

A transgressive segregation factor (RKN2) in Gossypium barbadense for nematode resistance clusters with gene rkn1 in G. hirsutum

Host plant resistance is an important strategy for managing root-knot nematode (Meloidogyne incognita) in cotton (Gossypium L.). Here we report evidence for enhanced resistance in interspecific crosses resulting from transgressive segregation of clustered gene loci. Recently, a major gene, rkn1, on chromosome 11 for resistance to M. incognita in cv. Acala NemX was identified using an intraspecific G. hirsutum cross with susceptible cv. Acala SJ-2. Using interspecific crosses of Acala NemXxsusceptible G. barbadense cv. Pima S-7, F1, F2, F2:3, backcross, and testcross Acala NemXxF1 (Pima S-7xSJ-2), parental entries and populations were inoculated in greenhouse tests with M. incognita. Genetic analyses based on nematode-induced root galling and nematode egg production on roots, and molecular marker analysis of the segregating interspecific populations revealed that gene rkn1 interacted with a gene (designated as RKN2) in susceptible Pima S-7 to produce a highly resistant phenotype. RKN2 did not confer resistance in Pima S-7, but when combined with rkn1 (genotype Aa or aa), high levels of resistance were produced in the F1 and segregating F2, F3, and BC1F1 populations. One SSR marker MUCS088 was identified tightly linked to RKN2 within 4.4 cM in a NemXxF1 (Pima S-7xSJ-2) testcross population. Using mapped SSR markers and interspecific segregating populations, MUCS088 linked to the transgressive gene from the susceptible parent and was located in the vicinity of rkn1 on chromosome 11. Diverse genome analyses among A and D genome diploid and tetraploid cottons revealed that marker MUCS088 (165 and 167 bp) is derived from G. arboreum, A2 diploid genome. These results demonstrated that a highly susceptible parent contributed to nematode resistance via transgressive segregation. Derived highly resistant lines can be used as improved resistance sources in cotton breeding, and MUCS088 can be used to monitor RKN2 introgression in diverse populations. The close genomic location of the transgressive resistance determinants provides an important model system for studying transgressive segregation and epistasis in plants.

Identification of Molecular Markers Associated with Root‐Knot Nematode Resistance in Upland Cotton

Cotton breeding for resistance to root-knot nematode (RKN) [Meloidogyne incognita (Kofoid and White) Chitwood] is hindered by the lack of convenient and reliable screening methods for resistant plants. The identification of molecular markers closely linked to RKN resistance will facilitate the development of RKN resistant cultivars through marker-assisted selection (MAS). Our objective was to identify and develop new DNA markers that are associated with RKN resistance in cotton. Using three pairs of near-isogenic (NIL) resistant (R) and susceptible (S) lines, two AFLP markers, two RAPD markers, and three RGA markers were identified to be polymorphic between the NIL-R and NIL-S lines. One RAPD marker was converted into a sequence-tagged site (STS) marker. In an F2 population of ‘ST 474’ × ‘Auburn 634 RNR’, the two RAPD markers and the STS marker were mapped to the same linkage group containing several markers that were previously reported to be linked with the RKN resistance gene rkn1 on chromosome 11 in ‘Acala NemX’. All these markers were found to be associated with a major RKN resistance gene, presumably Mi2 in the resistant line Auburn 634 RNR, suggesting that rkn1 and Mi2 are either allelic or closely linked. In addition, no susceptible recombinants were found in a resistance screen of 200 F2 plants from the cross Acala NemX × Auburn 634 RNR. The utility of the two RAPD markers and the converted STS marker were evaluated using 23 R and 8 S germplasm lines. The RAPD and STS markers, along with other previously reported markers associated with RKN resistance will be useful in germplasm screening, MAS for RKN resistance, and map-based cloning for RKN resistance genes.

Development of AFLP and derived CAPS markers for root-knot nematode resistance in cotton

Resistance to root-knot nematode (Meloidogyne incognita) is determined by a single major gene rkn1 in Gossypium hirsutum Acala NemX cotton. Bulked segregant analysis (BSA) combined with amplified fragment length polymorphism (AFLP) was used to identify molecular markers linked to rkn1. DNA pools from homozygous susceptible (S) and resistant (R) bulks of an F2:3 originating from the intraspecific cross NemX × SJ-2 were screened with 128 EcoR1/Mse1 primer combinations. Putative AFLP markers were then screened with 60 F2:7 RIL plants and four AFLP markers were found linked to rkn1. The linkage of AFLP markers to rkn1 was also confirmed in a F2 population. The closest AFLP marker was converted to a cleaved amplified polymorphic sequence (CAPS) marker (designated GHACC1) by aligning the sequences from both susceptible and resistant parents. GHACC1 linkage to rkn1 was confirmed in the F2 (1R:3S), F2:7 RIL (1R:1S) and the backcross population SJ-2 × F1 (NemX × SJ-2) (1 heterozygous: 1 homozygous). The four AFLP markers, GHACC1 plus two SSR markers (CIR316 and BNL1231) linked to rkn1 from previous work were mapped to intervals of 2.6–14.2 cM from the rkn1 locus, and the genomic region around rkn1 was spanned to about 28.2 cM in the F2:7 population. The PCR-based GHACC1 and CIR316 markers were tested on 21 nematode resistant and susceptible cotton breeding lines and cultivars. GHACC1 was suitable for nematode resistance screening within G.␣hirsutum, but not G. barbadense, whereas CIR316 was useful in both species, indicating their␣potential for utilization in marker-assisted selection.

A Fusarium Wilt Resistance Gene in Gossypium barbadense and Its Effect on Root-Knot Nematode-Wilt Disease Complex

ABSTRACT Fusarium wilt, caused by the soilborne pathogen Fusarium oxysporum f. sp. vasinfectum race 1, is a vascular disease in cotton (Gossypium spp.), and is a component of a disease complex with root-knot nematodes (Meloidogyne incognita). Genetic analysis of two interspecific crosses (G. barbadense Pima S-7 x G. hirsutum Acala NemX and Pima S-7 x Acala SJ-2) showed that one major gene (designated Fov1) with allele dosage effect conferred resistance to F. oxysporum f. sp. vasinfectum race 1 in Pima S-7. Two amplified fragment length polymorphism (AFLP) markers were linked to Fov1 in Pima S-7, with genetic distance from the gene of 9.3 and 14.6 centimorgans. Less severe wilt symptoms in Acala NemX than Acala SJ-2 indicated that Acala NemX possesses one or more minor genes contributing to delay of wilt symptoms. Highly resistant plants in F(2) and F(3) (Pima S-7 x NemX) families indicated transgressive segregation effects of minor genes in Acala NemX combined with Fov1 from Pima S-7. The effects of wilt and nematode resistance on the nematode-wilt disease complex were assayed with two inoculation methods. In the presence of both pathogens, wilt damage measured as shoot and root weight reductions was greatest on wilt- and nematode-susceptible Acala SJ-2 and least in root-knot nematode-resistant and wilt-susceptible Acala NemX. Intermediate damage occurred in wilt-resistant and root-knot nematode-susceptible Pima S-7. The results indicated that nematode resistance was more effective than wilt resistance in suppressing wilt symptoms when either resistance was present alone. Nematode resistance combined with intermediate wilt resistance, as in the F(1) (Pima S-7 x NemX), was highly effective in protecting plants from root-knot nematodes and race 1 of Fusarium wilt as a disease complex.

Identification and mapping of microsatellite markers linked to a root-knot nematode resistance gene (rkn1) in Acala NemX cotton (Gossypium hirsutum L.)

Host-plant resistance is the most economic and effective strategy for root-knot nematode (RKN) Meloidogyne incognita control in cotton (Gossypium hirsutum L.). Molecular markers linked to resistance are important for incorporating resistance genes into elite cultivars. To screen for microsatellite markers (SSR) closely linked to RKN resistance in G. hirsutum cv. Acala NemX, F1, F2, BC1F1, and F2:7 recombinant inbred lines (RILs) from intraspecific crosses and an F2 from an interspecific cross with G. barbadense cv. Pima S-7 were used. Screening of 284 SSR markers, which cover all the known identified chromosomes and most linkage groups of cotton, was performed by bulked segregant analysis, revealing informative SSRs. The informative SSRs were then mapped on the above populations. One co-dominant SSR marker CIR316 was identified tightly linked to a major resistance gene (designated as rkn1), producing amplified DNA fragments of approximately 221 bp (CIR316a) and 210 bp (CIR316c) in Acala NemX and susceptible Acala SJ-2, respectively. The linkage between CIR316a marker and resistance gene rkn1 in Acala NemX had an estimated distance of 2.1-3.3 cM depending on the population used. Additional markers, including BNL1231 with loose linkage to rkn1 (map distance 25.1-27.4 cM), BNL1066, and CIR003 allowed the rkn1 gene to be mapped to cotton linkage group A03. This is the first report in cotton with a closely linked major gene locus determining nematode resistance, and informative SSRs may be used for marker-assisted selection.

Resistance to Rotylenchulus reniformis and Meloidogyne incognita Race 3 in the Major Cotton Cultivars Planted Since 1950

The southern root-knot nematode, Meloidogyne incognita race 3 (Kofoid & White) Chitwood, and the reniform nematode, Rotylenchulus reniformis Linford & Oliveira, are the most widespread and economically important nematode pests of Upland cotton (Gossypium hirsutum L.) and Pima cotton (G. barbadense L.) in the USA. Progress in the development of nematode-resistant cotton cultivars is unclear. Plant breeders often have selected for nematode tolerance by measuring cotton yield in nematode-infested fields but seldom have selected for resistance by measuring nematode reproduction. The objective of this research was to measure nematode reproduction to assess progress in the development of nematode resistance in cotton. Fiftyfive Upland and four Pima cotton cultivars, comprising the most widely planted cultivars in the USA each decade since 1950, were evaluated in growth chambers for resistance to M. incognita and R. reniformis. The cultivar Deltapine 16 served as the susceptible and the germplasm line Auburn 623 RNR as the resistant control. Reproduction by M. incognita on the cultivars ranged from 4 to 294% of that on Deltapine 16. All cultivars were highly susceptible to R. reniformis. A small increase was detected in the general level of resistance to M. incognita between 1950 and 1996, but only the recently released cultivars, Stoneville ‘LA887’, Paymaster ‘1560’, and CPCSD ‘Acala Nem-X’, showed consistently high resistance. These cultivars were developed by measuring nematode reproduction in pots or root galling in field plantings, as well as yield and related agronomic traits.