Ascochyta Blight Resistance In Chickpea Research Articles

STMS markers related to Ascochyta blight resistance in chickpea

Chickpea (Cicer arietinum L.) is one of the important legume crops and is cultivated in large-scale throughout Türkiye as well as the world. Ascochyta blight, caused by the fungal phytopathogen Ascochyta rabiei, is the leading reason for the highest yield losses among the diseases known for chickpea. The pathogen exhibits high genetic diversity in Türkiye. Therefore, resistancy using Sequence Tagged Microsatellite Site (STMS) markers related with the genes that provide resistant against Ascochyta blight was investigated for the 205 chickpea breeding lines grown in different parts of Türkiye. The analysis for Ascochyta blight resistance was performed using Ta2, Ta146 and Ts54. It was demonstrated that Ta2, Ts54 and Ta146 were the STMS markers having distinguishable features for the detection of Ascochyta blight resistance and were shown to be used in credible fashion for the selection of resistant chickpea breeding lines.

Novel Genomic Regions Linked to Ascochyta Blight Resistance in Two Differentially Resistant Cultivars of Chickpea.

Ascochyta blight (AB), caused by the fungal pathogen Ascochyta rabiei, is a devastating foliar disease of chickpea (Cicer arietinum L.). The genotyping-by-sequencing (GBS)-based approach was deployed for mapping QTLs associated with AB resistance in chickpea in two recombinant inbred line populations derived from two crosses (AB3279 derived from ILC 1929 × ILC 3279 and AB482 derived from ILC 1929 × ILC 482) and tested in six different environments. Twenty-one different genomic regions linked to AB resistance were identified in regions CalG02 and CalG04 in both populations AB3279 and AB482. These regions contain 1,118 SNPs significantly associated with AB resistance (p ≤ 0.001), which explained 11.2–39.3% of the phenotypic variation (PVE). Nine of the AB resistance-associated genomic regions were newly detected in this study, while twelve regions were known from previous AB studies. The proposed physical map narrows down AB resistance to consistent genomic regions identified across different environments. Gene ontology (GO) assigned these QTLs to 319 genes, many of which were associated with stress and disease resistance, and with most important genes belonging to resistance gene families such as leucine-rich repeat (LRR) and transcription factor families. Our results indicate that the flowering-associated gene GIGANTEA is a possible key factor in AB resistance in chickpea. The results have identified AB resistance-associated regions on the physical genetic map of chickpea and allowed for the identification of associated markers that will help in breeding of AB-resistant varieties.

Inheritance and mapping of QTLs for Ascochyta blight resistance in chickpea (Cicer arietinum)

There are very limited studies on the QTLs governing Ascochyta blight resistance which is a major problem in chickpea (Cicer arietinum L.) grown in North West Plain Zone of India in the cooler climates and world over. An interspecific mapping population consisting of 157 F2:3 lines developed by crossing a resistant parent ILWC129 (C. reticulatum) with a susceptible variety GL769 (C. arietinum L.), was evaluated (2019–20) and genotyped (2021) for genetic studies and mapping of resistance to Ascochyta rabiei (syn. Phoma rabiei), isolate 8(3968). The inheritance pattern gave 1R:2H:1S goodness of fit ratio with χ2 value of 0.4 indicating that resistance against isolate 8 of the pathogen governed by a single gene. Out of 831 SSR markers screened, 86 were found polymorphic with 10.3% polymorphism from which only 41 markers segregated in 1:2:1 ratio and could be mapped. Eight linkage groups with a cumulative distance of 1042.31 cM were formed with an average distance of 25.42 cM. This is probably the first report of inheritance studies and tagging of A. rabei race 8 (arr8) resistance gene using molecular markers in an interspecific cross with C. reticulatum.

Application of Genomics Approaches for the Improvement in Ascochyta Blight Resistance in Chickpea

Advancements in high-throughput genotyping and sequencing technologies are enabling the development of a vast range of genomic tools and resources for a new revolution in plant breeding. Several genotyping-by-sequencing (GBS) methods including capture-based, genome complexity reduction and sequencing of cDNA (GBS-t) are available for application in trait dissection, association mapping, and genomic selection (GS) in crop plants. The aims of this study were to identify genomic regions conferring resistance to Ascochyta blight (AB) introgressed from the wild Cicer echinospernum into the domesticated C. arietinum, through a conventional recombinant inbred population genotyped using a variety of GBS methods. Evaluation of GBS methods revealed that capture-based approaches are robust and reproducible while GBS-t is rapid and flexible. A genetic linkage map consisting of 5886 polymorphic loci spanning 717.26 cM was generated. Using field phenotyping data from two years, a single genomic region on LG4 was identified with quantitative trait loci (QTL) mapping. Both GBS methods reported in this study are well suited for applications in genomics assisted plant breeding. Linked markers for AB resistance, identified in the current study, provide an important resource for the deployment into chickpea breeding programs for marker-assisted selection (MAS).

Correction to: Genome-wide SNP discovery for development of high-density genetic map and QTL mapping of ascochyta blight resistance in chickpea (Cicer arietinum L.).

Unfortunately there was an error in the name of the gene "Ca-AKL18" in the discussion section.

Genome-wide SNP discovery for development of high-density genetic map and QTL mapping of ascochyta blight resistance in chickpea (Cicer arietinum L.)

Key messageA high-density linkage map of chickpea using 3430 SNPs was constructed and used to identify QTLs and candidate genes for ascochyta blight resistance in chickpea.Chickpea cultivation in temperate conditions is highly vulnerable to ascochyta blight infection. Cultivation of resistant cultivars in combination with fungicide application within an informed disease management package is the most effective method to control ascochyta blight in chickpeas. Identifying new sources of resistance is critical for continued improvement in ascochyta blight resistance in chickpea. The objective of this study was to identify genetic loci and candidate genes controlling the resistance to ascochyta blight in recombinant inbred lines derived from crossing cultivars Amit and ICCV 96029. The RILs were genotyped using the genotyping-by-sequencing procedure and Illumina® GoldenGate array. The RILs were evaluated in the field over three site-years and in three independent greenhouse experiments. A genetic map with eight linkage groups was constructed using 3430 SNPs. Eight QTLs for resistance were identified on chromosomes 2, 3, 4, 5 and 6. The QTLs individually explained 7–40% of the phenotypic variations. The QTLs on chromosomes 2 and 6 were associated with the resistance at vegetative stage only. The QTLs on chromosomes 2 and 4 that were previously reported to be conserved across diverse genetic backgrounds and against different isolates of Ascochyta rabiei were confirmed in this study. Candidate genes were identified within the QTL regions. Their co-localization with the underlying QTLs was confirmed by genetic mapping. The candidate gene-based SNP markers would lead to more efficient marker-assisted selection for ascochyta blight resistance and would provide a framework for fine mapping and subsequent cloning of the genes associated with the resistance.

Integrated transcriptome, small RNA and degradome sequencing approaches provide insights into Ascochyta blight resistance in chickpea.

SummaryAscochyta blight (AB) is one of the major biotic stresses known to limit the chickpea production worldwide. To dissect the complex mechanisms of AB resistance in chickpea, three approaches, namely, transcriptome, small RNA and degradome sequencing were used. The transcriptome sequencing of 20 samples including two resistant genotypes, two susceptible genotypes and one introgression line under control and stress conditions at two time points (3rd and 7th day post inoculation) identified a total of 6767 differentially expressed genes (DEGs). These DEGs were mainly related to pathogenesis‐related proteins, disease resistance genes like NBS‐LRR, cell wall biosynthesis and various secondary metabolite synthesis genes. The small RNA sequencing of the samples resulted in the identification of 651 miRNAs which included 478 known and 173 novel miRNAs. A total of 297 miRNAs were differentially expressed between different genotypes, conditions and time points. Using degradome sequencing and in silico approaches, 2131 targets were predicted for 629 miRNAs. The combined analysis of both small RNA and transcriptome datasets identified 12 miRNA‐mRNA interaction pairs that exhibited contrasting expression in resistant and susceptible genotypes and also, a subset of genes that might be post‐transcriptionally silenced during AB infection. The comprehensive integrated analysis in the study provides better insights into the transcriptome dynamics and regulatory network components associated with AB stress in chickpea and, also offers candidate genes for chickpea improvement.

QTL sequencing strategy to map genomic regions associated with resistance to ascochyta blight in chickpea.

SummaryWhole‐genome sequencing‐based bulked segregant analysis (BSA) for mapping quantitative trait loci (QTL) provides an efficient alternative approach to conventional QTL analysis as it significantly reduces the scale and cost of analysis with comparable power to QTL detection using full mapping population. We tested the application of next‐generation sequencing (NGS)‐based BSA approach for mapping QTLs for ascochyta blight resistance in chickpea using two recombinant inbred line populations CPR‐01 and CPR‐02. Eleven QTLs in CPR‐01 and six QTLs in CPR‐02 populations were mapped on chromosomes Ca1, Ca2, Ca4, Ca6 and Ca7. The QTLs identified in CPR‐01 using conventional biparental mapping approach were used to compare the efficiency of NGS‐based BSA in detecting QTLs for ascochyta blight resistance. The QTLs on chromosomes Ca1, Ca4, Ca6 and Ca7 overlapped with the QTLs previously detected in CPR‐01 using conventional QTL mapping method. The QTLs on chromosome Ca4 were detected in both populations and overlapped with the previously reported QTLs indicating conserved region for ascochyta blight resistance across different chickpea genotypes. Six candidate genes in the QTL regions identified using NGS‐based BSA on chromosomes Ca2 and Ca4 were validated for their association with ascochyta blight resistance in the CPR‐02 population. This study demonstrated the efficiency of NGS‐based BSA as a rapid and cost‐effective method to identify QTLs associated with ascochyta blight in chickpea.

Combining Ascochyta blight and Botrytis grey mould resistance in chickpea through interspecific hybridization

Ascochyta blight (AB) caused by Ascochyta rabiei (Pass.) Labr. and Botrytis grey mould (BGM) caused by Botrytis cinerea (Pers. ex Fr.) are important diseases of the aerial plant parts of chickpea in most chickpea growing areas of the world. Although conventional approaches have contributed to reducing disease, the use of new technologies is expected to further reduce losses through these biotic stresses. Reliable screening techniques were developed: ‘field screening technique’ for adult plant screening, ‘cloth chamber technique’ and ‘growth chamber technique’ for the study of races of the pathogen and for segregating generations. Furthermore, the ‘cut twig technique’ for interspecific population for AB and BGM resistance was developed. For introgression of high levels of AB and BGM resistance in cultivated chickpea from wild relatives, accessions of seven annual wild Cicer spp. were evaluated and identified: C. judaicum accessions 185, ILWC 95 and ILWC 61, C. pinnatifidum accessions 188, 199 and ILWC 212 as potential donors. C. pinnatifidum accession188 was crossed with ICCV 96030 and 62 F9 lines resistant to AB and BGM were derived. Of the derived lines, several are being evaluated for agronomic traits and yield parameters while four lines, GL 29029, GL29206, GL29212, GL29081 possessing high degree of resistance were crossed with susceptible high yielding cultivars BG 256 to improve resistance and to undertake molecular studies. Genotyping of F2 populations with SSR markers from the chickpea genome was done to identify markers potentially linked with AB and BGM resistance genes. In preliminary studies, of 120 SSR markers used, six (Ta 2, Ta 110, Ta 139, CaSTMS 7, CaSTMS 24 and Tr 29) were identified with polymorphic bands between resistant derivative lines and the susceptible parent. The study shows that wild species of Cicer are the valuable gene pools of resistance to AB and BGM. The resistant derivative lines generated here can serve as good pre-breeding material and markers identified can assist in marker assisted selection for resistance breeding.

Allele-specific amplification for the detection of ascochyta blight resistance in chickpea

A new co-dominant molecular marker, CaETR, was developed based on allelic sequence length polymorphism in an ethylene receptor-like gene located in the genomic region of a QTL (QTLAR1) conferring ascochyta blight resistance in chickpea. This marker not only discriminated resistance and susceptible phenotypes of chickpea to ascochyta blight, but also easily detected heterozygous genotypes. Using the CaETR marker in combination with a previously developed co-dominant SCAR marker (SCY17590) linked to another QTL (QTLAR2) it was possible to detect resistance alleles in 90 % of resistant accessions in a collection of landraces, advances breeding lines and cultivars, and also detected susceptible alleles in all cases. The results of this study offer a scope for improving the efficiency of conventional chickpea breeding by carrying out negative selection for QTLAR1 and QTLAR2 in early generations without relaying directly on the phenotype. This PCR-based approach using both co-dominant markers is proposed as an efficient tool for selecting blight-resistant genotypes in breeding programs.

Characterization and genetic analysis of an EIN4-like sequence (CaETR-1) located in QTLAR1 implicated in ascochyta blight resistance in chickpea

Two alleles of a chickpea (Cicer arietinum L.) ethylene receptor-like sequence (CaETR-1) were sequence-characterized using synteny analysis with genome sequences of Medicago truncatula L. The full length of the sequence obtained in the accession FLIP84-92C resistant to ascochyta blight (CaETR-1a) span 4,428bp, including the polyadenylation signal in the 3'-untranslated region (UTR), whereas it has a 730bp deletion in the 3'-UTR region in the susceptible accession PI359075 (CaETR-1b). The deduced protein belongs to subfamily II of the ethylene receptors and contains all the domains that define EIN4 homologs in Arabidopsis. The EIN4-like sequence (CaETR-1) has been mapped using a recombinant inbred line (RIL) population derived from an intraspecific cross between ILC3279 and WR315, resistant and susceptible to blight, respectively. The locus was located in LGIVa of the genetic map, flanked by markers NCPGR91 and GAA47 (at distances of 11.3 and 17.9cM, respectively). This is the first potentially functional sequence identified under a QTL peak for ascochyta blight resistance in chickpea (QTL(AR1)). This EIN4-like (CaETR-1) sequence explained up to 33.8% of the total phenotypic variation. This sequence could be directly related to blight resistance, together with other QTLs that have been found to be involved in resistance to this major chickpea disease.

Peroxidase: a marker for Ascochyta blight resistance in chickpea

On the basis of incidence of appearance of Ascochyta blight symptoms after artificial inoculation of 25-day-old chickpea seedlings with 10 different pathotypes of Ascochyta rabiei, GL94011, PBG5 and C214 have been classified as resistant, moderately resistant and susceptible, respectively, to Ascochyta blight. In none of the pathotypes, peroxidase (PO) activity could be detected in culture medium and mycelium. Healthy tissues of GL94011 have almost three times the PO activity in comparison with that of C214. Resistant and moderately resistant genotypes showed 30–60% upregulation of PO activity against infection by A. rabiei whereas it was only 3–6% in susceptible genotype C214. These results indicate the possibility of using PO as a marker of Ascochyta blight resistance.

Genetic analyses and conservation of QTL for ascochyta blight resistance in chickpea (Cicer arietinum L.)

Ascochyta blight (AB) caused by Ascochyta rabiei (teleomorph, Didymella rabiei) Pass. Lab. is an important fungal disease of chickpea worldwide. Only moderate sources of resistance are available within the cultivated species and we hypothesized that the available sources may carry different genes for resistance, which could be pyramided to improve field resistance to AB. Four divergent moderately resistant cultivars CDC Frontier, CDC Luna, CDC Corinne, and Amit were each crossed to a highly susceptible germplasm ICCV 96029. Parents, F(1) and F(2) generations were evaluated under controlled conditions for their reactions to AB. A total of 144 simple sequence repeat (SSR) markers were first mapped to eight linkage groups (LG) for the CDC Frontier x ICCV 96029 population. Then based on the evidence from this population, 76, 61, and 42 SSR markers were systematically chosen and mapped in CDC Luna, CDC Corinne, and Amit populations, respectively. Frequency distributions of the AB rating in the F(2) generation varied among the four populations. Composite interval mapping revealed five QTLs (QTL1-5), one on each of LG 2, 3, 4, 6, and 8, respectively, distributed across different sources, controlling resistance to AB. CDC Frontier contained QTL2, 3, and 4 that simultaneously accounted for 56% of phenotypic variations. CDC Luna contained QTL 1 and 3. CDC Corinne contained QTL 3 and 5, while only QTL 2 was identified in Amit. Altogether these QTL explained 48, 38, and 14% of the estimated phenotypic variations in CDC Luna, CDC Corinne, and Amit populations, respectively. The results suggested that these QTLs could be combined into a single genotype to enhance field resistance to AB.

Mapping and validation of QTLs for resistance to an Indian isolate of Ascochyta blight pathogen in chickpea

Ascochyta blight (AB) caused by Ascochyta rabiei, is globally the most important foliar disease that limits the productivity of chickpea (Cicer arietinum L.). An intraspecific linkage map of cultivated chickpea was constructed using an F2 population derived from a cross between an AB susceptible parent ICC 4991 (Pb 7) and an AB resistant parent ICCV 04516. The resultant map consisted of 82 simple sequence repeat (SSR) markers and 2 expressed sequence tag (EST) markers covering 10 linkage groups, spanning a distance of 724.4 cM with an average marker density of 1 marker per 8.6 cM. Three quantitative trait loci (QTLs) were identified that contributed to resistance to an Indian isolate of AB, based on the seedling and adult plant reaction. QTL1 was mapped to LG3 linked to marker TR58 and explained 18.6% of the phenotypic variance (R2) for AB resistance at the adult plant stage. QTL2 and QTL3 were both mapped to LG4 close to four SSR markers and accounted for 7.7% and 9.3%, respectively, of the total phenotypic variance for AB resistance at seedling stage. The SSR markers which flanked the AB QTLs were validated in a half-sib population derived from the same resistant parent ICCV 04516. Markers TA146 and TR20, linked to QTL2 were shown to be significantly associated with AB resistance at the seedling stage in this half-sib population. The markers linked to these QTLs can be utilized in marker-assisted breeding for AB resistance in chickpea.

Discovery and detection of single nucleotide polymorphism (SNP) in coding and genomic sequences in chickpea (Cicer arietinum L.)

Chickpea genetic mapping has been hampered by insufficient amplicon length polymorphism for sequence based markers. To develop an alternative source of polymorphic markers, we determined naturally abundant single nucleotide polymorphism (SNP) in coding and genomic regions between FLIP 84-92C (C. arietinum) and PI 599072 (C. reticulatum) and identified an inexpensive method to detect SNP for mapping. In coding sequences, 110 single base changes or substitutions (47% transitions and 53% transversions) and 18 indels were found; while 50 single base changes (68% transitions and 33% transversions) and eight indels were observed in genomic sequences. SNP frequency in coding and genomic regions was 1 in 66 bp and 1 in 71 bp, respectively. In order to effectively use this high frequency of polymorphism, we used Cleaved Amplified Polymorphic Site (CAPS) and derived CAPS (dCAPS) marker systems to identify a restriction site at SNP loci. In this study, we developed six CAPS and dCAPS markers and fine mapped QTL1, a region previously identified as important for ascochyta blight resistance. One of the CAPS markers from a BAC end was identified to account for 56% of the variation for ascochyta blight resistance in chickpea. Conversion of naturally abundant SNPs to CAPS and dCAPS for chickpea mapping, where absence of amplicon length polymorphism is a constraint, has potential to generate high-density maps necessary for map-based cloning and integration of physical and genetic maps.

Detection of two quantitative trait loci for resistance to ascochyta blight in an intra-specific cross of chickpea (Cicer arietinum L.): development of SCAR markers associated with resistance

Two quantitative trait loci (QTLs), (QTL(AR1) and QTL(AR2)) associated with resistance to ascochyta blight, caused by Ascochyta rabiei, have been identified in a recombinant inbred line population derived from a cross of kabulixdesi chickpea. The population was evaluated in two cropping seasons under field conditions and the QTLs were found to be located in two different linkage groups (LG4a and LG4b). LG4b was saturated with RAPD markers and four of them associated with resistance were sequenced to give sequence characterized amplified regions (SCARs) that segregated with QTL(AR2). This QTL explained 21% of the total phenotypic variation. However, QTL(AR1), located in LG4a, explained around 34% of the total phenotypic variation in reaction to ascochyta blight when scored in the second cropping season. This LG4a region only includes a few markers, the flower colour locus (B/b), STMS GAA47, a RAPD marker and an inter-simple-sequence-repeat and corresponds with a previously reported QTL. From the four SCARs tagging QTL(AR2), SCAR (SCY17(590)) was co-dominant, and the other three were dominant. All SCARs segregated in a 1:1 (presence:absence) ratio and the scoring co-segregated with their respective RAPD markers. QTL(AR2) on LG4b was mapped in a highly saturated genomic region covering a genetic distance of 0.8 cM with a cluster of nine markers (three SCARs, two sequence-tagged microsatellite sites (STMS) and four RAPDs). Two of the four SCARs showed significant alignment with genes or proteins related to disease resistance in other species and one of them (SCK13(603)) was sited in the highly saturated region linked to QTL(AR2). STMS TA72 and TA146 located in LG4b were described in previous maps where QTL for blight resistance were also localized in both inter and intraspecific crosses. These findings may improve the precision of molecular breeding for QTL(AR2) as they will allow the choice of as much polymorphism as possible in any population and could be the starting point for finding a candidate resistant gene for ascochyta blight resistance in chickpea.

Markers associated with Ascochyta blight resistance in chickpea and their potential in marker-assisted selection

A chickpea recombinant inbred line (RIL) population F 6:7 was evaluated for disease reaction to blight caused by Ascochyta rabiei in three different environments. In Spain, disease was measured in the field by the area under the disease progress curve (AUDPC) and in the greenhouse by the linear infection index (LII). In Tunisia, disease was measured in the field by the 1–9 scale of Reddy and Singh [Plant Dis. 68 (1984) 900]. Twenty RILs showing extreme values for resistance and susceptibility were selected from field and greenhouse data obtained in Spain and assessed individually for DNA markers linked with disease resistance. Previously, 1028 DNA markers were found polymorphic in a collection of resistant and susceptible accessions of chickpea and 39 of them were associated with resistance. Both the more resistant and the more susceptible RILs, evaluated in Spain, were scored for the 39 chosen markers and 20 of them maintained a clear association with resistance or susceptibility. These loci formed a linkage group covering a map distance of 18.8 cM when the entire RIL population ( n=76) was analysed. The markers on the linkage group were significantly associated with disease reaction in the three environments, indicating the presence of a QTL for resistance to blight in this genomic region and explaining from 20 to 23.7% of the total phenotypic variation of the character. The resistant genotype for random amplified polymorphic DNA (RAPD) marker OPAC04/1200, in the middle of the group, was the most frequent (84%) in a set of cultivars and breeding lines selected as resistant to Ascochyta blight. The association of the other markers with resistance on each side of OPAC04/1200 decreased in frequency inversely with their distance from this locus. This result suggests that the QTL for resistance to blight would be close to OPAC04/1200 and we therefore propose to use this marker jointly with two others (OPAI09/1276 and OPM02/935) that flank it in MAS.

DAF marker tightly linked to a major locus for Ascochyta blight resistance in chickpea (Cicer arietinum L.)

Resistance of chickpea against the disease caused by the ascomycete Ascochyta rabiei is encoded by two or three quantitative trait loci, QTL1, QTL2 and QTL 3. A total of 94 recombinant inbred lines developed from a wide cross between a resistant chickpea line and a susceptible accession of Cicer reticulatum, a close relative of cultivated chickpea, was used to identify markers closely linked to QTL1 by DNA amplification fingerprinting in combination with bulked segregant analysis. Of 312 random 10mer oligonucleotides, 3 produced five polymorphic bands between the parents and bulks. Two of them were transferred to the population on which the recent genetic map of chickpea is based, and mapped to linkage group 4. These markers, OPS06-1 and OPS03-1, were linked at LOD-scores above 5 to markers UBC733B and UBC181A flanking the major ascochyta resistance locus. OPS06-1 mapped at the peak of the QTL between markers UBC733B (distance 4.1 cM) and UBC181A (distance 9.6 cM), while OPS03-1 mapped 25.1 cM away from marker UBC733B on the other flank of the resistance locus. STMS markers localised on this linkage group were transferred to the population segregating for ascochyta resistance. Three of these markers were closely linked to QTL1. Twelve of 14 STMS markers could be used in both populations. The order of STMS markers was essentially similar in both populations, with differences in map distances between them. The availability of flanking STMS markers for the major resistance locus QTL1 will help to elucidate the complex resistance against different Ascochyta pathotypes in future.

Screening for Ascochyta Blight Resistance in Chickpea Under Controlled Environment and Field Conditions

The occurrence and severity of Ascochyta blight of chickpea is influenced by environmental factors, which can complicate field screening. A controlled-environment plant growth room was developed at ICRISAT Asia Center to rapidly screen chickpea genotypes within 15 days for resistance to Ascochyta blight caused by Ascochyta rabiei. A positive correlation was observed between results from controlled-environment and field screenings in an area where the pathogen is endemic.