Preliminary investigation of QTLs associated with seedling resistance to ascochyta blight from <Emphasis Type="Italic">Cicer echinospermum</Emphasis>, a wild relative of chickpea

Accessions from Cicer echinospermum, a wild relative of chickpea (Cicer arietinum L.), contain resistance to the fungal disease ascochyta blight, a devastating disease of chickpea. A linkage map was constructed based on an interspecific F(2) population, derived from a cross between a susceptible chickpea cultivar (Lasseter) and a resistant C. echinospermum accession (PI 527930). The linkage map incorporated 83 molecular markers, that included RAPD, ISSR, STMS and RGA markers; eight markers remained unlinked. The map comprised eight linkage groups and covered a map distance of 570 cM. Six out of the eight linkage groups were correlated to linkage groups from the integrated Cicer map using STMS markers. Quantitative trait loci (QTLs) associated with ascochyta blight resistance were detected using interval mapping and single-point analysis. The F(2) population was evaluated for seedling and stem resistance in glasshouse trials. At least two QTLs were identified for seedling resistance, both of which were located within linkage group 4. Five markers were associated with stem resistance, four of which were also associated with seedling resistance. QTLs from previous studies also mapped to LG 4, suggesting that this linkage group is an important region of the Cicer genome for resistance to ascochyta blight.     

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 kabuli×desi 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 (SCY17₅₉₀) 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 (SCK13₆₀₃) 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.     

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

Whole‐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.     

Characterization and genetic analysis of an EIN4-like sequence (CaETR-1) located in QTL(AR1) 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 4,428?bp, including the polyadenylation signal in the 3'-untranslated region (UTR), whereas it has a 730?bp 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 17.9?cM, 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.     

Genetic mapping of ascochyta blight resistance in chickpea (Cicer arietinum L.) using a simple sequence repeat linkage map.

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Lab., is one of the most devastating diseases of chickpea (Cicer arietinum L.) worldwide. Research was conducted to map genetic factors for resistance to ascochyta blight using a linkage map constructed with 144 simple sequence repeat markers and 1 morphological marker (fc, flower colour). Stem cutting was used to vegetatively propagate 186 F2 plants derived from a cross between Cicer arietinum L. 'ICCV96029' and 'CDC Frontier'. A total of 556 cutting-derived plants were evaluated for their reaction to ascochyta blight under controlled conditions. Disease reaction of the F1 and F2 plants demonstrated that the resistance was dominantly inherited. A Fain's test based on the means and variances of the ascochyta blight reaction of the F3 families showed that a few genes were segregating in the population. Composite interval mapping identified 3 genomic regions that were associated with the reaction to ascochyta blight. One quantitative trait locus (QTL) on each of LG3, LG4, and LG6 accounted for 13%, 29%, and 12%, respectively, of the total estimated phenotypic variation for the reaction to ascochyta blight. Together, these loci controlled 56% of the total estimated phenotypic variation. The QTL on LG4 and LG6 were in common with the previously reported QTL for ascochyta blight resistance, whereas the QTL on LG3 was unique to the current population.     

Integration of sequence tagged microsatellite sites to the chickpea genetic map

Fifty sequence-tagged microsatellite site (STMS) markers and a resistant gene-analog (RGA) locus were integrated into a chickpea ( Cicer arietinum L., 2n = 2 x = 16 chromosomes) genetic map that was previously constructed using 142 F(6)-derived recombinant inbred lines (RILs) from a cross of C. arietinum x Cicer reticulatum Lad. The map covers 1,174.5 cM with an average distance of 7.0 cM between markers in nine linkage groups (LGs). Nine markers including the RGA showed distorted segregation ( P < 0.05). The majority of the newly integrated markers were mapped to marker-dense regions of the LGs. Six co-dominant STMS markers were integrated into two previously reported major quantitative trait loci (QTLs) conferring resistance to Ascochyta blight caused by Ascochyta rabiei (Pass.) Labr. Using common STMS markers as anchors, three maps developed from different mapping populations were joined, and genes for resistance to Ascochyta blight, Fusarium wilt (caused by Fusarium oxysporum Schlechtend.: Fr. f. sp. ciceris), and for agronomically important traits were located on the combined linkage map. The integration of co-dominant STMS markers improves the map of chickpea and makes it possible to consider additional fine mapping of the genome and also map-based cloning of important disease resistance genes.     

Genetic linkage map construction and QTL mapping of cadmium accumulation in radish (Raphanus sativus L.)

Cadmium (Cd) is a widespread soil pollutant and poses a significant threat to human health via the food chain. Large phenotypic variations in Cd concentration of radish roots and shoots have been observed. However, the genetic and molecular mechanisms of Cd accumulation in radish remain to be elucidated. In this study, a genetic linkage map was constructed using an F(2) mapping population derived from a cross between a high Cd-accumulating cultivar NAU-Dysx and a low Cd-accumulating cultivar NAU-Yh. The linkage map consisted of 523 SRAP, RAPD, SSR, ISSR, RAMP, and RGA markers and had a total length of 1,678.2 cM with a mean distance of 3.4 cM between two markers. All mapped markers distributed on nine linkage groups (LGs) having sizes between 134.7 and 236.8 cM. Four quantitative trait loci (QTLs) for root Cd accumulation were mapped on LGs 1, 4, 6, and 9, which accounted for 9.86 to 48.64 % of all phenotypic variance. Two QTLs associated with shoot Cd accumulation were detected on LG1 and 3, which accounted for 17.08 and 29.53 % of phenotypic variance, respectively. A major-effect QTL, qRCd9 (QTL for root Cd accumulation on LG9), was identified on LG 9 flanked by NAUrp011_754 and EM5me6_286 markers with a high LOD value of 23.6, which accounted for 48.64 % of the total phenotypic variance in Cd accumulation of F(2) lines. The results indicated that qRCd9 is a novel QTL responsible for controlling root Cd accumulation in radish, and the identification of specific molecular markers tightly linked to the major QTL could be further applied for marker-assisted selection (MAS) in low-Cd content radish breeding program.     

An intraspecific linkage map of the chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) genome based on sequence tagged microsatellite site and resistance gene analog markers

An intraspecific linkage map of the chickpea genome based on STMS as anchor markers, was established using an F(2) population of chickpea cultivars with contrasting disease reactions to Ascochyta rabiei (Pass.) Lab. At a LOD-score of 2.0 and a maximum recombination distance of 20 cM, 51 out of 54 chickpea-STMS markers (94.4%), three ISSR markers (100%) and 12 RGA markers (57.1%) were mapped into eight linkage groups. The chickpea-derived STMS markers were distributed throughout the genome, while the RGA markers clustered with the ISSR markers on linkage groups LG I, II and III. The intraspecific linkage map spanned 534.5 cM with an average interval of 8.1 cM between markers. Sixteen markers (19.5%) were unlinked, while l1 chickpea-STMS markers (20.4%) deviated significantly ( P < 0.05) from the expected Mendelian segregation ratio and segregated in favor of the maternal alleles. However, ten of the distorted chickpea-STMS markers were mapped and clustered mostly on LG VII, suggesting the association of these loci in the preferential transmission of the maternal germ line. Preliminary comparative mapping revealed that chickpea may have evolved from Cicer reticulatum, possibly via inversion of DNA sequences and minor chromosomal translocation. At least three linkage groups that spanned a total of approximately 79.2 cM were conserved in the speciation process.     

Construction of an intraspecific linkage map of lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> ssp. <Emphasis Type="Italic">culinaris</Emphasis>)

The first intraspecific linkage map of the lentil genome was constructed with 114 molecular markers (100 RAPD, 11 ISSR and three RGA) using an F₂ population developed from a cross between lentil cultivars ILL5588 and ILL7537 which differed in resistance for ascochyta blight. Linkage analysis at a LOD score of 4.0 and a maximum recombination fraction of 0.25 revealed nine linkage groups comprising between 6 and 18 markers each. The intraspecific map spanned a total length of 784.1 cM. The markers were distributed throughout the genome, however markers were clustered in the middle or near the middle of the linkage groups, suggesting the location of centromeres. Of 114 mapped markers, 16 (14.0%) were distorted, usually at the end or middle of the linkage groups. The utility of ISSR and RGA markers for mapping in lentil was explored, and the primer with an (AC) repeat motif was found to be useful.Communicated by H.C. Becker     

Construction of a linkage map based on a <Emphasis Type="Italic">Lathyrus sativus</Emphasis> backcross population and preliminary investigation of QTLs associated with resistance to ascochyta blight

A linkage map of the Lathyrus sativus genome was constructed using 92 backcross individuals derived from a cross between an accession resistant (ATC 80878) to ascochyta blight caused by Mycosphaerella pinodes and a susceptible accession (ATC 80407). A total of 64 markers were mapped on the backcross population, including 47 RAPD, seven sequence-tagged microsatellite site and 13 STS/CAPS markers. The map comprised nine linkage groups, covered a map distance of 803.1 cM, and the average spacing between markers was 15.8 cM. Quantitative trait loci (QTL) associated with ascochyta blight resistance were detected using single-point analysis and simple and composite interval mapping. The backcross population was evaluated for stem resistance in temperature-controlled growth room trials. One significant QTL, QTL1, was located on linkage group 1 and explained 12% of the phenotypic variation in the backcross population. A second suggestive QTL, QTL2, was detected on linkage group 2 and accounted for 9% of the trait variation. The L. sativus R-QTL regions detected may be targeted for future intergenus transfer of the trait into accessions of the closely related species Pisum sativum.     

Genetic mapping of pear sawfly (<Emphasis Type="Italic">Caliroa cerasi</Emphasis>) and pear blister mite (<Emphasis Type="Italic">Eriophyes pyri</Emphasis>) resistance in an interspecific pear family

Whilst minor pests of pear, both sawfly larvae (pear slug) and pear blister mite can at times cause sufficient damage in commercial and particularly in organic pear production for treatment to be required. In the course of breeding new pear cultivars, resistance to both pests was identified in an interspecific pear family raised from a cross between ‘PremP003’ and ‘Moonglow’. The replicated seedling family was subjected to uninhibited insect development for both pests in an insect-proof cage, providing ample infestations for resistance segregation. Using an existing genetic map for the family, one major quantitative trait locus (QTL) for resistance to pear blister mite was located to linkage group 13 (LG13) of ‘PremP003’. For pear slug, we mapped three QTLs for oviposition antixenosis, one each on LG7 and LG9 of ‘Moonglow’ and another on LG10 of ‘PremP003’, and one resistance QTL for leaf damage to LG9 of ‘Moonglow’ at a distance of 8.1 cM below the oviposition QTL. Incorporating these resistances into future cultivars could contribute to a reduction in pesticide use in pear production, especially in combination with the resistances for pear psylla (Cacopsylla pyri) and fire blight (Erwinia amylovora) recently mapped in the same population using marker-assisted selection.     

Genetic marker discovery,intraspecific linkage map construction and quantitative trait locus analysis of ascochyta blight resistance in chickpea (Cicer arietinum L.)

Ascochyta blight, caused by the fungus Ascochyta rabiei (Pass.) Labr., is a highly destructive disease of chickpea (Cicer arietinum L.) on a global basis, and exhibits considerable natural variation for pathogenicity. Different sources of ascochyta blight resistance are available within the cultivated species, suitable for pyramiding to improve field performance. Robust and closely linked genetic markers are desirable to facilitate this approach. A total of 4,654 simple sequence repeat (SSR) and 1,430 single nucleotide polymorphism (SNP) markers were identified from a chickpea expressed sequence tag (EST) database. Subsets of 143 EST–SSRs and 768 SNPs were further used for validation and subsequent high-density genetic mapping of two intraspecific mapping populations (Lasseter × ICC3996 and S95362 × Howzat). Comparison of the linkage maps to the genome of Medicago truncatula revealed a high degree of conserved macrosynteny. Based on field evaluation of ascochyta blight incidence performed over 2 years, two genomic regions containing resistance determinants were identified in the Lasseter × ICC3996 family. In the S95362 × Howzat population, only one quantitative trait locus (QTL) region was identified for both phenotypic evaluation trials, which on the basis of bridging markers was deduced to coincide with one of the Lasseter × ICC3996 QTLs. Of the two QTL-containing regions identified in this study, one (ab_QTL1) was predicted to be in common with QTLs identified in prior studies, while the other (ab_QTL2) may be novel. Markers in close linkage to ascochyta blight resistance genes that have been identified in this study can be further validated and effectively implemented in chickpea breeding programs.     

Quantitative trait loci for lodging resistance,plant height and partial resistance to mycosphaerella blight in field pea (Pisum sativum L.)

With the development of genetic maps and the identification of the most-likely positions of quantitative trait loci (QTLs) on these maps, molecular markers for lodging resistance can be identified. Consequently, marker-assisted selection (MAS) has the potential to improve the efficiency of selection for lodging resistance in a breeding program. This study was conducted to identify genetic loci associated with lodging resistance, plant height and reaction to mycosphaerella blight in pea. A population consisting of 88 recombinant inbred lines (RILs) was developed from a cross between Carneval and MP1401. The RILs were evaluated in 11 environments across the provinces of Manitoba, Saskatchewan and Alberta, Canada in 1998, 1999 and 2000. One hundred and ninety two amplified fragment length polymorphism (AFLP) markers, 13 random amplified polymorphic DNA (RAPD) markers and one sequence tagged site (STS) marker were assigned to ten linkage groups (LGs) that covered 1,274 centi Morgans (cM) of the pea genome. Six of these LGs were aligned with the previous pea map. Two QTLs were identified for lodging resistance that collectively explained 58% of the total phenotypic variation in the mean environment. Three QTLs were identified each for plant height and resistance to mycosphaerella blight, which accounted for 65% and 36% of the total phenotypic variation, respectively, in the mean environment. These QTLs were relatively consistent across environments. The AFLP marker that was associated with the major locus for lodging resistance was converted into the sequence-characterized amplified-region (SCAR) marker. The presence or absence of the SCAR marker corresponded well with the lodging reaction of 50 commercial pea varieties.Communicated by H. F. Linskens     

Pathotype-specific genetic factors in chickpea (Cicer arietinum L.) for quantitative resistance to ascochyta blight

Ascochyta blight in chickpea (Cicer arietinum L.) is a devastating fungal disease caused by the necrotrophic pathogen, Ascochyta rabiei (Pass.) Lab. To elucidate the genetic mechanism of pathotype-dependent blight resistance in chickpea, F₇-derived recombinant inbred lines (RILs) from the intraspecific cross of PI 359075(1) (blight susceptible) × FLIP84-92C(2) (blight resistant) were inoculated with pathotypes I and II of A. rabiei. The pattern of blight resistance in the RIL population varied depending on the pathotype of A. rabiei. Using the same RIL population, an intraspecific genetic linkage map comprising 53 sequence-tagged microsatellite site markers was constructed. A quantitative trait locus (QTL) for resistance to pathotype II of A. rabiei and two QTLs for resistance to pathotype I were identified on linkage group (LG)4A and LG2+6, respectively. A putative single gene designated as Ar19 (or Ar21d) could explain the majority of quantitative resistance to pathotype I. Ar19 (or Ar21d) appeared to be required for resistance to both pathotypes of A. rabiei, and the additional QTL on LG4A conferred resistance to pathotype II of A. rabiei. Further molecular genetic approach is needed to identify individual qualitative blight resistance genes and their interaction for pathotype-dependent blight resistance in chickpea.     

Linkage analysis in tetraploid potato and association of markers with quantitative resistance to late blight (Phytophthora infestans)

We have constructed a partial linkage map in tetraploid potato which integrates simplex, duplex and double-simplex AFLP markers. The map consists of 231 maternal and 106 paternal markers with total map lengths of 990.9?cM and 484.6?cM. The longer of the two cumulative map lengths represents approximately 25% coverage of the genome. In tetraploids, much of the polymorphism between parental clones is masked by `dosage' which significantly reduces the number of individual markers that can be scored in a population. Consequently, the major advantage of using AFLPs – their high multiplex ratio – is reduced to the point where the use of alternative multi-allelic marker types would be significantly more efficient. The segregation data and map information have been used in a QTL analysis of late blight resistance, and a multi-allelic locus at the proximal end of chromosome VIII has been identified which contributes significantly to the expression of resistance. No late blight resistance genes or QTLs have previously been mapped to this location.     

Genetic analyses and conservation of QTL for ascochyta blight resistance in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> 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, F₁ and F₂ 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 × 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 F₂ 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. Y. Anbessa and B. Taran contributed equally to this work.     

Major-effect QTLs for stem and foliage resistance to late blight in the wild potato relatives <Emphasis Type="Italic">Solanum sparsipilum</Emphasis> and <Emphasis Type="Italic">S. spegazzinii</Emphasis> are mapped to chromosome X

To find out new resistance sources to late blight in the wild germplasm for potato breeding, we examined the polygenic resistance of Solanum sparsipilum and S. spegazzinii by a quantitative trait locus (QTL) analysis. We performed stem and foliage tests under controlled conditions in two diploid mapping progenies. Four traits were selected for QTL detection. A total of 30 QTLs were mapped, with a large-effect QTL region on chromosome X detected in both potato relatives. The mapping of literature-derived markers highlighted colinearities with published late blight QTLs or R-genes. Results showed (a) the resistance potential of S. sparsipilum and S. spegazzinii for late blight control, and (b) the efficacy of the stem test as a complement to the foliage test to break down the complex late blight resistance into elementary components. The relationships of late blight resistance QTLs with R-genes and maturity QTLs are discussed.     

Genetic Linkage Mapping and Analysis of Muscle Fiber-Related QTLs in Common Carp (Cyprinus carpio L.)

A genetic linkage map of common carp (Cyprinus carpio L.) was constructed using Type I and Type II microsatellite markers and a pseudo-testcross mapping strategy. The microsatellite markers were isolated from microsatellite-enriched genomic libraries and tested for their segregation in a full-sib mapping panel containing 92 individuals. A total of 161 microsatellite loci were mapped into 54 linkage groups. The total lengths of the female, male and consensus maps were 2,000, 946, and 1,852?cM, with an average marker spacing of approximately 13, 7, and 11?cM, respectively. Muscle fiber-related traits, including muscle fiber cross-section area and muscle fiber density, were mapped to the genetic map. Three QTLs for muscle fiber cross-section area and two QTLs for muscle fiber density were identified when considering both significant and suggestive QTL effects. The QTLs with largest effects for muscle fiber cross-section area and muscle fiber density were 21.9% and 18.9%, and they were located in LG3, respectively.