Identification of QTLs Associated with Improved Resistance to ...

Published October 6, 2016

RESEARCH

Identification of QTLs Associated with Improved Resistance to Ascochyta Blight in an Interspecific Pea Recombinant Inbred Line Population Ambuj Bhushan Jha, Bunyamin Tar’an, Robert Stonehouse, and Thomas D. Warkentin*

ABSTRACT Ascochyta blight of pea (Pisum sp.), caused by Peyronellaea pinodes, often results in serious yield losses. Some accessions of Pisum fulvum Sibth. & Sm. previously showed high levels of resistance and could be used as sources of new resistance genes. The objectives of this study were to develop a single nucleotide polymorphism (SNP)-based linkage map and identify genomic regions associated with Ascochyta blight resistance. A population (PR-19) consisting of 144 recombinant inbred lines (RILs) developed from a cross between P651 (P. fulvum) and Alfetta (Pisum sativum L.) was used. These RILs were evaluated for Ascochyta blight resistance under field conditions in Saskatchewan, as well as under greenhouse conditions. The RILs were genotyped using an Illumina GoldenGate array panel of 1536 SNPs. A wide range of variation was observed in Ascochyta blight scores and other agronomic traits. Disease scores were positively correlated (P < 0.001) with lodging and were negatively correlated (P < 0.001) with days to flower, plant height, days to maturity, and grain yield. A total of 733 SNP markers were assigned to six linkage groups covering 682.1 cM of the pea genome. Nine quantitative trait loci (QTLs) were identified for Ascochyta blight resistance, which individually explained 7.5 to 28% of the total phenotypic variation. In addition, five QTLs each were identified for plant height, days to maturity, and grain yield, four QTLs for days to flower, and two QTLs for lodging. The QTL abIII-1 was consistent across locations and years, whereas abI-IV-2 was significant at both locations in 2014. These QTLs can be utilized for development of molecular markers associated with Ascochyta blight resistance.

A.B. Jha, B. Tar’an, R. Stonehouse, and T.D. Warkentin, Crop Development Centre/Dep. of Plant Sciences, Univ. of Saskatchewan, 51 Campus Dr., Saskatoon, SK S7N 5A8, Canada. Received 1 Jan. 2016. Accepted 22 Aug. 2016. *Corresponding author (tom.warkentin@usask. ca). Assigned to Associate Editor Hussein Abdel-Haleem. Abbreviations: AFLP, amplified fragment length polymorphism; DTF, days to flower; DTM, days to maturity; HOVTEST, homogeneity of variance test; LG, linkage group; QTL, quantitative trait locus; RAPD, random amplified polymorphic DNA; RCBD, randomized complete block design; RIL, recombinant inbred line; SNP, single nucleotide polymorphism; SSR, simple sequence repeat.

P

pinodes (Berk. & A. Bloxam) Aveskamp, Gruyter, & Verkley, formerly known as Didymella pinodes (Berk. & A. Bloxam) Petrak or Mycosphaerella pinodes (Berk. & A. Bloxam) Vestergren (Aveskamp et al., 2010), is an important pathogen that causes Ascochyta blight disease in field pea (Pisum sativum L.) in many temperate regions of the world, including Europe, North America, and Australia (Wallen, 1965; Lawyer, 1984; Kraft et al., 1998). It can cause up to 50% grain-yield loss under favorable conditions (Xue et al., 1997). In western Canada, recent disease surveys indicated the presence of this disease in all fields examined in Alberta (Chatterton et al., 2015), Manitoba (McLaren et al., 2015), and Saskatchewan (Peluola et al., 2015). Spread of the disease under field conditions is greatly affected by environmental conditions, as well as by the morphological and physiological characteristics of the plants (Wroth, 1999). Significant correlation of Ascochyta blight with lodging and plant height was reported in previous studies (Tar’an et al., 2003; Banniza et al., 2005; Jha et al., 2013). Among various strategies, genetic resistance is the most economically and ecologically sound approach to control Ascochyta eyronellaea

Published in Crop Sci. 56:2926–2939 (2016). doi: 10.2135/cropsci2016.01.0001 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

2926

www.crops.org

crop science, vol. 56, november– december 2016

blight in field pea (Fondevilla et al., 2011b). A moderate level of resistance was observed in a few accessions on evaluation of large collections of cultivated pea accessions (Kraft et al., 1998; Zhang et al., 2006); however, that is not sufficient to control this disease. Zhang et al. (2007) observed the importance of additive genetic variance and suggested that resistance can be improved through breeding. Inheritance studies indicated the quantitative nature of this disease (Wroth, 1999) and the importance of additive and dominance effects for conferring resistance (Fondevilla et al., 2007). Various studies have shown high levels of resistance to P. pinodes in Pisum fulvum Sibth. & Sm., a wild relative of field pea (Clulow et al., 1991; Wroth, 1998; Fondevilla et al., 2005). Several linkage maps have been developed in pea using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and simple sequence repeat (SSR) markers for the identification of genomic regions associated with Ascochyta blight resistance and other agronomic traits (Tar’an et al., 2003; Prioul et al., 2004; Fondevilla et al., 2008). Recently, a transcriptome sequencing approach using next-generation sequencing (NGS) technology produced a library of single nucleotide polymorphisms (SNPs) for linkage mapping in pea (Leonforte et al., 2013; Duarte et al., 2014; Sindhu et al., 2014). Quantitative trait locus (QTL) analyses in various P. sativum mapping populations have resulted in the identification of several genomic regions associated with Ascochyta blight resistance (Timmerman-Vaughan et al., 2002, 2004; Tar’an et al., 2003; Prioul et al., 2004). Timmerman-Vaughan et al. (2002, 2004) identified a large number of QTLs for disease resistance on all seven linkage groups (LGs) in two pea mapping populations, and Tar’an et al. (2003) reported three QTLs on LGs II, IV, and VI under field conditions. Prioul et al. (2004) identified six QTLs at the seedling stage on LGs III, Va, VI, and VII under controlled conditions and 10 QTLs at the adult stage on LGs II, III, Va, and VII under field conditions. Recently, QTLs were identified for Ascochyta blight resistance in a cross involving wild P. sativum ssp. syriacum accession P665 (Fondevilla et al., 2008; Fondevilla et al., 2011a). Fondevilla et al. (2008) reported six QTLs on LGs II, III, IV, and V, which collectively explained 31 to 75% of the phenotypic variation, whereas Fondevilla et al. (2011a) detected three new QTLs on LGs III and VI after enriching their previous map with additional SSR markers. More recently, QTLs and candidate genes controlling cellular mechanisms involved in Ascochyta blight resistance were identified in P665 (Carrillo et al., 2014). Using a Medicago truncatula Gaertn. microarray (Fondevilla et al., 2011b) and deepSuperSAGE transcription profiling (Fondevilla et al., 2014), differentially expressed genes involved in defense responses to P. pinodes were observed in P665. Candidate genes including RGAs (resistance gene analogs), PsDof1 (a crop science, vol. 56, november– december 2016

putative transcription factor), and DRR230-b (a pea defensin) colocated with previously described QTLs for Ascochyta blight resistance (Prioul-Gervais et al., 2007). Further, SNPs detected within candidate genes PsDof1 (PsDof1p308) and RGA-G3A (RGA-G3Ap103) showed significant associations with Ascochyta blight scores (Jha et al., 2015). Promising wild pea accessions (P. fulvum and P. sativum ssp. elatius) identified in previous research have potential for further improvement of Ascochyta blight resistance through molecular breeding (Jha et al., 2012). P651 (P. fulvum) showed the highest level of resistance against P. pinodes isolates on evaluation of 78 wild Pisum accessions (Fondevilla et al., 2005). Thus, the objectives of this research were (i) to generate a genetic linkage map of a pea recombinant inbred line (RIL) population (PR-19) derived from a cross between P651 (P. fulvum) and Alfetta (P. sativum) and (ii) to identify QTLs in PR-19 associated with Ascochyta blight resistance and potentially correlated agronomic traits.

MATERIALS AND METHODS Plant Material PR-19 was developed from a cross between the resistant accession P651 (P. fulvum) and the susceptible field pea cultivar Alfetta (P. sativum). IFPI3232, originated in Syria, was the original code for P651, which was obtained by CSIC (Cordoba, Spain) from ICARDA (Syria). P651 was chosen as the resistance source after an evaluation study was performed under greenhouse and field conditions using 44 wild pea accessions obtained from the USDA (Pullman, WA) and the CSIC (Cordoba, Spain), which included P. fulvum (26), P. sativum ssp. elatius (6), P. sativum ssp. transcaucasicum (4), P. sativum ssp. abyssinicum (4), P. sativum ssp. asiaticum (3), and P. sativum ssp. sativum var. arvense (1) ( Jha et al., 2012). A single F1 plant was used to generate approximately 300 F2 seeds through clonal propagation. In this method, several cuttings were made using a sharp knife from the top-growing branch just below the leaf node. The cut end was dipped into 0.1% indole-3-butyric acid rooting powder (Plant-Prod Stim-Root No. 1, Plant Products Co. Ltd., Brampton, ON), then inserted into a peat moss plug placed on a tray, which was transferred to a misting chamber until roots were developed. Rooted cuttings were transferred to potting soil in individual pots in the greenhouse and allowed to self. Generation F6 RILs were generated by self-pollination using a single seed descent from the F2 generation. Seeds of the F7 for conducting the 2013 field trial were generated by bulking the seeds produced from three F6 greenhouse-grown plants, and the resulting F7:8 seeds were used for evaluation of Ascochyta blight resistance in 2014 under greenhouse and field conditions.

Assessment of Ascochyta Blight Resistance and Other Agronomic Traits under Field Conditions Depending on the availability of seeds, a total of 127 RILs of PR-19 were evaluated for Ascochyta blight resistance in two replicates under field conditions in 2013 at Saskatoon, and in 2014 at Saskatoon and Rosthern, with three replicates at each

www.crops.org 2927

location. Plots were not inoculated with P. pinodes, as natural infection levels were sufficient. The experimental design was a randomized complete block design (RCBD) with three-row plots of 1.0 ´ 1.0 m, a plant density of 75 plants m−2, and row spacing of 0.25 m. Plants were scored for Ascochyta blight severity on a plot basis using a scale of zero (no disease) to nine (whole plant severely blighted), based on Xue et al. (1996). The first disease score was recorded at the midflowering stage, and subsequent assessments were taken at 7- to 10-d intervals until physiological maturity (80% of the pods in the plot turned brown). Recombinant inbred lines were also evaluated for agronomic traits, including days to flower (DTF), plant height, lodging, days to maturity (DTM), and grain yield on a plot basis. Days to flower and DTM were calculated as the number of days from planting to 50% bloom and physiological maturity, respectively. Plant height was measured from the soil level to the tip of the central stem, while lodging was assessed on a one (upright) to nine (completely lodged) scale at physiological maturity.

Assessment of Ascochyta Blight Resistance under Greenhouse Conditions PR-19 RILs (144) were evaluated for Ascochyta blight resistance under greenhouse conditions in 2014, in four repeated experiments using an RCBD with four plant replicates. Plants were grown at 22 ± 3°C day/20 ± 3°C night temperatures under an 18-h photoperiod with approximately 60% relative humidity, and an integrated photosynthetic active radiation of 210 mmol m−2 s−1. Three-week-old plants were wrapped with translucent plastic cones and inoculated with spore suspensions of P. pinodes isolate PP25 collected from a commercial field in Saskatchewan at a concentration of 5 ´ 104 spores mL−1, similar to that of Jha et al. (2012). Plants were scored for Ascochyta blight severity 1 wk (AB1-GH) and 2 wk (AB2-GH) after inoculation using the zero-to-nine scale, as described earlier.

Genotyping, Linkage Mapping, and QTL Analysis Genotypic data generated through 1536 Illumina GoldenGate assays by Sindhu et al. (2014) were used for linkage mapping and QTL analysis. GenomeStudio software 2010.3 (Illumina, 2010) was used for data clustering and allele calling. The linkage map, using genotyping from 133 RILs, was constructed using Carthagene 1.2.2 software (de Givry et al., 2005). Out of the 144 RILs genotyped, 11 RILs were removed from linkage map construction due to poor allele calling. The Kosambi map function was used to calculate map distances. The frame of the map was created using the markers not showing distorted segregation, and then adding those showing distorted segregation using a combination of the Build, Annealing, Taboo, and Flips commands. Linkage groups were assigned on the basis of comparison of shared markers with the pea consensus map generated by Sindhu et al. (2014). Quantitative trait loci were identified by composite interval mapping (CIM) using Windows QTL Cartographer 2.5 (Wang et al., 2012). The significance threshold (P < 0.05) was used to declare the presence of QTLs for each trait by performing 1000 permutations of the data (Churchill and Doerge, 1994). Linkage maps and QTLs were graphically shown using MapChart 2.2 (Voorrips, 2002). 2928

Statistical Analysis Data were analyzed using PROC MIXED implemented in SAS 9.3 (SAS Institute, 2011). Line (genotype) was treated as a fixed effect and replication as random across the RILs. Homogeneity of variance test (HOVTEST) was used to assess the homogeneity of variance among replications.

RESULTS Ascochyta Blight Resistance and Other Agronomic Traits under Field Conditions Data across years and locations can be combined when the variance is not significant at P ³ 0.05 in HOVTEST. Since location and year were significant in HOVTEST, datasets were not combined for analysis of variance (ANOVA) or QTL analysis. A wide range of variation was observed for Ascochyta blight scores and for the other agronomic traits evaluated under field conditions (Table 1, Fig. 1 and 2). The effect of line (RIL) was significant (P < 0.001) for all measured traits at all station–years. Ascochyta blight scores ranged from zero to four at midflowering, one to five at late flowering, one to six at pod development, two to eight at pod filling, and two to nine at physiological maturity (0–9 scale). Days to flower ranged from 31 to 54 d, and plant height ranged from 10 to 170 cm. Lodging scores varied from one to nine on the one-to-nine scale. Days to maturity varied from 75 to 101 d, and grain yield varied from 3 to 4209 kg ha−1. Disease scores in 2014 were positively correlated (r = 0.74, P < 0.001) with scores in 2013. Similarly, disease scores at Rosthern in 2014 had a positive correlation (r = 0.71, P < 0.001) with scores at Saskatoon. Ascochyta blight score at the pod filling stage was positively correlated with lodging (r = 0.49, P < 0.001) and negatively correlated with DTF (r = −0.43, P < 0.001), plant height (r = −0.64, P < 0.001), DTM (r = −0.63, P < 0.001), and grain yield (r = −0.67, P < 0.001) (Table 2). A wide range of variation was observed in Ascochyta blight scores and agronomic traits (Fig. 1 and 2).

Ascochyta Blight Resistance under Greenhouse Conditions Similar to field conditions, the effect of line (RIL) was significant (P < 0.001) for each greenhouse experiment. Homogeneity of variance tests for the greenhouse experiments indicated that the results from these experiments should not be combined. Ascochyta blight scores of RILs for AB1-GH ranged from zero to six for the first and third experiments and one to six for the second and fourth experiments (Table 3). The Ascochyta blight severity of RILs at 2 wk ranged from two to nine for the first and one to nine for the third experiment, whereas the scores ranged from three to eight for the second and three to nine for the fourth experiment. P651 disease scores ranged

www.crops.org


Table 1. F-values, coefficients of variations (CV) of statistical analyses, and means with standard deviations (SD) of Ascochyta blight scores and other agronomic assessments for 127 PR-19 recombinant inbred lines evaluated under field conditions in 2013 at Saskatoon and in 2014 at Saskatoon and Rosthern, SK. AB1† Saskatoon 2013

Saskatoon 2014

Rosthern 2014

Line Range

AB2

AB3

AB4

AB5

——————————————— 0–9 scale ——————————————— 1.8*** 3.9*** 4.1*** 6.3*** 10.8*** 1.0–4.0 1.0–4.0 2.0–6.0 2.0–8.0 3.0–9.0

DTF

Plant height

Lodging

DTM

d 7.7*** 31–49

cm 59.4*** 11–101

1–9 scale 7.9*** 2.0–9.0

d 6.9*** 75–101

Mean ± SD 2.2 ± 0.5 CV (%) 24.9 Line 3.2*** Range 1.0–4.0 Mean ± SD 2.6 ± 0.7 CV (%) 26.4 Line 2.2*** Range 0–3.0

2.8 ± 0.7 24.5 2.8*** 2.0–5.0 3.3 ± 0.6 17.6 2.2*** 1.0–4.0

3.2 ± 0.8 24.2 4.1*** 2.0–6.0 3.5 ± 0.7 19.1 4.1*** 1.0–5.0

4.6 ± 1.3 28.5 6.2*** 2.0–6.0 4.0 ± 0.8 20.4 7.1*** 2.0–6.0

5.8 ± 1.3 21.9 11.7*** 2.0–8.0 5.0 ± 1.2 23.1 11.4*** 2.0–8.0

40.9 ± 3.8 37.7 ± 18.1 9.2 48.0 13.6*** 29.6*** 46–54 13–170 49.5 ± 1.6 54.7 ± 28.5 3.3 52.1 15.7*** 23.1*** 46–54 10–137

Mean ± SD 1.4 ± 0.6 CV (%) 38.8

2.1 ± 0.5 25.8

2.8 ± 0.7 26.7

3.7 ± 0.9 23.1

5.3 ± 1.1 21.3

49.0 ± 1.9 60.2 ± 29.6 5.2 ± 1.7 3.9 49.1 33.2

Grain yield kg ha−1 10.5*** 3–4209

6.1 ± 1.6 90.0 ± 8.3 837 ± 109 26.6 9.2 87.4 8.01*** 505.1*** 17.3*** 1.0–9.0 80–98 10–4138 6.3 ± 1.7 92.8 ± 5.0 814 ± 98 27.7 5.4 82.4 8.3*** 230.5*** 6.9*** 1.0–9.0 85–99 3–3412 90.6 ± 4.6 845 ± 91 5.1 73.5

*** Significant at the 0.001 level. † AB1, AB2, AB3, AB4, and AB5 denote Ascochyta blight scores at the midflowering, late flowering, pod development, pod filling, and physiological maturity stages, respectively; DTF, days to flower; DTM, days to maturity.

from zero to four (AB1-GH) and two to six (AB2-GH), whereas Alfetta scores ranged from one to five (AB1-GH) and five to nine (AB2-GH). The disease scores of P651 and Alfetta never overlapped at any stage of evaluation. In all four repeated experiments, mean Ascochyta blight score was greater at AB2 than AB1, and the coefficient of variation for Ascochyta blight score was less for AB2 than AB1. Figure 3 shows the frequency distributions of 144 RILs based on least squares means. No definite trend was observed in correlation between disease scores from the greenhouse and field experiments.

Linkage Map Out of 1536 SNP markers tested, 768 were polymorphic between the parents, Alfetta and P651. Among them, 733 were utilized in mapping, while 35 were unlinked. A total of 386 (52.7%) markers exhibited segregation distortion at the P < 0.01 significance level. Each of the 733 SNPs was mapped to one of six LGs that, in total, covered 682.1 cM of the pea genome, with an average marker interval of 0.93 cM (Fig. 4). In this study, six LGs instead of the seven typically reported for pea were observed, because LGs I and IV were joined, and this linkage group was therefore named LG I-IV. Linkage groups were assigned on the basis of the consensus map reported by Sindhu et al. (2014). Linkage group I-IV was the largest, with 209 markers and an average marker interval of 0.93 cM, whereas LG VI was the smallest, with 92 markers and the shortest average marker interval of 0.85 cM.

Identification of QTLs for Ascochyta Blight Resistance and Other Agronomic Traits Six QTLs (abI-IV-1, abI-IV-2, abI-IV-3, abI-IV-4, abIII1, and abVII-1) were identified for Ascochyta blight crop science, vol. 56, november– december 2016

resistance at different stages on LGs I-IV, III, and VII under field conditions (Table 4). Individually, these QTLs explained 7.5 (abI-IV-2) to 28% (abIII-1) of the total phenotypic variation. Three additional QTLs (abI-IV-5, abIII-2, and abVII-2) were identified at AB1-GH stage under greenhouse conditions and, individually, these QTLs contributed 11 to 12.5%, of the total phenotypic variation. For QTLs abI-IV-1, abI-IV-4, and abIII-2, parent Alfetta contributed alleles for Ascochyta blight resistance, whereas for the other six QTLs, the wild pea parent P651 contributed alleles. In addition, five QTLs each were identified for plant height, DTM, and grain yield, four QTLs for DTF, and two QTLs for lodging. In the majority of cases, P651 alleles decreased the value of the traits (Table 4).

DISCUSSION In this research, a high density genetic linkage map constructed from gene-based SNPs was used to identify QTLs for Ascochyta blight resistance in a mapping population utilizing wild pea accession P651 (P. fulvum). Previous studies have identified several QTLs associated with Ascochyta blight resistance in P. sativum and wild P. sativum ssp. syriacum using mainly AFLP, RAPD, and SSR markers (Tar’an et al., 2003; Prioul et al., 2004; Fondevilla et al., 2008). Accession P651 utilized in this research was the most promising for resistance breeding, as it had a relatively low disease score under field, as well as greenhouse, conditions (Jha et al., 2012). Earlier, Fondevilla et al. (2005) observed that P651 (P. fulvum) had the highest level of resistance to P. pinodes, followed by P670 (P. sativum ssp. elatius) and P665 (P. sativum ssp. syriacum). Further, P651 was the most resistant against the isolates of P. pinodes collected from France, Spain, Poland, Canada, and Japan (Fondevilla et al., 2005). In histological studies on wild peas including P651, Carrillo et al. (2013) indicated that

www.crops.org 2929

Fig. 1. Frequency distribution of 127 PR-19 recombinant inbred lines (RILs) using least squares means of 2013 Saskatoon, 2014 Saskatoon, and 2014 Rosthern for the reaction to Ascochyta blight resistance at different stages under field conditions.

resistance to P. pinodes could be due to reduced lesion size and colony development, and that these events were associated with epidermal cell death and protein crosslinking. Various researchers have emphasized the need for efficient utilization of wild pea accessions in breeding for resistance to P. pinodes, but success has been hampered by the polygenic nature of resistance (Wroth, 1999; Prioul et al., 2004; Muehlbauer and Chen, 2007; Fondevilla et al., 2008; Fondevilla et al., 2011a). 2930

In recent years, several gene-based linkage maps have been developed in pea (Deulvot et al., 2010; Leonforte et al., 2013; Carrillo et al., 2014; Duarte et al., 2014). Recently, a pea consensus map consisting of 939 SNPs was created using data from five RIL populations, including 303 SNPs from PR-19, genotyped using Illumina GoldenGate technology (Sindhu et al., 2014). In the current research, the same genotyping data was used to generate a stand-alone linkage map

www.crops.org


Fig. 2. Frequency distribution of 127 recombinant inbred lines (RILs) of PR-19 using least squares means of 2013 Saskatoon, 2014 Saskatoon, and 2014 Rosthern for days to flower, plant height, lodging, days to maturity, and grain yield under field conditions. Table 2. Pearson correlation coefficients for traits of 127 PR-19 recombinant inbred lines evaluated in 2013 at Saskatoon and in 2014 at Saskatoon and Rosthern, SK. AB4 Plant height Lodging DTM Grain yield

DTF†

AB4

Plant height

Lodging

DTM

−0.43*** 0.19* −0.21* 0.40*** 0.25**

−0.64*** 0.49*** −0.63*** −0.67***

−0.04NS 0.64*** 0.33***

−0.17NS −0.67***

0.27**

* Significant at the 0.05 level; ** significant at the 0.01 level; *** significant at the 0.001 level; NS, not significant. † DTF, days to flower; AB4, Ascochyta blight score at the pod filling stage; DTM, days to maturity crop science, vol. 56, november– december 2016

of PR-19 consisting of 733 SNP markers and covering a genetic distance of 682.1 cM. This map is more saturated than previous maps used for QTL analysis, with an average marker interval of 0.93 cM (Tar’an et al., 2003; Prioul et al., 2004; Timmerman-Vaughan et al., 2004; Fondevilla et al., 2008). Population PR-19 was part of the consensus map of Sindhu et al. (2014), in which they only used 303 highquality polymorphic SNP markers. There were many markers not used in the consensus map because they exhibited varying degrees of segregation distortion. In

www.crops.org 2931

Table 3. F-values, coefficients of variations (CV) of statistical analyses, and means with standard deviations (SD) of Ascochyta blight scores for 144 PR-19 recombinant inbred lines evaluated under greenhouse conditions in 2014. Trait AB1-GH

AB2-GH

GH1†

GH2

GH3

GH4

Line Range

4.2*** 0.0–6.0

3.0*** 1.0–6.0

3.9*** 0.0–6.0

3.9*** 1.0–6.0

Mean ± SD CV (%) Line Range

2.9 ± 1.1 37.5 5.3*** 2.0–9.0

3.5 ± 1.0 28.9 3.5*** 3.0–8.0

2.3 ± 1.1 50.6 6.2*** 1.0–9.0

4.1 ± 1.1 27.0 5.0*** 3.0–9.0

Mean ± SD CV (%)

5.2 ± 1.3 24.3

5.6 ± 1.2 20.8

4.5 ± 1.6 34.5

6.3 ± 1.1 17.5

*** Significant at the 0.001 level. † GH1, GH2, GH3, and GH4 denote four repeated experiments under greenhouse conditions; AB1-GH and AB2-GH denote Ascochyta blight scores at 7 and 14 d after inoculation, respectively.

this study, a large number of markers (52.7%) exhibited segregation distortion at the P < 0.01 significance level. The map presented here includes many of these distorted markers; however, the frame of the map was created using only the high-quality markers. The rest of the markers were then added using Carthagene commands, as described in Materials and Methods, and only included if their positions compared well with the other individual and consensus pea maps. Distorted segregation affects the estimated recombination fractions between loci and the resulting estimated linkage map distance, and reduces the power of detecting QTL (Wang et al., 2005; Xu, 2008). Distorted segregation is frequently noticed in progeny of inter- and intraspecific hybrids, due to competition among gametes or abortion of the gamete or zygote (Faris et al., 1998).

Fig. 3. Frequency distribution of 144 recombinant inbred lines (RILs) of PR-19 using least squares means of the four repeated experiments (A–D) for reaction to Ascochyta blight resistance under greenhouse conditions in 2014.

2932

www.crops.org


Fig. 4a. Linkage map of 133 recombinant inbred lines (RILs) of PR-19 (Alfetta ´ P651) developed from 733 single nucleotide polymorphism (SNP) markers. Abbreviations LG I-IV to LG III represent three of the six linkage groups. Markers with segregation distortion at P < 0.01 are shown in italics. These groups were assigned on the basis of the consensus map reported by Sindhu et al. (2014). Red dotted lines indicate the location where LGs I and IV are joined. The left side of each linkage group shows the genetic distances in cM. Locations of quantitative trait loci (QTLs) for a given trait are shown by vertical bars, which indicate 1-LOD (logarithm of odds) support intervals. Abbreviations S13, S14, R14, and GH associated with QTL names denote 2013 Saskatoon, 2014 Saskatoon, 2014 Rosthern, and Greenhouse, respectively. Underlined markers represent the closest marker to the identified QTL with maximum LOD value.


www.crops.org 2933

Fig 4b. Continued linkage map of 133 recombinant inbred lines (RILs) of PR-19 (Alfetta ´ P651) developed from 733 single nucleotide polymorphism (SNP) markers. Abbreviations LG V to LG VII represent the three of the six linkage groups. Markers with segregation distortion at P < 0.01 are shown in italics. These groups were assigned on the basis of the consensus map reported by Sindhu et al. (2014). The left side of each linkage group shows the genetic distances in cM. Locations of quantitative trait loci (QTLs) for a given trait are shown by vertical bars, which indicate 1-LOD (logarithm of odds) support intervals. Abbreviations S13, S14, R14, and GH associated with QTL names denote 2013 Saskatoon, 2014 Saskatoon, 2014 Rosthern, and Greenhouse, respectively. Underlined markers represent the closest marker to the identified QTL with maximum LOD value.

2934

www.crops.org


Table 4. Quantitative trait loci (QTLs) detected for reaction to Ascochyta blight resistance, days to flower (DTF), plant height, lodging, days to maturity (DTM), and grain yield at Saskatoon under field conditions in 2013 and under greenhouse conditions in 2014, and at Saskatoon and Rosthern under field conditions in 2014. Trait

Year

Location

Linkage group†

Locus‡

abIII-2 abVII-1 abVII-2 phI-IV-1

AB2, AB3, AB4# AB1, AB2, AB3 AB1, AB2, AB3 AB1, AB2 AB3, AB4 AB1-GH1, AB1-GH4 AB2, AB3, AB4, AB5 AB3, AB4, AB5 AB3, AB4, AB5 AB1-GH2, AB1-GH3 AB2 AB1-GH2 Plant height

phI-IV-2 phI-IV-3

Plant height Plant height

phIII-1

Plant height

phVII-1

Plant height

lodgIII-1

Lodging

lodgVII-1 dtfI-IV-1 dtfI-IV-2

Lodging DTF DTF

2013 2014 2014 2014 2013 2014 2013 2014 2014 2014 2014 2014 2013 2014 2013 2014 2014 2013 2014 2014 2013 2014 2014 2013 2014 2014 2013 2014 2014 2013 2014 2014 2013 2013 2014 2014 2013 2014 2014 2013 2014 2014 2013 2014 2014 2014 2014 2014 2014 2014

Saskatoon Rosthern Saskatoon Rosthern Saskatoon Greenhouse Saskatoon Rosthern Saskatoon Greenhouse Rosthern Greenhouse Saskatoon Rosthern Saskatoon Rosthern Saskatoon Saskatoon Rosthern Saskatoon Saskatoon Saskatoon Rosthern Saskatoon Rosthern Saskatoon Saskatoon Rosthern Saskatoon Saskatoon Rosthern Saskatoon Saskatoon Saskatoon Saskatoon Rosthern Saskatoon Rosthern Saskatoon Saskatoon Saskatoon Rosthern Saskatoon Rosthern Saskatoon Rosthern Saskatoon Rosthern Saskatoon Saskatoon

I-IV I-IV I-IV I-IV I-IV I-IV III III III III VII VII I-IV I-IV I-IV I-IV I-IV III III III VII VII VII III III VII I-IV I-IV I-IV III III III V I-IV I-IV I-IV III III III V VII VII I-IV I-IV I-IV I-IV II VI VI VII

PsC20818p367 PsC8031p219 PsC8031p219 PsC7497p542 PsC13000p248 PsC12819p70 PsC8780p118 PsC22609p103 PsC22609p103 PsC12032p118 PsC4701p407 PsC7233p842 PsC20818p367 PsC20818p367 PsC21835p265 PsC943p541 PsC4233p498 PsC16976p219 PsC12032p118 PsC12032p118 PsC19517p115 PsC4701p407 PsC6738p294 PsC8780p118 PsC22609p103 PsC4701p407 PsC20787p97 PsC19558p107 PsC4233p498 PsC8780p118 PsC22609p103 PsC22609p103 PsC5495p478 PsC19763p599 PsC4233p498 PsC4233p498 PsC8780p118 PsC22609p103 PsC22609p103 PsC5495p478 PsC4701p407 PsC4701p407 PsC12819p70 PsC20818p367 PsC20818p367 PsC21343p133 PsC19573p398 PsC14811p215 PsC8338p234 PsC14648p140

QTL abI-IV-1 abI-IV-2 abI-IV-3 abI-IV-4 abI-IV-5 abIII-1

dtfIII-1

DTF

dtfV-1 dtmI-IV-1 dtmI-IV-2

DTF DTM DTM

dtmIII-1

DTM

dtmV-1 dtmVII-1

DTM DTM

gyI-IV-1

Grain yield

gyI-IV-2 gyII-1 gyVI-1

Grain yield Grain yield Grain yield

gyVII-1

Grain yield

Max. LOD Additive genetic % Variation§ value effect¶ 5.6 6.3 3.2 4 4.9 4.1 10.3 3.6 4.8 3.9 4.5 3.8 10.3 4.4 4 6.1 4.8 18.8 8.6 11.2 3.4 3.6 3.1 6.8 4.5 3.8 3.2 4.8 4.4 35.9 2.5 2.8 14.1 6.8 4.4 4.3 36.1 2.5 3.1 10.9 3.0 3.1 5.1 5.7 5.2 4.5 3.9 6.8 4.8 3.7

12.6 16.2 7.5 10.3 11.9 11.2 27.8 9.2 11.9 12.5 10.9 10.9 16.6 9.2 5.8 12.9 11.2 37.5 20.8 28.2 5.2 7.4 6.2 18.9 12.2 9.7 5.4 11.1 10.5 59.0 5.6 6.3 20.9 11.6 11.1 10.8 55.5 5.6 6.8 15.5 6.5 6.6 11.6 11.8 10.9 9.3 8.1 14.4 10.9 7.8

−0.3 0.4 0.5 0.3 −0.5 0.4 3.1 1.0 1.2 −0.4 0.3 0.4 7.7 10.4 4.5 17.5 16.4 −13.1 −17.5 −19.9 4.9 11.7 10.2 3.1 1.9 1.0 1.5 7.9 7.8 19.5 9.3 10.0 −20.2 5.4 15.7 14.7 42.0 17.1 19.4 −38.5 10.5 10.3 42.0 30.9 32.0 27.8 26.7 33.5 32.0 30.2

† Linkage group was assigned on the basis of consensus map reported by Sindhu et al. (2014). ‡ Closest marker to the identified QTL with maximum LOD (logarithm of odds) value. § Percentage of total variability explained by the QTL detected for the trait. ¶ The value associated with the Alfetta allele; a negative value means that the Alfetta allele decreases the value of the trait, while a positive value means that the Alfetta allele increases the value of the trait. # AB1, AB2, AB3, AB4, and AB5 denote Ascochyta blight scores at the midflowering, late flowering, pod development, pod filling, and physiological maturity stages, respectively. AB1-GH1, AB1-GH2, AB1-GH3, and AB1-GH4 denote Ascochyta blight scores at 7 d after inoculation for four repeated experiments. crop science, vol. 56, november– december 2016

www.crops.org 2935

Xu (2008) reported that the presence of a few segregation distortion loci (SDL) can cause the entire chromosome to distort from Mendelian segregation but can be useful to QTL mapping when distortion of a locus is random. Previous reports have typically presented maps of seven or more linkage groups representing the seven chromosomes of pea (Tar’an et al., 2003; Prioul et al., 2004; Fondevilla et al., 2008; Duarte et al., 2014; Sindhu et al., 2014). The PR-19 map is depicted here as six linkage groups (Fig. 4). Linkage group I-IV is presented here as one linkage group, rather than two, because standard mapping analysis was unable to separate them. This inability to define break points between known distinct linkage groups is often due to a translocation event that exists in one of the parents relative to the other. Markers from the PR-19 map were compared with several maps from cultivated crosses, the consensus map of Sindhu et al. (2014), as well as a linkage map developed from a cross between P651 and another wild pea accession, W6 15017 (P. fulvum) (Jha et al., unpublished data, 2016). The markers defining the translocation block were only polymorphic in the PR-19 population, and these comparisons allowed for characterization of the translocation region, but did not allow for assignment of a map location in cultivated or wild pea. Once the physical map of pea is available, it could be compared with the PR-19 map to localize the translocation region. Indeed, several translocations between P. fulvum and P. sativum have been reported based on genetic and cytological analyses (Lamprecht, 1964; Ben-Ze’ev and Zohary, 1973; Errico et al., 1991). Conicella and Errico (1985) and Errico et al. (1991) demonstrated two independent, reciprocal interchanges in P. fulvum that involved chromosome pairs I and VII and III and V, relative to P. sativum. Lamprecht (1964) observed three translocations in P. fulvum in comparison with cultivated pea, whereas Ben Ze’ev and Zohary (1973) reported two translocations in P. fulvum when these were crossed with P. sativum. Errico et al. (1991) observed two translocations in meiotic data from F1 plants obtained by crosses between P. sativum with P. fulvum. In this research, a total of nine QTLs were identified for Ascochyta blight resistance. Six QTLs were identified under field conditions, and three additional QTLs were detected under greenhouse conditions. Individually, these QTLs explained 7.5 to 28% of the total phenotypic variation. Wild parent P651 contributed the alleles for resistance for six QTLs, whereas Alfetta contributed the allele for three QTLs. One reason for observing different QTLs under field and greenhouse conditions could be that, while plants in the greenhouse were inoculated with a single isolate (PP25), lines in the field were exposed to the natural variation of Ascochyta isolates. Indication of pathogenic variation among isolates was reported based on differential reactions of pea cultivars to P. pinodes isolates (Xue et al., 1998; Zhang et al., 2003). Xue et al. 2936

(1998) reported 22 pathotypes of P. pinodes from 275 isolates collected from infected fields from Manitoba (147), Saskatchewan (76), and Alberta (52), Canada. Among the 275 isolates tested on 21 pea genotypes, 47 showed a specific genotype–isolate interaction. Similarly, Zhang et al. (2003) identified 15 pathotypes from 58 isolates collected from western Canada, New Zealand, France, Australia, the United Kingdom, and Ireland. Thirty-three out of 58 isolates were collected from infected fields in Manitoba, Saskatchewan, and Alberta, Canada. According to the virulence effect of the isolates, they observed that 57.2 and 42.8% of the total variation were due to differences among populations and molecular diversity within populations, respectively. Under field conditions, QTLs abI-IV-2 (PsC8031p219), abI-IV-3 (PsC7497p542), and abI-IV-4 (PsC13000p248) could account for resistance, whereas abI-IV-1 (PsC20818p367), abIII-1 (PsC8780p118), and abVII-1 (PsC4701p407) could account for disease avoidance and/or resistance, as these loci were concurrently associated with other traits including plant height and/or lodging. Alternatively, resistance under field conditions could be due to physiological resistance (Khan et al., 2013). Canopy architecture features such as branching, lodging resistance, stem height, and leaf area index could affect the impact of Ascochyta blight disease under field conditions to a greater extent than under greenhouse conditions, as these affect microclimate within the canopy and splash dispersal of P. pinodes conidia (Schoeny et al., 2008; Le May et al., 2009). Differences in the growth stages when the plants were rated in the field and greenhouse experiments would be an important factor, and the effects of differences in height and lodging would not be important factors for disease development under greenhouse conditions. Plants were evaluated for disease resistance during early growth stages under greenhouse conditions, whereas evaluation was done during the midflowering stage and onward under field conditions, and that could also be one of the possible reasons for not observing the same QTLs under different conditions. Out of nine QTLs for disease resistance, five were on LG I-IV and two each were on LG III and VII. Under field conditions, QTLs abI-IV-1, abI-IV-2, and abIII-1 were present in data from three out of five evaluation growth stages in either 2013 or 2014, whereas under greenhouse conditions, QTLs abI-IV-5 and abIII-2 were detected in two out of four experiments. The QTL abIII-1 that was present on LG III in data from four out of five growth stages under field conditions in both years and locations in 2014 was most promising. This QTL explained 28% of the phenotypic variation, and P651 contributed the allele for resistance. Previously, several QTLs were identified for Ascochyta blight resistance on LG III in pea mapping populations (Timmerman-Vaughan et al., 2002, 2004; Prioul et al., 2004; Fondevilla et al., 2008, 2011a; Carrillo et al.,

www.crops.org


2014), and LG III has harbored the most QTLs for Ascochyta resistance identified to date. Using common SSR markers, Fondevilla et al. (2011a) confirmed that three QTLs identified in P. sativum ssp. syriacum, MpIII.1, MpIII.3, and MpIII.2, were corresponding to the QTLs mpIII-1, mpIII3, and mpIII-5 detected in P. sativum by Prioul et al. (2004). Further, a comparative analysis revealed that QTL MpIII.1 for disease resistance under growth chamber, as well as field conditions, explained up to 29% of the phenotypic variation (Fondevilla et al., 2008) was located on the same distal part of LG III where Prioul et al. (2004) reported QTL mpIII-1, which explained up to 42% of the phenotypic variance. Colocalization of RGAs and defense-related genes with QTLs for resistance to P. pinodes were observed in pea (Timmerman-Vaughan et al., 2002; Prioul-Gervais et al., 2007). Prioul-Gervais et al. (2007) observed colocations of candidate genes PsDof1 and DRR230-b with QTLs mpIII-1 and mpIII-4 associated with Ascochyta blight resistance, found by Prioul et al. (2004) on LG III in pea. In the same region on LG III, Timmerman-Vaughan et al. (2002) and Fondevilla et al. (2008) found QTLs Asc3.1 and MpIII.1 for Ascochyta blight resistance in different mapping populations, respectively. In this study, five QTLs were present on LGI-IV and two QTLs were present on LG VII. Similar to our findings, a number of QTLs were identified on these linkage groups by various investigators in P. sativum and P. sativum ssp. syriacum. Of note, Timmerman-Vaughan et al. (2002, 2004) identified several QTLs on LGs I, IV, and VII, and Tar’an et al. (2003) detected one QTL on LG IV in P. sativum. Further, Prioul et al. (2004) detected two QTLs on LG VII in P. sativum, whereas Fondevilla et al. (2008, 2011a) identified one QTL on LG IV in P. sativum ssp. syriacum. Timmerman-Vaughan et al. (2002) reported colocalization between RGAs, RGA1.1, RGA2.97, and RGA-G3A and QTLs for Ascochyta blight resistance on LG VII. At the same locus, Prioul-Gervais et al. (2007) reported colocalization of RGAs (RGA2, RGA3, RGA-G3A, IJB174, and IJB91) with the QTL mpVII-1 (Prioul et al., 2004) for Ascochyta blight resistance. More recently, Jha et al. (2015) reported significant associations between Ascochyta blight scores and SNPs detected within candidate genes PsDof1 (PsDof1p308) and RGA-G3A (RGA-G3Ap103). On the basis of shared anchored markers used in Sindhu et al. (2014) with previous pea maps, the location of QTLs identified in the present research was compared with QTLs reported in P. sativum (Prioul et al., 2004; Prioul-Gervais et al., 2007) and P. sativum ssp. syriacum (Fondevilla et al., 2008, 2011a; Carrillo et al., 2014). None of the currently identified QTLs was located in regions where these investigators reported QTLs for Ascochyta blight resistance. In the present research, five QTLs each were identified for plant height, DTM, and grain yield, four QTLs for DTF, and two QTLs for lodging. crop science, vol. 56, november– december 2016

The P. fulvum parent P651 alleles decreased the value of traits in the majority of QTLs. Previous researchers also reported several QTLs for agronomic traits in pea (TimmermanVaughan et al., 2002, 2004; Tar’an et al., 2003; Prioul et al., 2004; Burstin et al., 2007; Fondevilla et al., 2008). The current study identified four loci on each of the LGs I-IV, III, V, and VII associated with multiple traits. The locus PsC8780p118 on LG III was associated not only with Ascochyta blight resistance, but also with lodging, DTF, and DTM. Parent P651 contributed an allele for all traits, and this locus explained 19 (lodging) to 55.5% (DTM) of total phenotypic variation. Ascochyta blight scores were positively correlated with lodging and negatively correlated with DTF and DTM. Similar to this study, previous researchers had also reported correlations between Ascochyta blight, lodging, plant height, and DTM (Tar’an et al., 2003; Banniza et al., 2005; Conner et al., 2007; Jha et al., 2013). A locus associating with multiple traits could be explained by genetic linkage or pleiotropic effects of genes (Prioul et al., 2004; Timmerman-Vaughan et al., 2004; Fondevilla et al., 2008) and could contribute to correlations between different traits (Tar’an et al., 2003). Overlapping QTLs associated with Ascochyta blight resistance and plant maturity were observed on LGs II, IIb, III, and V in pea (Timmerman-Vaughan et al., 2004). A common locus (PsC20818p367) on LG I-IV was associated with Ascochyta blight resistance, plant height, and grain yield. Parent Alfetta alleles increased disease resistance, plant height, and grain yield. Similarly, locus PsC4701p407 on LG VII was associated with Ascochyta blight resistance, plant height, lodging, and DTM. P651 alleles increased resistance and decreased plant height, lodging, and DTM values. Colocalizations between QTLs for Ascochyta blight resistance and QTLs for plant height and flowering date were identified on LGs II, III, and VI in pea (Prioul et al., 2004). Markers can be designed within identified QTL regions and used in breeding programs to help develop pea cultivars with improved disease resistance. Further, QTL abIII-1 was consistent across locations and years, whereas abI-IV-2 was significant at both locations in 2014. These QTLs were utilized for development of heterogeneous inbred family populations, which will be used for fine mapping to identify closely linked markers and/or potential candidate genes for Ascochyta blight resistance.

Conflict of Interest The authors declare that there is no conflict of interest. Acknowledgments The Saskatchewan Ministry of Agriculture, Saskatchewan Pulse Growers, and Western Grains Research Foundation are gratefully acknowledged for financial support. We are thankful to Dr. Sabine Banniza for critically reviewing the manuscript. We are also thankful to Kamal Bandara, Brent Barlow, and other pulse crop field lab members for technical assistance.

www.crops.org 2937

References Aveskamp, M.M., J. de Gruyter, J.H.C. Woudenberg, G.J.M. Verkley, and P.W. Crous. 2010. Highlights of the Didymellaceae: A polyphasic approach to characterise Phoma and related pleosporalean genera. Stud. Mycol. 65:1–60. doi:10.3114/ sim.2010.65.01 Banniza, S., P. Hashemi, T.D. Warkentin, A. Vandenberg, and A.R. Davis. 2005. The relationship among lodging, stem anatomy, degree of lignification and susceptibility to mycosphaerella blight in field pea (Pisum sativum). Can. J. Bot. 83:954–967. doi:10.1139/b05-044 Ben-Ze’ev, N., and D. Zohary. 1973. Species relationships in the genus Pisum L. Isr. J. Bot. 22:73–91. Burstin, J., P. Marget, M. Huart, A. Moessner, B. Mangin, C. Duchene et al. 2007. Developmental genes have pleiotropic effects on plant morphology and source capacity, eventually impacting on seed protein content and productivity in pea. Plant Physiol. 144:768–781. doi:10.1104/pp.107.096966 Carrillo, E., D. Rubiales, A. Pe’rez-de-Luque, and S. Fondevilla. 2013. Characterization of mechanisms of resistance against Didymella pinodes in Pisum spp. Eur. J. Plant Pathol. 135:761– 769. doi:10.1007/s10658-012-0116-0 Carrillo, E., Z. Satovic, G. Aubert, K. Boucherot, D. Rubiales, and S. Fondevilla. 2014. Identification of quantitative trait loci and candidate genes for specific cellular resistance responses against Didymella pinodes in pea. Plant Cell Rep. 33:1133–1145. doi:10.1007/s00299-014-1603-x Chatterton, S., M. Harding, R. Bowness, S. Strydhorst, C. Cleland, Q. Storozynsky, T. Dubitz, J. Nielsen, and M. Olson. 2015. Survey of root rot in Alberta field pea in 2014. Can. Plant Dis. Surv. 95:170–172. Churchill, G.A., and R.W. Doerge. 1994. Empirical threshold values for quantitative trait mapping. Genetics 138:963–971. Clulow, S.A., B.G. Lewis, and P. Matthews. 1991. A pathotype classification for Ascochyta pinodes. J. Phytopathol. 131:322– 332. doi:10.1111/j.1439-0434.1991.tb01203.x Conicella, C., and A. Errico. 1985. Identification of the chromosomes involved in translocations of P. abyssinicum and P. fulvum. In: Proceedings of the EUCARPIA Meeting on Pea Breeding, Sorrento, Italy, 10–15 June. University of Naples/ ENEA, Naples, Italy. p. 86–101. Conner, R.L., S.F. Hwang, S.M. Woods, K.F. Chang, D.J. Bing, Y. Dongfang et al. 2007. Influence of agronomic traits on the expression of tissue-specific resistance to mycosphaerella blight in field pea. Can. J. Plant Sci. 87:157–165. doi:10.4141/P05-213 de Givry, S., M. Bouchez, P. Chabrier, D. Milan, and T. Schiex. 2005. CARTHAGENE: Multipopulation integrated genetic and radiated hybrid mapping. Bioinformatics 21:1703–1704. doi:10.1093/bioinformatics/bti222 Deulvot, C., H. Charrel, A. Marty, F. Jacquin, C. Donnadieu, I. Lejeune-He’naut, and G. Aubert. 2010. Highly-multiplexed SNP genotyping for genetic mapping and germplasm diversity studies in pea. BMC Genomics 11:468. doi:10.1186/14712164-11-468 Duarte, J., N. Rivière, A. Baranger, G. Aubert, J. Burstin, L. Cornet et al. 2014. Transcriptome sequencing for high throughput SNP development and genetic mapping in Pea. BMC Genomics 15:126. doi:10.1186/1471-2164-15-126 Errico, A., C. Conicella, and G. Venora. 1991. Karyotype studies on Pisum fulvum and Pisum sativum, using a chromosome image analysis system. Genome 34:105–108. doi:10.1139/g91-017

2938

Faris, J.D., B. Laddomada, and B.S. Gill. 1998. Molecular mapping of segregation distortion loci in Aegilops tauschii. Genetics 149:319–327. Fondevilla, S., N.F. Almeida, Z. Satovic, D. Rubiales, M.C.V. Patto, J.I. Cubero, and A.M. Torres. 2011a. Identification of common genomic regions controlling resistance to Mycosphaerella pinodes, earliness and architectural traits in different pea genetic backgrounds. Euphytica 182:43–52. doi:10.1007/ s10681-011-0460-8 Fondevilla, S., C.M. Avila, J.I. Cubero, and D. Rubiales. 2005. Response to Ascochyta pinodes in a germplasm collection of Pisum spp. Plant Breed. 124:313–315. doi:10.1111/j.14390523.2005.01104.x Fondevilla, S., J.I. Cubero, and D. Rubiales. 2007. Inheritance of resistance to Ascochyta pinodes in two wild accessions of Pisum. Eur. J. Plant Pathol. 119:53–58. doi:10.1007/s10658-007-9146-4 Fondevilla, S., H. Küster, F. Krajinski, J.I. Cubero, and D. Rubiales. 2011b. Identification of genes differentially expressed in a resistant reaction to Ascochyta pinodes in pea using microarray technology. BMC Genomics 12:28. Fondevilla, S., B. Rotter, N. Krezdorn, R. Jüngling, P. Winter, and D. Rubiales. 2014. Identification of genes involved in resistance to Didymella pinodes in pea by deepSuperSAGE transcriptome profiling. Plant Mol. Biol. Report. 32:258–269. doi:10.1007/s11105-013-0644-6 Fondevilla, S., Z. Satovic, D. Rubiales, M.T. Moreno, and A.M. Torres. 2008. Mapping of quantitative trait loci for resistance to Ascochyta pinodes in Pisum sativum subsp. syriacum. Mol. Breed. 21:439–454. doi:10.1007/s11032-007-9144-4 Illumina. 2010. GenomeStudio genotyping module. Release 2010.3. Illumina, Inc., San Diego, CA. Jha, A.B., G. Arganosa, B. Tar’an, A. Diederichsen, and T.D. Warkentin. 2013. Characterization of 169 diverse pea germplasm accessions for agronomic performance, Mycosphaerella blight resistance and nutritional profile. Genet. Resour. Crop Evol. 60:747–761. doi:10.1007/s10722-012-9871-1 Jha, A.B., B. Tar’an, M. Diapari, A. Sindhu, A. Shunmugam, K. Bett, and T.D. Warkentin. 2015. Allele diversity analysis to identify SNPs associated with ascochyta blight resistance in pea. Euphytica 202:189–197. doi:10.1007/s10681-014-1254-6 Jha, A.B., T.D. Warkentin, V. Gurusamy, B. Tar’an, and S. Banniza. 2012. Identification of Mycosphaerella blight resistance in wild Pisum species for use in pea breeding. Crop Sci. 52:2462–2468. doi:10.2135/cropsci2012.04.0242 Khan, T.N., G.M. Timmerman-Vaughan, D. Rubiales, T.D. Warkentin, K.H.M. Siddique, W. Erskine, and M.J. Barbetti. 2013. Didymella pinodes and its management in field pea: Challenges and opportunities. Field Crops Res. 148:61–77. doi:10.1016/j.fcr.2013.04.003 Kraft, J.M., B. Dunne, D. Goulden, and S. Armstrong. 1998. A search for resistance in peas to Ascochyta pinodes. Plant Dis. 82:251–253. doi:10.1094/PDIS.1998.82.2.251 Lamprecht, H. 1964. Partielle sterilitat und chromosomes ruktur bei Pisum. (In German.) Agri. Hort. Genet. 22:56–148. Lawyer, S.A. 1984. Diseases caused by Ascochyta spp. In: D.J. Hargedon, editor, Compendium of pea diseases. APS Press, St. Paul, MN. p. 11–15. Le May, C., B. Ney, E. Lemarchand, A. Schoeny, and B. Tivoli. 2009. Effect of pea plant architecture on spatiotemporal epidemic development of ascochyta blight (Mycosphaerella pinodes) in the field. Plant Pathol. 58:332–343. doi:10.1111/j.13653059.2008.01947.x

www.crops.org


Leonforte, A., S. Sudheesh, N.O. Cogan, P.A. Salisbury, M.E. Nicolas, M. Materne et al. 2013. SNP marker discovery, linkage map construction and identification of QTLs for enhanced salinity tolerance in field pea (Pisum sativum L.). BMC Plant Biol. 13:161. doi:10.1186/1471-2229-13-161 McLaren, D.L., D.J. Hausermann, M.A. Henriquez, K.F. Chang, and T.J. Kerley. 2015. Field pea diseases in Manitoba in 2014. Can. Plant Dis. Surv. 95:173–175. Muehlbauer, F., and W. Chen. 2007. Resistance to ascochyta blights of cool season food legumes. Eur. J. Plant Pathol. 119:135–141. doi:10.1007/s10658-007-9180-2 Peluola, C.O., F.L. Dokken-Bouchard, D.T. Stephens, T. Sliva, S. Kassir, and B. Aryal. 2015. Diseases diagnosed on crop samples submitted in 2014 to the Saskatchewan Ministry of Agriculture Crop Protection Laboratory. Can. Plant Dis. Surv. 95:20–24. Prioul, S., A. Frankewitz, G. Deniot, G. Morin, and A. Baranger. 2004. Mapping of quantitative trait loci for partial resistance to Ascochyta pinodes in pea (Pisum sativum L.) at the seedling and adult plant stages. Theor. Appl. Genet. 108:1322–1334. doi:10.1007/s00122-003-1543-2 Prioul-Gervais, S., G. Deniot, E.M. Receveur, A. Frankewitz, M. Fourmann, C. Rameau et al. 2007. Candidate genes for quantitative resistance to Ascochyta pinodes in pea (Pisum sativum L.). Theor. Appl. Genet. 114:971–984. doi:10.1007/s00122-0060492-y SAS Institute. 2011. SAS system for Windows. Release 9.3. SAS Institute Inc., Cary, NC. Schoeny, A., J. Menat, A. Darsonval, F. Rouault, S. Jumel, and B. Tivoli. 2008. Effect of pea canopy architecture on splash dispersal of Mycosphaerella pinodes conidia. Plant Pathol. 57:1073– 1085. doi:10.1111/j.1365-3059.2008.01888.x Sindhu, A., L. Ramsay, L.A. Sanderson, R. Stonehouse, R. Li, J. Condie et al. 2014. Gene-based SNP discovery and genetic mapping in pea. Theor. Appl. Genet. 127:2225–2241. doi:10.1007/s00122-014-2375-y Tar’an, B., T. Warkentin, D.J. Somers, D. Miranda, A. Vandenberg, S. Balde et al. 2003. Quantitative trait loci for lodging resistance, plant height and partial resistance to mycosphaerella blight in field pea (Pisum sativum L.). Theor. Appl. Genet. 107:1482–1491. doi:10.1007/s00122-003-1379-9 Timmerman-Vaughan, G.M., T.J. Frew, R. Butler, S. Murray, M. Gilpin, K. Falloon et al. 2004. Validation of quantitative trait loci for Ascochyta blight resistance in pea (Pisum sativum L.), using populations from two crosses. Theor. Appl. Genet. 109:1620–1631. doi:10.1007/s00122-004-1779-5 Timmerman-Vaughan, G.M., T.J. Frew, A.C. Russell, T. Khan, R. Butler, M. Gilpin et al. 2002. QTL mapping of partial resistance to field epidemics of Ascochyta blight of pea. Crop Sci. 42:2100–2111. doi:10.2135/cropsci2002.2100


Voorrips, R.E. 2002. MapChart: Software for the graphical presentation of linkage maps and QTLs. J. Hered. 93:77–78. doi:10.1093/jhered/93.1.77 Wallen, V.R. 1965. Field evaluation of the importance of the Ascochyta complex of peas. Can. J. Plant Sci. 45:27–33. doi:10.4141/cjps65-004 Wang, C., C. Zhu, H. Zhai, and J. Wan. 2005. Mapping segregation distortion loci and quantitative trait loci for spikelet sterility in rice (Oryza sativa L.). Genet. Res. 86:97–106. doi:10.1017/S0016672305007779 Wang, S., C.J. Basten, and Z.B. Zeng. 2012. Windows QTL Cartographer 2.5. North Carolina State University, Raleigh, NC. Wroth, J.M. 1998. Possible role for wild genotypes of Pisum spp. to enhance ascochyta blight resistance in pea. Aust. J. Exp. Agric. 38:469–479. doi:10.1071/EA98024 Wroth, J.M. 1999. Evidence suggests that Mycosphaerella pinodes infection of Pisum sativum is inherited as a quantitative trait. Euphytica 107:193–204. doi:10.1023/A:1003688430893 Xu, S. 2008. Quantitative trait locus mapping can benefit from segregation distortion. Genetics 180:2201–2208. doi:10.1534/ genetics.108.090688 Xue, A.G., T.D. Warkentin, B.D. Gossen, P.A. Burnett, A. Vandenberg, and K.Y. Rashid. 1998. Pathogenic variation of western Canadian isolates of Mycosphaerella pinodes on selected Pisum genotypes. Can. J. Plant Pathol. 20:189–193. doi:10.1080/07060669809500426 Xue, A.G., T.D. Warkentin, M.T. Greeniaus, and R.C. Zimmer. 1996. Genotypic variability in seed borne infection of field pea by Ascochyta pinodes and its relation to foliar disease severity. Can. J. Plant Pathol. 18:370–374. doi:10.1080/07060669609500590 Xue, A.G., T.D. Warkentin, and E.O. Kenaschuk. 1997. Effect of timings of inoculation with Ascochyta pinodes on yield and seed infection on field pea. Can. J. Plant Sci. 77:685–689. doi:10.4141/P96-150 Zhang, J.X., W.G.D. Fernando, and A.G. Xue. 2003. Virulence and genetic variability among isolates of Mycosphaerella pinodes. Plant Dis. 87:1376–1383. doi:10.1094/PDIS.2003.87.11.1376 Zhang, R., S.F. Hwang, K.F. Chang, B.D. Gossen, S.E. Strelkov, G.D. Turnbull, and S.F. Blade. 2006. Genetic resistance to Ascochyta pinodes in 558 field pea accessions. Crop Sci. 46:2409–2414. doi:10.2135/cropsci2006.02.0089 Zhang, R., S.F. Hwang, B.D. Gossen, K.F. Chang, and D.G. Turnbull. 2007. A quantitative analysis of resistance to Mycosphaerella blight in field pea. Crop Sci. 47:162–167. doi:10.2135/ cropsci2006.05.0305

www.crops.org 2939