María J. Cobos's research while affiliated with Institute for Sustainable Agriculture and other places

In Mediterranean environments, with mild winters and dry summers, peas are planted in autumn or early winter to profit from winter rain and to avoid terminal drought and high summer temperatures. The root parasitic weed broomrape (Orobanche crenata) appears as a major limiting factor under these conditions. To address such specific growing conditions and associated constraints, targeted breeding is needed. We present here recent achievements in the development of pea lines arising from a wide hybridization program incorporating resistance to broomrape and to powdery mildew (Erysiphe pisi) from landraces and wild relatives. Their adaption to autumn sowings under Mediterranean rain fed conditions, and their agronomic performance and resistance to prevailing diseases is compared with those of check cultivars in a multi-environment field test with nine trials performed over three seasons. HA-GGE biplots were a powerful tool for comparison among accessions in terms of performance and stability for each trait assessed. Like this, breeding lines NS22, NS34, NS8, NS39, NS35, NS21 and NS83 over-yielded all check cultivars. Grain yield was strongly affected by broomrape infection, with little influence of powdery mildew and ascochyta blight. All breeding lines studied showed high to moderate resistance to broomrape, whereas all check cultivars were severely infected. Broomrape infection was not correlated with days to flowering, whereas powdery mildew infection was favored by long cycles. Broomrape infection was enhanced by mild winter temperatures before flowering and spring rain, whereas high spring temperatures hampered broomrape development.

BACKGROUND Pea (Pisum sativum) is one of the most important temperate grain legumes in the world, and its production is severely constrained by the pea aphid (Acyrthosiphon pisum). Wild relatives, such as P. fulvum, are valuable sources of allelic diversity to improve the genetic resistance of cultivated pea species against A. pisum attack. To unravel the genetic control underlying resistance to the pea aphid attack, a quantitative trait loci (QTL) analysis was performed using the previously developed high density integrated genetic linkage map originated from an intraspecific recombinant inbred line (RIL) population (P. fulvum: IFPI3260 × IFPI3251). RESULTS We accurately evaluated specific resistance responses to pea aphid that allowed the identification, for the first time, of genomic regions that control plant damage and aphid reproduction. Eight QTLs associated with tolerance to pea aphid were identified in LGs I, II, III, IV and V, which individually explained from 17.0% to 51.2% of the phenotypic variation depending on the trait scored, and as a whole from 17.0% to 88.6%. The high density integrated genetic linkage map also allowed the identification of potential candidate genes co‐located with the QTLs identified. CONCLUSIONS Our work shows how the survival of P. fulvum after the pea aphid attack depends on the triggering of a multi‐component protection strategy that implies a quantitative tolerance. The genomic regions associated with the tolerance responses of P. fulvum during A. pisum infestation have provided six potential candidate genes that could be useful in marker‐assisted selection (MAS) and genomic assisted breeding (GAB) after functional validation in the future. © 2019 Society of Chemical Industry

Pea weevil (Bruchus pisorum) is a damaging insect pest affecting pea (Pisum sativum) production worldwide. No resistant cultivars are available, although some levels of incomplete resistance have been identified in Pisum germplasm. To decipher the genetic control underlying the resistance previously identify in P. sativum ssp. syriacum, a recombinant inbred line (RIL F8:9) population was developed. The RIL was genotyped through Diversity Arrays Technology PL’s DArTseq platform and screened under field conditions for weevil seed infestation and larval development along 5 environments. A newly integrated genetic linkage map was generated with a subset of 6,540 markers, assembled into seven linkage groups, equivalent to the number of haploid pea chromosomes. An accumulated distance of 2,503 cM was covered with an average density of 2.61 markers cM−1. The linkage map allowed the identification of three QTLs associated to reduced seed infestation along LGs I, II and IV. In addition, a QTL for reduced larval development was also identified in LGIV. Expression of these QTLs varied with the environment, being particularly interesting QTL BpSI.III that was detected in most of the environments studied. This high-saturated pea genetic map has also allowed the identification of seven potential candidate genes co-located with QTLs for marker-assisted selection, providing an opportunity for breeders to generate effective and sustainable strategies for weevil control.

em>Pisum sativum is the cool season grain legume most cultivated in Europe and the second in the world [1]. However, its yield is still relatively unstable and low due to the limited adaptability to extreme environmental conditions of available cultivars and their susceptibility to diseases and pests. We studied in the past resistance to a number of fungal diseases and parasitic weeds. Here we cover resistance to the biotrophic fungus Uromyces pisi, the causal agent of pea rust, and to the insect pests pea aphid ( Acyrthosiphon pisum ) and the pea weevil ( Bruchus pisorum ) [2] [3] [4]. Several studies have been performed, ranging from the screening of pea cultivars to the assessment of the mechanisms involved in the resistance. No complete resistance has been reported against these biotic stresses so far, but different levels of moderately incomplete resistance are available especially in the wild Pisum species. To unravel the genetic control of the resistance, a quantitative trait loci (QTL) analysis was performed using two recombinant inbred line (RIL F7:8) populations. The first one originated from a cross between two P. fulvum accessions, P660 and P651, while the second one originated from the cross between P. sativum ssp . syriacum accession P665 and P. sativum cv . Messire. Both RILs populations were phenotyped for resistance under controlled and field conditions, and genotyped through Diversity Arrays Technology PL’s DArTseq platform (Australia) [2]. The newly constructed integrated genetic linkage maps of wild peas includes high-quality DArT-Seq and SNPs derived-markers, as well as several previously mapped ”anchor” markers, which assembled in both cases in 7 Linkage Groups (LGs) and showed an average density lower than 2 markers cM-1. Genomic regions involved in resistance to rust, aphid and weevil will be presented and critically discussed.

Summary and description of the trimmed sequences of the P. fulvum DArT markers with correspondence to Medicago truncatula genome and their correspondence to P. sativum linkage groups.

Pisum fulvum, a wild relative of pea is an important source of allelic diversity to improve the genetic resistance of cultivated species against fungal diseases of economic importance like the pea rust caused by Uromyces pisi. To unravel the genetic control underlying resistance to this fungal disease, a recombinant inbred line (RIL) population was generated from a cross between two P. fulvum accessions, IFPI3260 and IFPI3251, and genotyped using Diversity Arrays Technology. A total of 9,569 high-quality DArT-Seq and 8,514 SNPs markers were generated. Finally, a total of 12,058 markers were assembled into seven linkage groups, equivalent to the number of haploid chromosomes of P. fulvum and P. sativum. The newly constructed integrated genetic linkage map of P. fulvum covered an accumulated distance of 1,877.45 cM, an average density of 1.19 markers cM⁻¹ and an average distance between adjacent markers of 1.85 cM. The composite interval mapping revealed three QTLs distributed over two linkage groups that were associated with the percentage of rust disease severity (DS%). QTLs UpDSII and UpDSIV were located in the LGs II and IV respectively and were consistently identified both in adult plants over 3 years at the field (Córdoba, Spain) and in seedling plants under controlled conditions. Whenever they were detected, their contribution to the total phenotypic variance varied between 19.8 and 29.2. A third QTL (UpDSIV.2) was also located in the LGIVand was environmentally specific as was only detected for DS % in seedlings under controlled conditions. It accounted more than 14% of the phenotypic variation studied. Taking together the data obtained in the study, it could be concluded that the expression of resistance to fungal diseases in P. fulvum originates from the resistant parent IFPI3260.

Summary and description of the DArT-derived markers used to generate the P. fulvum map including marker identity, sequence description, linkage group assignment in the map and the position in the linkage group.

The development of chickpea cultivars with high quality grains for human consumption is an important objective in breeding programs. Genotype and environment effects on seed quality traits (sensorial, nutritional and physical) were studied in chickpea dry grain. Twenty genotypes were grown in winter and spring sowings over two campaigns in four different locations in southern Spain. Significant differences were observed in oil, acid detergent fiber (ADF) and protein content between sowing times (S). In winter, oil and ADF content were higher, while protein content was lower. Although, in general, highly significant variation was detected for genotype (G), environment (E) and single interactions (GE, GS and ES), the genotype effect was stronger for ADF, neutral detergent fiber (NDF), oil, starch and protein content, and for physical and sensory traits (r2>27%). In contrast, environment played an important role in variation in the content of amylose and amylopectin (r2=71.7%). No high relationships were found between the sensory and nutritional or physical characteristics studied. In general, our results suggest a high genetic gain for seed quality in nutritional, physical and sensory traits in chickpea. Genotypes with good seed sensory quality should be selected in the final stages of the breeding program, because it is not feasible to evaluate very large numbers of samples. However, in some cases, moderate correlations were found between sensory and either nutritional or physical traits. Therefore, indirect selection to increase the frequency of genes for sensory traits in an early stage should be considered.

Didymella pinodes is the principal causal agent of ascochyta blight, one of the most important fungal diseases of pea (Pisum sativum) worldwide. Understanding its host specificity has crucial implications in epidemiology and management; however, this has not been clearly delineated yet. In this study we attempt to clarify the host range of D. pinodes and to compare it with that of other close Didymella spp. D. pinodes was very virulent on pea accessions, although differences in virulence were identified among isolates. On the contrary, studied isolates of D. fabae, D. rabiei, and D. lentil showed a reduced ability to infect pea not causing macroscopically visible symptoms on any of the pea accessions tested. D. pinodes isolates were also infective to some extend on almost all species tested including species such as Hedysarum coronarium, Lathyrus sativus, Lupinus albus, Medicago spp., Trifolium spp., Trigonella foenum-graecum, and Vicia articulata which were not mentioned before as hosts of D. pinodes. On the contrary, D. lentil and D. rabiei were more specific, infecting only lentil and chickpea, respectively. D. fabae was intermediate, infecting mainly faba bean, but also slightly other species such as Glycine max, Phaseolus vulgaris, Trifolium spp., Vicia sativa, and V. articulata. DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region (ITS) was performed to confirm identity of the isolates studies and to determine phylogenetic relationship among the Didymella species, revealing the presence of two clearly distinct clades. Clade one was represented by two supported subclusters including D. fabae isolates as well as D. rabiei with D. lentil isolates. Clade two was the largest and included all the D. pinodes isolates as well as Phoma medicaginis var. pinodella. Genetic distance between D. pinodes and the other Didymella spp. isolates was not correlated with overall differences in pathogenicity. Based on evidences presented here, D. pinodes is not specialized on pea and its host range is larger than that of D. fabae, D. lentil, and D. rabiei. This has relevant implications in epidemiology and control as these species might act as alternative hosts for D. pinodes.