Genome-wide association study, combined with bulk segregant analysis, identify plant receptors and defense related genes as candidate genes for downy mildew resistance in quinoa.

BACKGROUND: Downy mildew is the most relevant disease of quinoa and the most widespread. Though, little is known about the genetics of resistance to this disease. The objective of this study was to identify the genomic regions controlling downy mildew resistance in quinoa and candidate genes for this trait. With this aim we carried out a GWAS analysis in a collection formed by 211 quinoa accessions from different origins. This approach was combined with inheritance studies and Bulk Segregant Analysis (BSA) in a segregating population. RESULTS: GWAS analysis identified 26 genomic regions associated with the trait. Inheritance studies in a F2 population segregating for resistance revealed the existence of a major single dominant gene controlling downy mildew complete resistance in quinoa accession PI614911. Through BSA, this gene was found to be located in chromosome 4, in a region also identified by GWAS. Furthermore, several plant receptors and resistance genes were found to be located into the genomic regions identified by GWAS and are postulated as candidate genes for resistance. CONCLUSIONS: Until now, little was known about the genetic control of downy mildew resistance in quinoa. A previous inheritance study suggested that resistance to this disease was a quantitative polygenic trait and previous GWAS analyses were unable to identify accurate markers for this disease. In our study we demonstrate the existence of, at least, one major gene conferring resistance to this disease, identify the genomic regions involved in the trait and provide plausible candidate genes involved in defense. Therefore, this study significantly increases our knowledge about the genetics of downy mildew resistance and provides relevant information for breeding for this important trait.

Site-directed genotype screening for elimination of antinutritional saponins in quinoa seeds identifies TSARL1 as a master controller of saponin biosynthesis selectively in seeds.

Climate change may result in a drier climate and increased salinization, threatening agricultural productivity worldwide. Quinoa (Chenopodium quinoa) produces highly nutritious seeds and tolerates abiotic stresses such as drought and high salinity, making it a promising future food source. However, the presence of antinutritional saponins in their seeds is an undesirable trait. We mapped genes controlling seed saponin content to a genomic region that includes TSARL1. We isolated desired genetic variation in this gene by producing a large mutant library of a commercial quinoa cultivar and screening the library for specific nucleotide substitutions using droplet digital PCR. We were able to rapidly isolate two independent tsarl1 mutants, which retained saponins in the leaves and roots for defence, but saponins were undetectable in the seed coat. We further could show that TSARL1 specifically controls seed saponin biosynthesis in the committed step after 2,3-oxidosqualene. Our work provides new important knowledge on the function of TSARL1 and represents a breakthrough for quinoa breeding.

Molecular Characterization of Peronospora variabilis Isolates Infecting Chenopodium quinoa and Chenopodium album in Spain.

Quinoa is an expanding crop in southern Spain. Downy mildew, caused by Peronospora variabilis, is the most important quinoa disease in Spain and worldwide. In Spain, this disease has also been observed on the weed Chenopodium album. The objectives of this study were to unravel the origin of the P. variabilis isolates currently infecting quinoa in southern Spain and to study their genetic diversity. We hypothesized that P. variabilis isolates infecting quinoa in Spain could have been introduced through the seeds of the quinoa varieties currently grown in the country or, alternatively, that these isolates are endemic isolates, originally infecting C. album, that jumped to quinoa. In order to test these hypotheses, we sequenced the internal transcribed spacer (ITS), cytochrome c oxidase subunit 1 (cox1), and cox2 regions of 33 P. variabilis isolates infecting C. quinoa and C. album in southern Spain and analyzed their phylogenetic relationship with isolates present in other countries infecting Chenopodium spp. cox1 gene sequences from all of the Spanish P. variabilis isolates were identical and exhibited nine single-nucleotide polymorphisms (SNPs) compared with a single P. variabilis cox1 sequence found at GenBank. Phylogenetic analyses based on the ITS ribosomal DNA region were not suitable to differentiate isolates according to their geographical origin or host. The cox2 sequences from P. variabilis Spanish isolates collected from C. quinoa and C. album were all identical and had a distinctive SNP in the last of four polymorphic sites that distinguished Spanish isolates from isolates from other countries. These results suggest that P. variabilis infecting quinoa in southern Spain could be native isolates that originally infected C. album.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Bulked segregant transcriptome analysis in pea identifies key expression markers for resistance to Peyronellaea pinodes.

Peyronellaea pinodes is a devastating pathogen of pea crop. Quantitative trait loci (QTL) associated with resistance have been identified, as well as genes differentially expressed between resistant and susceptible pea lines. The key question is which of these many genes located into these QTLs, or differentially expressed, are the key genes that distinguish resistant from susceptible plants and could be used as markers. To identify these key genes, in the present study we applied MACE (Massive Analysis of cDNA Ends) -Seq to a whole Recombinant Inbred Line population segregating for resistance to this disease and their parental lines and identified those genes which expression was more correlated with the level of resistance. We also compared gene expression profiles between the most resistant and the most susceptible families of the RIL population. A total of 6780 transcripts were differentially expressed between the parental lines after inoculation. Of them, 803 showed the same expression pattern in the bulks formed by the most resistant and most susceptible RIL families. These genes, showing a consistent expression pattern, could be used as expression markers to distinguish resistant from susceptible plants. The analysis of these genes also discovered the crucial mechanisms acting against P. pinodes.

Identification of genes differentially expressed in a resistant reaction to Mycosphaerella pinodes in pea using microarray technology.

BACKGROUND: Ascochyta blight, caused by Mycosphaerella pinodes is one of the most important pea pathogens. However, little is known about the genes and mechanisms of resistance acting against M. pinodes in pea. Resistance identified so far to this pathogen is incomplete, polygenic and scarce in pea, being most common in Pisum relatives. The identification of the genes underlying resistance would increase our knowledge about M. pinodes-pea interaction and would facilitate the introgression of resistance into pea varieties. In the present study differentially expressed genes in the resistant P. sativum ssp. syriacum accession P665 comparing to the susceptible pea cv. Messire after inoculation with M. pinodes have been identified using a M. truncatula microarray. RESULTS: Of the 16,470 sequences analysed, 346 were differentially regulated. Differentially regulated genes belonged to almost all functional categories and included genes involved in defense such as genes involved in cell wall reinforcement, phenylpropanoid and phytoalexins metabolism, pathogenesis- related (PR) proteins and detoxification processes. Genes associated with jasmonic acid (JA) and ethylene signal transduction pathways were induced suggesting that the response to M. pinodes in pea is regulated via JA and ET pathways. Expression levels of ten differentially regulated genes were validated in inoculated and control plants using qRT-PCR showing that the P665 accession shows constitutively an increased expression of the defense related genes as peroxidases, disease resistance response protein 39 (DRR230-b), glutathione S-transferase (GST) and 6a-hydroxymaackiain methyltransferase. CONCLUSIONS: Through this study a global view of genes expressed during resistance to M. pinodes has been obtained, giving relevant information about the mechanisms and pathways conferring resistance to this important disease. In addition, the M. truncatula microarray represents an efficient tool to identify candidate genes controlling resistance to M. pinodes in pea.

In planta Identification of Putative Pathogenicity Factors from the Chickpea Pathogen Ascochyta rabiei by De novo Transcriptome Sequencing Using RNA-Seq and Massive Analysis of cDNA Ends.

The most important foliar diseases in legumes worldwide are ascochyta blights. Up to now, in the Ascochyta-legume pathosystem most studies focused on the identification of resistance genes in the host, while very little is known about the pathogenicity factors of the fungal pathogen. Moreover, available data were often obtained from fungi growing under artificial conditions. Therefore, in this study we aimed at the identification of the pathogenicity factors of Ascochyta rabiei, causing ascochyta blight in chickpea. To identify potential fungal pathogenicity factors, we employed RNA-seq and Massive Analysis of cDNA Ends (MACE) to produce comprehensive expression profiles of A. rabiei genes isolated either from the fungus growing in absence of its host or from fungi infecting chickpea leaves. We further provide a comprehensive de novo assembly of the A. rabiei transcriptome comprising 22,725 contigs with an average length of 1178 bp. Since pathogenicity factors are usually secreted, we predicted the A. rabiei secretome, yielding 550 putatively secreted proteins. MACE identified 596 transcripts that were up-regulated during infection. An analysis of these genes identified a collection of candidate pathogenicity factors and unraveled the pathogen's strategy for infecting its host.

Pinolide, a new nonenolide produced by Didymella pinodes , the causal agent of ascochyta blight on Pisum sativum.

An aggressive isolate of Didymella pinodes isolated from pea ( Pisum sativum ) produced four different metabolites in vitro. The metabolites isolated from the culture filtrates were characterized by spectroscopic and optical methods. A new nonenolide, named pinolide, was isolated and characterized as (2S*,7R*,8S*,5E,9R*)-2,7,8-trihydroxy-9-propyl-5-nonen-9-olide. Pinolidoxin, the main toxin produced by D. pinodes, was also isolated together with two other closely related nonenolides, identified as herbarumin II and 2-epi-herbarumin II. Herbarumin II and 2-epi-herbarumin II have been previously isolated from the fungi Phoma herbarum and Paraphaeosphaeria recurvifoliae , respectively, but described here to be isolated for the first time from D. pinodes. When tested on leaves of the host plant and other legumes and weeds, pinolidoxin was phytotoxic in all of the plant species, whereas the other three nonenolides did not produce any symptoms. The importance of the stereochemistry of the hydroxy group at C-7 on phytotoxicity also is discussed.

Two-dimensional electrophoresis based proteomic analysis of the pea (Pisum sativum) in response to Mycosphaerella pinodes.

Responses to Mycosphaerella pinodes in pea were studied by using a proteomics approach. Two-dimensional electrophoresis (2-DE) was used in order to compare the leaf proteome of two pea cultivars displaying different phenotypes (susceptible and partial resistance to the fungus), as well as in response to the inoculation. Multivariate statistical analysis identified 84 differential protein spots under the experimental conditions (cultivars/treatments). All of these 84 protein spots were subjected to MALDI-TOF/TOF mass spectrometry to deduce their possible functions. A total of 31 proteins were identified using a combination of peptide mass fingerprinting (PMF) and MSMS fragmentation. Most of the identified proteins corresponded to enzymes belonging to photosynthesis, metabolism, transcription/translation and defense and stress categories. Results are discussed in terms of responses to pathogens.

Breeding approaches for crenate broomrape (Orobanche crenata Forsk.) management in pea (Pisum sativum L.).

BACKGROUND: Pea cultivation is strongly hampered in Mediterranean and Middle East farming systems by the occurrence of Orobanche crenata Forsk. Strategies of control have been developed, but only marginal successes have been achieved. Most control methods are either unfeasible, uneconomical, hard to achieve or result in incomplete protection. The integration of several control measures is the most desirable strategy. RESULTS: [corrected] Recent developments in control are presented and re-evaluated in light of recent developments in crop breeding and molecular genetics. These developments are placed within a framework that is compatible with current agronomic practices. CONCLUSION: The current focus in applied breeding is leveraging biotechnological tools to develop more and better markers to speed up the delivery of improved cultivars to the farmer. To date, however, progress in marker development and delivery of useful markers has been slow. The application of knowledge gained from basic genomic research and genetic engineering will contribute to more rapid pea improvement for resistance against O. crenata and/or the herbicide.