A Distinct Genetic Cluster in Cultivated Chickpea as Revealed by Genome-wide Marker Discovery and Genotyping

The accurate description of plant biodiversity is of utmost importance to efficiently address efforts in conservation genetics and breeding. Herein, we report the successful application of a genotyping-by-sequencing (GBS) approach in chickpea ( L.), resulting in the characterization of a cultivated germplasm collection with 3187 high-quality single nucleotide polymorphism (SNP) markers. Genetic structure inference, principal component analysis, and hierarchical clustering all indicated the identification of a genetic cluster corresponding to black-seeded genotypes traditionally cultivated in Southern Italy. Remarkably, this cluster was clearly distinct at both genetic and phenotypic levels from germplasm groups reflecting commercial chickpea classification into and seed types. Fixation index estimates for individual polymorphisms pointed out loci and genomic regions that might be of significance for the diversification of agronomic and commercial traits. Overall, our findings provide information on genetic relationships within cultivated chickpea and highlight a gene pool of great interest for the scientific community and chickpea breeding, which is limited by the low genetic diversity available in the primary gene pool

Functional characterization of a syntaxin involved in tomato (Solanum lycopersicum) resistance against powdery mildew

Specific syntaxins, such as Arabidopsis AtPEN1 and its barley ortholog ROR2, play a major role in plant defense against powdery mildews. Indeed, the impairment of these genes results in increased fungal penetration in both host and non-host interactions. In this study, a genome-wide survey allowed the identification of 21 tomato syntaxins. Two of them, named SlPEN1a and SlPEN1b, are closely related to AtPEN1. RNAi-based silencing of SlPEN1a in a tomato line carrying a loss-of-function mutation of the susceptibility gene SlMLO1 led to compromised resistance toward the tomato powdery mildew fungus Oidium neolycopersici. Moreover, it resulted in a significant increase in the penetration rate of the non-adapted powdery mildew fungus Blumeria graminis f. sp. hordei. Codon-based evolutionary analysis and multiple alignments allowed the detection of amino acid residues that are under purifying selection and are specifically conserved in syntaxins involved in plant-powdery mildew interactions. Our findings provide both insights on the evolution of syntaxins and information about their function which is of interest for future studies on plant–pathogen interactions and tomato breeding.</p

Identification and functional inference on the MLO-family in viridiplantae

Powdery mildew (PM) is a widespread plant disease of temperate climates caused by ascomycete fungi of the order Erysiphales. PM is an important agricultural issue since it can cause significant economic losses. Specific members of the MLO gene family act as susceptibility factors towards the PM disease. A step towards the stability of crop productions would be thus the characterization of MLO genes at the genomic level. We carried out a genome-wide characterization of the MLO gene family in twenty-three plant and two algal genomes providing manual curated MLO protein catalogues. In total,180 novel proteins containing the MLO domain were identified. Evolutionary history and phylogenetic relationships were studied through maximum likelihood analysis. This highlighted eight different clades,including a new monocot-specific clade (VIII) identified for the first time. In addition,15 and 67 putative PM susceptibility genes,clustering in clade IV and V,respectively,were identified. Results of this work may help to address further biological questions concerning MLOs involved in PM susceptibility. In follow-up studies,it could be investigated whether the silencing or loss-of-function mutations in one or more of these candidate genes may lead to PM resistance

Genome-Wide Study of the Tomato SlMLO Gene Family and Its Functional Characterization in Response to the Powdery Mildew Fungus Oidium neolycopersici

The MLO (Mildew Locus O) gene family encodes plant-specific proteins containing seven transmembrane domains and likely acting in signal transduction in a calcium and calmodulin dependent manner. Some members of the MLO family are susceptibility factors towards fungi causing the powdery mildew disease. In tomato, for example, the loss-of-function of the MLO gene SlMLO1 leads to a particular form of powdery mildew resistance, called ol-2, which arrests almost completely fungal penetration. This type of penetration resistance is characterized by the apposition of papillae at the sites of plant-pathogen interaction. Other MLO homologs in Arabidopsis regulate root response to mechanical stimuli (AtMLO4 and AtMLO11) and pollen tube reception by the female gametophyte (AtMLO7). However, the role of most MLO genes remains unknown. In this work, we provide a genome-wide study of the tomato SlMLO gene family. Besides SlMLO1, other fifteen SlMLO homologs were identified and characterized with respect to their structure, genomic organization, phylogenetic relationship, and expression profile. In addition, by analysis of transgenic plants, we demonstrated that simultaneous silencing of SlMLO1 and two of its closely related homologs, SlMLO5 and SlMLO8, confer higher level of resistance than the one associated with the ol-2 mutation.The outcome of this study provides evidence for functional redundancy among tomato homolog genes involved in powdery mildew susceptibility. Moreover, we developed a series of transgenic lines silenced for individual SlMLO homologs, which lay the foundation for further investigations aimed at assigning new biological functions to the MLO gene family

CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew

Background: The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene P owdery M ildew R esistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On). Results: To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants. Conclusion: CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.</p

Characterization of low-strigolactone germplasm in pea (Pisum sativum L.) resistant to crenate broomrape (Orobanche crenata Forsk.)

Crenate broomrape (Orobanche crenata Forsk.) is a devastating parasitic weed threatening the cultivation of legumes around the Mediterranean and in theMiddle East. So far, only moderate levels of resistance were reported to occur in pea (Pisum sativum L.) natural germplasm, and most commercial cultivars are prone to severe infestation. Here, we describe the selection of a pea line highly resistant to O. crenata, following the screening of local genetic resources. Time series observations show that delayed emergence of the parasite is an important parameter associated with broomrape resistance. High performance liquid chromatography connected to tandem mass spectrometry analysis and in vitro broomrape germination bioassays suggest that the resistance mechanism might involve the reduced secretion of strigolactones, plant hormones exuded by roots and acting as signaling molecules for the germination of parasitic weeds. Two years of replicated trials in noninfested fields indicate that the resistance is devoid of pleiotropic effects on yield, in contrast to pea experimental mutants impaired in strigolactone biosynthesis and, thus, is suitable for use in breeding programs.</p

Solanum lycopersicum callose synthase 12 gene, partial cds

Abstract Background The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene PowderyMildewResistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On). Results To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants. Conclusion CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations