Development of SNP markers linked to purple blotch resistance for marker-assisted selection in onion (Allium cepa L.) breeding.

Purple blotch (PB), caused by Alternaria porri (Ellis) Cifferi, is one of the most destructive diseases of onion worldwide. Rapid development and deployment of resistant onion varieties is the most effective approach to control this disease. A single dominant gene, ApR1 was previously linked to PB resistance in onion cultivar 'Arka Kalyan'. In this study, an advanced RIL population derived from a cross between the resistant (Arka Kalyan) and susceptible (Agrifound Rose) cultivar of onion was used to fine map the resistant locus with SNP markers. Twenty plants from the RIL population, ten each with disease resistance and susceptibility trait, were subjected to restriction site-associated DNA sequencing (RAD-Seq) and generated 7388 single nucleotide polymorphisms (SNPs). Correlation analysis between marker genotypes and PB disease phenotype on the 20 plants identified 27 SNPs as candidate markers linked to ApR1 gene for PB resistance. Six candidate SNPs were converted to Kompetitive Allele-Specific PCR (KASP) markers designated as ApRsnip5, ApRsnip8, ApRsnip14, ApRsnip21, ApRsnip23 and ApRsnip25. Marker-trait association based on disease phenotyping and KASP genotyping data on 153 RILs confirmed that all six KASP markers were tightly associated with ApR1 gene within the genetic distance of 1.3 CentiMorgan (cM). ApRsnip14 co-segregated with the ApR1 locus. Further, the six KASP markers were tested on 27 onion lines with different genetic backgrounds. ApRsnip14, ApRsnip21, ApRsnip5 and ApRsnip23 not only showed the correct resistance allele in 3 resistance genotypes, but also clustered together in the remaining 24 susceptible lines. Alternatively, ApRsnip8 and ApRsnip25 exhibited false positives in two onion lines which do not have the R-gene. Overall, our results suggest that ApRsnip14 and ApRsnip23 with their close linkage to ApR1 locus and greater applicability on breeding germplasm are recommended in marker-assisted selection for PB resistance in onion breeding program. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03562-7.

Cross-kingdom small RNA communication between plants and fungal phytopathogens-recent updates and prospects for future agriculture.

Small RNAs (sRNAs) are short non-coding regulatory RNA sequences that silence the complementary expressive transcripts through an endogenous RNA mediated interference mechanism (RNAi). These sRNAs typically move through plasmodesmata and phloem in plants to support disease resistance, and also through septal pores and vesicles in fungi to act as effector of pathogenicity. Notably, recent reports have shown the occurrence of a bidirectional trafficking of these sRNAs between the host plants and the attacking fungal phytopathogen which have significant implication in the nature of the infection. While the trans-species sRNAs from the pathogen can silence the host mRNAs and inhibit the host immunity genes, the sRNA modules from the host plants can silence the mRNA in the pathogen by impeding the expression of the pathogenicity-related genes. In the present review, we discuss the current state of sRNA trafficking between the plant and the pathogen with special emphasis on the mechanism of cross-kingdom communication which could contribute to the development of pathogen and pest control in future agriculture.

A single transcript CRISPR/Cas9 mediated mutagenesis of CaERF28 confers anthracnose resistance in chilli pepper (Capsicum annuum L.).

MAIN CONCLUSION: T-DNA-free homozygous mutant lines developed through a single transcript CRISPR/Cas9 system harboring the desired modification in the CaERF28 locus exhibited significantly enhanced resistance to the anthracnose pathogen Colletotrichum truncatum coupled with the improved expression of defense responsive genes. Anthracnose, caused by Colletotrichum species, is a major disease of chilli (Capsicum annuum) accounting for significant pre- and post-harvest yield losses across the tropical and subtropical regions of the world. Management of chilli anthracnose using traditional methods have not met with noticeable success. In the present study, we have demonstrated an enhanced anthracnose resistance through a single transcript unit CRISPR/Cas9 mediated alteration of the susceptibility gene CaERF28 in C. annuum. A construct with a single Pol II promoter-driven expression of Cas9, sgRNA and a hammerhead ribozyme (RZ) was designed to modify the CaERF28 gene in the susceptible chilli genotype Arka Lohit. Fourty-five C-ERF28-induced mutant lines (72.5%) were identified from 62 T0 transgenic plants. Further, simultaneously targeted multiple sites within CaERF28 showed increased mutation (85.7%) efficiency. DNA sequence analysis showed that these plants harboured multiple InDels at the target site. The allelic mutants of C-ERF28 were transferred to the following generations by simple Mendelian inheritance. Segregation in the T1 and T2 generations resulted in the identification of T-DNA free and marker-free C-ERF28 mutant lines. Five homozygous mutants demonstrated enhanced resistance to anthracnose compared to wild type as demonstrated by reduced spore count and fungal growth as well as induced expression of defense-related genes. Our results demonstrated that the STU-CRISPR/Cas9 mediated editing of the CaERF28 gene is a rapid, safe and versatile approach for enhancing anthracnose resistance in chilli pepper and pave way for its utilization in the improvement of other solanaceous crops.

Genome wide identification and expression analysis of pepper C2H2 zinc finger transcription factors in response to anthracnose pathogen Colletotrichum truncatum.

Although, the C2H2 zinc finger (ZF) family of plant transcription factors have been implicated in multiple biological processes, they are yet to be characterized in the economically important chilli pepper (Capsicum annuum). In this study, a total of 79 C2H2 ZF genes were identified in the pepper genome. Phylogenetic analysis categorized the pepper C2H2 ZF (CaZF) members into five subfamilies each with unique conserved domains and functions. Genomic organization revealed that CaZF genes have variable number of introns consistent with the characteristics defined by the evolutionary analysis. Segmental duplication-based purifying selection contributed to the expansion of CaZF genes in pepper. Additionally, 11 CaZF genes were identified as targets for 38 miRNAs indicating their role in post-transcriptional silencing-mediated genetic regulation. Gene expression analysis revealed that 18 CaZF genes were differentially expressed post-infection with the anthrocnose pathogen Colletotrichum truncatum, uncovering their potential function in pepper response to biotic stresses. Moreover, CaZFs were significantly induced post-treatment with methyl jasmonate and ethylene indicating their role in defense signaling. Notably, the MeJA responsive cis-elements were detected in the promoter regions of majority of CaZF genes, suggesting that CaZFs may be implicated in defense-responsive signal cross talking. Additionally, 18 CaZF genes were differentially expressed under drought and heat treatment, indicating their involvement in plant response to abiotic stresses. Overall, a comprehensive analysis of CaZF gene family in pepper provided significant insights into the understanding of C2H2 ZF-mediated stress regulation network, which would benefit the genetic improvement of pepper and other allied plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02601-x.

Genome wide identification and functional assignments of C2H2 Zinc-finger family transcription factors in Dichanthelium oligosanthes.

Transcription factors (TFs) are biological regulators of gene function in response to various internal and external stimuli. C2H2 zinc finger proteins (C2H2-ZFPs) are a large family of TFs that play crucial roles in plant growth and development, hormone signalling and response to biotic and abiotic stresses. While C2H2-ZFPs have been well characterized in many model and crop plants, they are yet to be ascertained in the evolutionarily important C3 plant Dichanthelium oligosanthes (Heller's rosette grass). In the present study, we report 32 C2H2-ZF genes (DoZFs) belonging to three different classes-Q type, C-type and Z-type based on structural elucidation and phylogenetic analysis. Sequence comparisons revealed paralogs within the DoZFs and orthologs among with rice ZF genes. Motif assignment showed the presence of the distinctive C2H2-ZF conserved domain "QALGGH" in these proteins. Cis-element analysis indicated that majority of the predicted C2H2-ZFPs are associated with hormone signalling and abiotic stress responses. Further, their role in nucleic acid binding and transcriptional regulation was also observed using predicted functional assignment. Thus, we report an overview of the C2H2-ZF gene family in D. oligosanthes that could serve as the basis for future experimental studies on isolation and functional implication of these genes in different biological mechanism of C3 plants.

Analysis of microRNAs and their targets from onion (Allium cepa) using genome survey sequences (GSS) and expressed sequence tags (ESTs).

MicroRNAs are small non-coding RNAs of 21-24 nucleotides in length that acts as important modulators of gene expression related to numerous biological processes including development and defense response in eukaryotes. However, only a limited report on onion (Allium cepa) miRNAs is available and their associated role in growth and development of onion is not yet clear. Therefore, it is of interest to identify miRNAs and their targets in Allium cepa using the genome survey sequences (GSSs) and expressed sequence tags (ESTs) and deduce the functions of the target genes using gene ontology (GO) terms. We report 14 potential miRNAs belonging to 13 different families (miR162, miR168, miR172c, miR172e, miR398, miR400, miR414, miR1134, miR1223, miR6219, miR7725, miR8570, miR8703 and miR8752). BLAST analysis using psRNATarget server predicted 39 potential targets for the identified miRNAs majority of which were transcription factors implicated in plant growth, development, hormone signaling and stress responses. These data forms the basis for further analysis and verification towards understanding the miRNA mediated regulatory mechanism in Allium cepa.