Plant genomics
Targeting‐induced local lesions in genomes (TILLING) is a powerful reverse‐genetics tool that enables high‐throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis‐based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)‐mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.
Commercial varieties of upland cotton (Gossypium hirsutum) have undergone extensive breeding for agronomic traits, such as fiber quality, disease resistance, and yield. Cotton breeding programs have widely used Chinese upland cotton source germplasm (CUCSG) with excellent agronomic traits. A better understanding of the genetic diversity and genomic characteristics of these accessions could accelerate the identification of desirable alleles. Here, we analyzed 10,522 high‐quality single‐nucleotide polymorphisms (SNP) with the CottonSNP63K microarray in 137 cotton accessions (including 12 hybrids of upland cotton). These data were used to investigate the genetic diversity, population structure, and genomic characteristics of each population and the contribution of these loci to heterosis. Three subgroups were identified, in agreement with their known pedigrees, geographical distributions, and times since introduction. For each group, we identified lineage‐specific genomic divergence regions, which potentially harbor key alleles that determine the characteristics of each group, such as early maturity‐related loci. Investigation of the distribution of heterozygous loci, among 12 commercial cotton hybrids, revealed a potential role for these regions in heterosis. Our study provides insight into the population structure of upland cotton germplasm. Furthermore, the overlap between lineage‐specific regions and heterozygous loci, in the high‐yield hybrids, suggests a role for these regions in cotton heterosis.
Trichoderma harzianum is a plant‐beneficial fungus that secretes small cysteine‐rich proteins that induce plant defense responses; however, the molecular mechanism involved in this induction is largely unknown. Here, we report that the class II hydrophobin ThHyd1 acts as an elicitor of induced systemic resistance (ISR) in plants. Immunogold labeling and immunofluorescence revealed ThHyd1 localized on maize (Zea mays) root cell plasma membranes. To identify host plant protein interactors of Hyd1, we screened a maize B73 root cDNA library. ThHyd1 interacted directly with ubiquilin 1‐like (UBL). Furthermore, the N‐terminal fragment of UBL was primarily responsible for binding with Hyd1 and the eight‐cysteine amino acid of Hyd1 participated in the protein‐protein interactions. Hyd1 from T. harzianum (Thhyd1) and ubl from maize were co‐expressed in Arabidopsis thaliana, they synergistically promoted plant resistance against Botrytis cinerea. RNA‐sequencing analysis of global gene expression in maize leaves 24 h after spraying with Curvularia lunata spore suspension showed that Thhyd1‐induced systemic resistance was primarily associated with brassinosteroid signaling, likely mediated through BAK1. Jasmonate/ethylene (JA/ET) signaling was also involved to some extent in this response. Our results suggest that the Hyd1‐UBL axis might play a key role in inducing systemic resistance as a result of Trichoderma‐plant interactions.
