Identification of novel candidate genes for Ascochyta blight resistance in chickpea.

Ascochyta blight, caused by the necrotrophic fungus Ascochyta rabiei, is a major threat to chickpea production worldwide. Resistance genes with broad-spectrum protection against virulent A. rabiei strains are required to secure chickpea yield in the US Northern Great Plains. Here, we performed a genome-wide association (GWA) study to discover novel sources of genetic variation for Ascochyta blight resistance using a worldwide germplasm collection of 219 chickpea lines. Ascochyta blight resistance was evaluated at 3, 9, 11, 13, and 14 days post-inoculation. Multiple GWA models revealed eight quantitative trait nucleotides (QTNs) across timepoints mapped to chromosomes 1, 3, 4, 6, and 7. Of these eight QTNs, only CM001767.1_28299946 on Chr 4 had previously been reported. QTN CM001766.1_36967269 on Chr 3 explained up to 33% of the variation in disease severity and was mapped to an exonic region of the pentatricopeptide repeat-containing protein At4g02750-like gene (LOC101506608). This QTN was confirmed across all models and timepoints. A total of 153 candidate genes, including genes with roles in pathogen recognition and signaling, cell wall biosynthesis, oxidative burst, and regulation of DNA transcription, were observed surrounding QTN-targeted regions. Further gene expression analysis on the QTNs identified in this study will provide insights into defense-related genes that can be further incorporated into breeding of new chickpea cultivars to minimize fungicide applications required for successful chickpea production in the US Northern Great Plains.

Dariva FD ，Arman A ，Morales M ，Navasca H ，Shah R ，Atanda SA ，Piche L ，Worral H ，Raymon G ，McPhee K ，Coyne C ，Flores P ，Ebert MK ，Bandillo N ... -  《Scientific Reports》

Discarded sequencing reads uncover natural variation in pest resistance in Thlaspi arvense.

Understanding the genomic basis of natural variation in plant pest resistance is an important goal in plant science, but it usually requires large and labor-intensive phenotyping experiments. Here, we explored the possibility that non-target reads from plant DNA sequencing can serve as phenotyping proxies for addressing such questions. We used data from a whole-genome and -epigenome sequencing study of 207 natural lines of field pennycress (Thlaspi arvense) that were grown in a common environment and spontaneously colonized by aphids, mildew, and other microbes. We found that the numbers of non-target reads assigned to the pest species differed between populations, had significant SNP-based heritability, and were associated with climate of origin and baseline glucosinolate contents. Specifically, pennycress lines from cold and thermally fluctuating habitats, presumably less favorable to aphids, showed higher aphid DNA load, i.e., decreased aphid resistance. Genome-wide association analyses identified genetic variants at known defense genes but also novel genomic regions associated with variation in aphid and mildew DNA load. Moreover, we found several differentially methylated regions associated with pathogen loads, in particular differential methylation at transposons and hypomethylation in the promoter of a gene involved in stomatal closure, likely induced by pathogens. Our study provides first insights into the defense mechanisms of Thlaspi arvense, a rising crop and model species, and demonstrates that non-target whole-genome sequencing reads, usually discarded, can be leveraged to estimate intensities of plant biotic interactions. With rapidly increasing numbers of large sequencing datasets worldwide, this approach should have broad application in fundamental and applied research.

Galanti D ，Jung JH ，Müller C ，Bossdorf O ... -  《eLife》

QTL mapping and genome-wide association analysis reveal genetic loci and candidate gene for resistance to gray leaf spot in tropical and subtropical maize germplasm.

Using QTL mapping and GWAS, two candidate genes (Zm00001d051039 and Zm00001d051147) were consistently identified across the three different environments and BLUP values. GWAS analysis identified the candidate gene, Zm00001d044845. These genes were subsequently validated to exhibit a significant association with maize gray leaf spot (GLS) resistance. Gray leaf spot (GLS) is a major foliar disease of maize (Zea mays L.) that causes significant yield losses worldwide. Understanding the genetic mechanisms underlying gray leaf spot resistance is crucial for breeding high-yielding and disease-resistant varieties. In this study, eight tropical and subtropical germplasms were crossed with the temperate germplasm Ye107 to develop a nested association mapping (NAM) population comprising 1,653 F2:8 RILs, consisting of eight recombinant inbred line (RIL) subpopulations, using the single-seed descent method. The NAM population was evaluated for GLS resistance in three different environments, and genotyping by sequencing of the NAM population generated 593,719 high-quality single-nucleotide polymorphisms (SNPs). Linkage analysis and genome-wide association studies (GWASs) were conducted to identify candidate genes regulating GLS resistance in maize. Both analyses identified 25 QTLs and 149 SNPs that were significantly associated with GLS resistance. Candidate genes were screened 20 Kb upstream and downstream of the significant SNPs, and three novel candidate genes (Zm00001d051039, Zm00001d051147, and Zm00001d044845) were identified. Zm00001d051039 and Zm00001d051147 were located on chromosome 4 and co-localized in both linkage (qGLS4-1 and qGLS4-2) and GWAS analyses. SNP-138,153,206 was located 0.499 kb downstream of the candidate gene Zm00001d051039, which encodes the protein IN2-1 homolog B, a homolog of glutathione S-transferase (GST). GSTs and protein IN2-1 homolog B scavenge reactive oxygen species under various stress conditions, and GSTs are believed to protect plants from a wide range of biotic and abiotic stresses by detoxifying reactive electrophilic compounds. Zm00001d051147 encodes a probable beta-1,4-xylosyltransferase involved in the biosynthesis of xylan in the cell wall, enhancing resistance. SNP-145,813,215 was located 2.69 kb downstream of the candidate gene. SNP-5,043,412 was consistently identified in three different environments and BLUP values and was located 8.788 kb downstream of the candidate gene Zm00001d044845 on chromosome 9. Zm00001d044845 encodes the U-box domain-containing protein 4 (PUB4), which is involved in regulating plant immunity. qRT-PCR analysis showed that the relative expression levels of the three candidate genes were significantly upregulated in the leaves of the TML139 (resistant) parent, indicating that these three candidate genes could be associated with resistance to GLS. The findings of this study are significant for marker-assisted breeding aimed at enhancing resistance to GLS in maize and lay the foundation for further elucidation of the genetic mechanisms underlying resistance to gray leaf spot in maize and breeding of new disease-resistant varieties.

Pan Y ，Jiang F ，Shaw RK ，Sun J ，Li L ，Yin X ，Bi Y ，Kong J ，Zong H ，Gong X ，Ijaz B ，Fan X ... -  《-》

Small RNA sequencing analysis provides novel insights into microRNA-mediated regulation of defense responses in chickpea against Fusarium wilt infection.

Small RNA sequencing analysis in two chickpea genotypes, JG 62 (Fusarium wilt-susceptible) and WR 315 (Fusarium wilt-resistant), under Fusarium wilt stress led to identification of 544 miRNAs which included 406 known and 138 novel miRNAs. A total of 115 miRNAs showed differential expression in both the genotypes across different combinations. A miRNA, Car-miR398 targeted copper chaperone for superoxide dismutase (CCS) that, in turn, regulated superoxide dismutase (SOD) activity during chickpea-Foc interaction. Fusarium wilt (FW) of chickpea (Cicer arietinum L.) caused by Fusarium oxysporum f. sp. ciceris (Foc) is a destructive soil-borne disease that severely reduces the chickpea yield and quality globally. In the present study, we have investigated microRNAs and the microRNA/target gene crosstalk involved in chickpea resistance to FW. The control and stress samples from two genotypes, JG 62 (FW-susceptible) and WR 315 (FW-resistant), collected at 10 days post-inoculation (dpi), were selected for small RNA sequencing. A total of 12 libraries were constructed and sequenced using Illumina HiSeq 2500 platform. The sequencing and in silico analyses revealed the identification of 544 miRNAs which included 406 known and 138 novel miRNAs. A total of 50 miRNAs were physically co-localized with Foc-resistance QTLs present on chromosome 2 (also known as Foc hotspot). A total of 115 miRNAs showed differential expression in both the genotypes across different combinations. Prediction and functional annotation of miRNA targets revealed their role in transcription regulation, disease resistance, defense response, metabolism, etc. Ten miRNAs and their targets were validated using poly(A)-based qRT-PCR in two genotypes grown under lab and field conditions. Many miRNAs and their targets showed genotype-specific expression. The expression profiling also highlighted, both, similar and different expression patterns for the same sets of miRNA and mRNA at different stages of Foc infection. A high correlation in expression patterns of the miRNAs and their targets in lab- and field-grown plant samples was observed. Interestingly, Car-miR398 targeted copper chaperone for superoxide dismutase (CCS) that, in turn, regulated superoxide dismutase (SOD) activity during chickpea-Foc interaction. The cleavage site in targets was mapped for three miRNAs by analyzing publicly available degradome data for chickpea. The study, for the first time, provides novel insights into microRNA-mediated regulation of resistance and susceptibility mechanisms in chickpea against FW and opens up avenues for the development of the wilt-resistant cultivars in chickpea.

Priyadarshini P ，Kalwan G ，Kohli D ，Kumar D ，Bharadwaj C ，Gaikwad K ，Jain PK ... -  《-》

Can a Liquid Biopsy Detect Circulating Tumor DNA With Low-passage Whole-genome Sequencing in Patients With a Sarcoma? A Pilot Evaluation.

A liquid biopsy is a test that evaluates the status of a disease by analyzing a sample of bodily fluid, most commonly blood. In recent years, there has been progress in the development and clinical application of liquid biopsy methods to identify blood-based, tumor-specific biomarkers for many cancer types. However, the implementation of these technologies to aid in the treatment of patients who have a sarcoma remains behind other fields of cancer medicine. For this study, we chose to evaluate a sarcoma liquid biopsy based on circulating tumor DNA (ctDNA). All human beings have normal cell-free DNA (cfDNA) circulating in the blood. In contrast with cfDNA, ctDNA is genetic material present in the blood stream that is derived from a tumor. ctDNA carries the unique genomic fingerprint of the tumor with changes that are not present in normal circulating cfDNA. A successful ctDNA liquid biopsy must be able to target these tumor-specific genetic alterations. For instance, epidermal growth factor receptor (EGFR) mutations are common in lung cancers, and ctDNA liquid biopsies are currently in clinical use to evaluate the status of disease in patients who have a lung cancer by detecting EGFR mutations in the blood. As opposed to many carcinomas, sarcomas do not have common recurrent mutations that could serve as the foundation to a ctDNA liquid biopsy. However, many sarcomas have structural changes to their chromosomes, including gains and losses of portions or entire chromosomes, known as copy number alterations (CNAs), that could serve as a target for a ctDNA liquid biopsy. Murine double minute 2 (MDM2) amplification in select lipomatous tumors or parosteal osteosarcoma is an example of a CNA due to the presence of extra copies of a segment of the long arm of chromosome 12. Since a majority of sarcomas demonstrate a complex karyotype with numerous CNAs, a blood-based liquid biopsy strategy that searches for these CNAs may be able to detect the presence of sarcoma ctDNA. Whole-genome sequencing (WGS) is a next-generation sequencing technique that evaluates the entire genome. The depth of coverage of WGS refers to how detailed the sequencing is, like higher versus lower power on a microscope. WGS can be performed with high-depth sequencing (that is, > 60×), which can detect individual point mutations, or low-depth sequencing (that is, 0.1× to 5×), referred to as low-passage whole-genome sequencing (LP-WGS), which may not detect individual mutations but can detect structural chromosomal changes including gains and losses (that is, CNAs). While similar strategies have shown favorable early results for specific sarcoma subtypes, LP-WGS has not been evaluated for applicability to the broader population of patients who have a sarcoma. Does an LP-WGS liquid biopsy evaluating for CNAs detect ctDNA in plasma samples from patients who have sarcomas representing a variety of histologic subtypes? This was a retrospective study conducted at a community-based, tertiary referral center. Nine paired (plasma and formalin-fixed paraffin-embedded [FFPE] tissue) and four unpaired (plasma) specimens from patients who had a sarcoma were obtained from a commercial biospecimen bank. Three control specimens from individuals who did not have cancer were also obtained. The paired and unpaired specimens from patients who had a sarcoma represented a variety of sarcoma histologic subtypes. cfDNA was extracted, amplified, and quantified. Libraries were prepared, and LP-WGS was performed using a NextSeq 500 next-generation sequencing machine at a low depth of sequencing coverage (∼1×). The ichorCNA bioinformatics algorithm, which was designed to detect CNAs from low-depth genomic sequencing data, was used to analyze the data. In contrast with the gold standard for diagnosis in the form of histopathologic analysis of a tissue sample, this test does not discriminate between sarcoma subtypes but detects the presence of tumor-derived CNAs within the ctDNA in the blood that should not be present in a patient who does not have cancer. The liquid biopsy was positive for the detection of cancer if the ichorCNA algorithm detected the presence of ctDNA. The algorithm was also used to quantitatively estimate the percent ctDNA within the cfDNA. The concentration of ctDNA was then calculated from the percent ctDNA relative to the total concentration of cfDNA. The CNAs of the paired FFPE tissue and plasma samples were graphically visualized using aCNViewer software. This LP-WGS liquid biopsy detected ctDNA in 9 of 13 of the plasma specimens from patients with a sarcoma. The other four samples from patients with a sarcoma and all serum specimens from patients without cancer had no detectable ctDNA. Of those 9 patients with positive liquid biopsy results, the percent ctDNA ranged from 6% to 11%, and calculated ctDNA quantities were 0.04 to 5.6 ng/mL, which are levels to be expected when ctDNA is detectable. In this small pilot study, we were able to detect sarcoma ctDNA with an LP-WGS liquid biopsy searching for CNAs in the plasma of most patients who had a sarcoma representing a variety of histologic subtypes. These results suggest that an LP-WGS liquid biopsy evaluating for CNAs to identify ctDNA may be more broadly applicable to the population of patients who have a sarcoma than previously reported in studies focusing on specific subtypes. Large prospective clinical trials that gather samples at multiple time points during the process of diagnosis, treatment, and surveillance will be needed to further assess whether this technique can be clinically useful. At our institution, we are in the process of developing a large prospective clinical trial for this purpose.

Anderson CJ ，Yang H ，Parsons J ，Ahrens WA ，Jagosky MH ，Hsu JH ，Patt JC ，Kneisl JS ，Steuerwald NM ... -  《-》