Identification of candidate virulence loci in Striga hermonthica, a devastating parasite of African cereal crops

Parasites have evolved suites of proteins, Virulence Factors (VFs), that are delivered into host plants to facilitate colonization. Whilst VFs mediating plant-microbe and plant-nematode interactions have been characterised extensively, less is known about VFs mediating parasitic plant interactions with their hosts. Striga hermonthica is an obligate, root-parasitic plant capable of parasitizing multiple cereal hosts in sub-Saharan Africa, causing devastating losses in yields. An understanding of the molecular nature and allelic variation of VFs in S. hermonthica is essential for breeding durable resistance and delaying the evolution of parasite virulence. To address this issue, we assembled a genome for Striga hermonthica and identified candidate VFs by combining in silico prediction of secreted proteins with pooled sequencing of parasites growing on a susceptible and a strongly resistant rice host. Consistent with predictions for parasites, like S. hermonthica, that can interact with multiple hosts, we identified multiple loci, potentially with a wide range of functions, implicated in overcoming host resistance. Extremely different allele frequencies were observed at 152 non-secreted and 38 putatively secreted VFs between S. hermonthica parasitising the resistant and susceptible rice varieties. Our candidate, secreted VFs encompassed functions such as host cell wall modification, protease inhibitors, oxidoreductase and kinase activities, as well as several with unknown functions. Consistent with maintenance of variation at virulence loci by balancing selection the candidate loci had significantly higher Tajima’s D on average than the genomic background. Our results show that diverse strategies are used by S. hermonthica to overcome different layers of host resistance. Understanding the maintenance of variation at virulence loci by balancing selection will be critical to managing the evolution of virulence as a part of a sustainable control strategy.

Characterization and Mapping of retr04, retr05 and retr06 Broad-Spectrum Resistances to Turnip Mosaic Virus in Brassica juncea, and the Development of Robust Methods for Utilizing Recalcitrant Genotyping Data

Turnip mosaic virus (TuMV) induces disease in susceptible hosts, notably impacting cultivation of important crop species of the Brassica genus. Few effective plant viral disease management strategies exist with the majority of current approaches aiming to mitigate the virus indirectly through control of aphid vector species. Multiple sources of genetic resistance to TuMV have been identified previously, although the majority are strain-specific and have not been exploited commercially. Here, two Brassica juncea lines (TWBJ14 and TWBJ20) with resistance against important TuMV isolates (UK 1, vVIR24, CDN 1, and GBR 6) representing the most prevalent pathotypes of TuMV (1, 3, 4, and 4, respectively) and known to overcome other sources of resistance, have been identified and characterized. Genetic inheritance of both resistances was determined to be based on a recessive two-gene model. Using both single nucleotide polymorphism (SNP) array and genotyping by sequencing (GBS) methods, quantitative trait loci (QTL) analyses were performed using first backcross (BC1) genetic mapping populations segregating for TuMV resistance. Pairs of statistically significant TuMV resistance-associated QTLs with additive interactive effects were identified on chromosomes A03 and A06 for both TWBJ14 and TWBJ20 material. Complementation testing between these B. juncea lines indicated that one resistance-linked locus was shared. Following established resistance gene nomenclature for recessive TuMV resistance genes, these new resistance-associated loci have been termed retr04 (chromosome A06, TWBJ14, and TWBJ20), retr05 (A03, TWBJ14), and retr06 (A03, TWBJ20). Genotyping by sequencing data investigated in parallel to robust SNP array data was highly suboptimal, with informative data not established for key BC1 parental samples. This necessitated careful consideration and the development of new methods for processing compromised data. Using reductive screening of potential markers according to allelic variation and the recombination observed across BC1 samples genotyped, compromised GBS data was rendered functional with near-equivalent QTL outputs to the SNP array data. The reductive screening strategy employed here offers an alternative to methods relying upon imputation or artificial correction of genotypic data and may prove effective for similar biparental QTL mapping studies.

Peptide-centric analyses of human plasma enable increased resolution of biological insights into non-small cell lung cancer relative to protein-centric analysis

Comprehensive assessment of the human proteome remains challenging due to multiple forms of a protein, or proteoforms, arising from alternative splicing, allelic variation, and protein modifications. As proteoforms can serve distinct functions and act as functional link between genotype and phenotype, proteoform-level knowledge is critical in understanding the molecular mechanisms underlying health and disease. However, identification of proteoforms requires unbiased protein coverage at amino acid resolution. Scalable, deep, and unbiased proteomics studies have been impractical due to cumbersome and lengthy workflows required for complex samples, like blood plasma. Here, we demonstrate the power of the Proteograph™ Product Suite in enabling unbiased, deep, and rapid proteomics at scale in a proof-of-concept proteoform analysis to dissect differences between protein isoforms in plasma samples from 80 healthy controls and 61 patients with early-stage non-small-cell lung cancer (NSCLC). Processing the 141 plasma samples with Proteograph yielded 22,993 peptides corresponding to 2,569 protein groups at a confidence of 1% false discovery rate. We extracted four proteins with peptides with significant abundance differences (p < 0.05; Benjamini-Hochberg corrected) in healthy control and cancer plasma samples. For one, the abundance variation can be explained by underlying annotated protein isoforms. For a second, we find evidence for differentially transcribed isoforms in the broader sequence data, but not in the known annotated protein isoforms. The others may be explained by novel isoforms or post-translational modifications. In addition, we sought to identify protein variants arising from allelic variation. To this end, we first performed whole exome sequencing on buffy coat samples from 29 individuals in the NSCLC study. Then, we created personalized mass spectrometry search databases for each individual subject from the exome sequences. From these libraries, we identified 422 protein variants, one of which has previously been shown to relate to lung cancer. In conclusion, our results demonstrate that Proteograph can generate unbiased and deep plasma proteome profiles that enable identification of proteoforms present in plasma at a scale sufficient to enable population-scale proteomic studies powered to reveal novel mechanistic and biomedical insights.

Allelic variation for alpha-Glucan Water Dikinase is associated with starch phosphate content in tetraploid potato

Key message Association analysis resulted in the identification of specific StGWD alleles causing either an increase or decrease in starch phosphate content which was verified in diploid and tetraploid potato mapping populations. Abstract Potatoes are grown for various purposes like French fries, table potatoes, crisps and for their starch. One of the most important aspects of potato starch is that it contains a high amount of phosphate ester groups which are considered to be important for providing improved functionalization after derivatization processes. Little is known about the variation in phosphate content as such in different potato varieties and thus we studied the genetic diversity for this trait. From other studies it was clear that the phosphate content is controlled by a quantitative trait locus (QTL) underlying the candidate gene α-Glucan Water Dikinase (StGWD) on chromosome 5. We performed direct amplicon sequencing of this gene by Sanger sequencing. Sequences of two StGWD amplicons from a global collection of 398 commercial cultivars and progenitor lines were used to identify 16 different haplotypes. By assigning tag SNPs to these haplotypes, each of the four alleles present in a cultivar could be deduced and linked to a phosphate content. A high value for intra-individual heterozygosity was observed (Ho = 0.765). The average number of different haplotypes per individual (Ai) was 3.1. Pedigree analysis confirmed that the haplotypes are identical-by-descent (IBD) and offered insight in the breeding history of elite potato germplasm. Haplotypes originating from introgression of wild potato accessions carrying resistance genes could be traced. Furthermore, association analysis resulted in the identification of specific StGWD alleles causing either an increase or decrease in starch phosphate content varying from 12 nmol PO4/mg starch to 38 nmol PO4/mg starch. These allele effects were verified in diploid and tetraploid mapping populations and offer possibilities to breed and select for this trait.

Dynamic evolution of small signaling peptide compensation in plant stem cell control

Gene duplications are a hallmark of plant genome evolution and a foundation for genetic interactions that shape phenotypic diversity. Compensation is a major form of paralog interaction, but how compensation relationships change as allelic variation accumulates is unknown. Here, we leveraged genomics and genome editing across the Solanaceae family to capture the evolution of compensating paralogs. Mutations in the stem cell regulator CLV3 cause floral organs to overproliferate in many plants. In tomato, this phenotype is partially suppressed by transcriptional upregulation of a closely related paralog. Tobacco lost this paralog, resulting in no compensation and extreme clv3 phenotypes. Strikingly, the paralogs of petunia and groundcherry nearly completely suppress clv3, indicating a potent ancestral state of compensation. Cross-species transgenic complementation analyses show this potent compensation partially degenerated in tomato due to a single amino acid change in the paralog and cis-regulatory variation that limits its transcriptional upregulation. Our findings show how genetic interactions are remodeled following duplications, and suggest that dynamic paralog evolution is widespread over short time scales and impacts phenotypic variation from natural and engineered mutations.

Genetic Diversity of Fig (Ficus Carica L.) Germplasm From The Mediterranean Basin As Revealed by SSR Markers

Fig (Ficus carica L.) tree is cultivated worldwide and is highly appreciated for its fruit, which is consumed fresh or dried, having high nutritional and pharmaceutical value and for these reasons there is an increasing interest for its cultivation. In the present study, an ex situ collection of 60 fig accessions (41 indigenous Greek and 19 from other Mediterranean countries) was established and its diversity was analyzed using eight simple sequence repeat (SSR) loci. Greek fig genotypes showed relatively low allelic variation (average number of SSR alleles per locus was 3.3), an excess of heterozygosity (mean He = 0.449 and Ho = 0.537), and extensive outbreeding (mean F index -0.184). Cluster analysis showed that the established fig population exhibited weak genetic structure with the majority of the genetic variation (69%) being present within individual members of the clusters. Both cluster and principal coordinate analysis confirmed that there is no correlation between genetic makeup and geographical origin of the fig accessions. Polymorphism information content (PIC) with an average of 0.398 was reasonably informative. An identification key scheme for fig cultivars that will be useful in cultivar discrimination and intellectual property protection was developed. This work will contribute to a sustainable fig production regionally and worldwide, through the establishment and conservation of a reference fig collection, providing germplasm for future breeding efforts.

THE USE OF ARCHIVED GIEMSA-STAINED BLOOD SMEARS AND RDT FOR PCR-BASED GENOTYPING OF Plasmodium vivax MEROZOITE SURFACE PROTEIN-1 IN CENTRAL KALIMANTAN PROVINCE, INDONESIA: PCR-Based Genotyping of Pvmsp-1 in Central Kalimantan Province, Indonesia

Background: Plasmodium vivax is transmitted most across the country of Indonesia. The country has set out a malaria elimination program by 2030. The information on genetic diversity of malarial parasites relates to malaria transmission in an endemic area may provide the information that can help the malaria control program to achieve the target. Therefore, the purpose of this study was to determine the genetic diversity of the Pvmsp-1 gene in Central Kalimantan Province. Materials and Methods: Samples were 140 of archived Giemsa-stained blood smear and rapid detection test. Samples were divided into the indigenous and migrant populations. After confirmation by single-step PCR, only P. vivax and mixed infection samples were amplified to nested PCR for genotyping of Pvmsp-1 allelic variation in segments F1, F2, and F3. Results: Genotyping of 23 PCR positive samples resulted in 13 genotypes. In segment F1, three allelic variants type A containing subtype A1 (1,050 bp), A2 (350 bp), A3 (150 bp), and type B (100 bp). In segment F2, mono genotypes were detected as variant type A (1,050 bp) and type B3 (150 bp), multiple genotypes were detected as type B containing subtype B1 (250 bp), B2 (200 bp), and B3 (150bp). In segment F3, three allelic variants generated from four mono genotypes were type A (350 bp), type B (300 bp), and two type C (250 bp). Conclusion: The low allelic variation of Pvmsp-1 gene may reflect the actual situation of the low malaria endemic status of the study sites.

Allelic Variation Analysis at the Vernalization Response and Photoperiod Genes in Russian Wheat Varieties Identified Two Novel Alleles of Vrn-B3

Heading time is an important agronomic trait affecting the adaptability and productivity of common wheat. In this study, 95 common wheat varieties from Russia and the late-maturing breeding line ‘Velut’ were tested for allelic diversity of genes having the strongest effect on heading. In this research, allelic variation at the Ppd-D1, Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 loci was tested. The Vrn-B1 and Vrn-B3 loci provided the largest contribution to genetic diversity. We found two novel allelic variants of the Vrn-B3 gene in the studied varieties. Ten varieties carried a 160 bp insertion in the promoter region, and the breeding line ‘Velut’ carried a 1617 bp insertion. These alleles were designated Vrn-B3e and Vrn-B3d, respectively. The analysis of the sequences showed the recent insertion of a retrotransposon homologous to the LTR retrotransposon (RLX_Hvul_Dacia_ RND-1) in the Vrn-B3d allele. Plants with the Vrn-B3e and the ‘Velut’ line with the Vrn-B3d allele headed later than the plants with the wild-type allele; among these plants, ‘Velut’ is the latest maturing wheat variety. Analysis of the gene expression of two groups of lines differing by the Vrn-B3 alleles (Vrn-B3d or vrn-B3) from the F2 population with ‘Velut’ as a parental line did not reveal a significant difference in the expression level between the groups. Additional research is required to study the reasons for the late maturation of the ‘Velut’ line. However, the studied wheat varieties could be used as a potential source of natural variation in genes controlling heading times.

Study of Parental Polymorphism and Allelic Variation for Grain Quality and Yield Traits in Rice (Oryza sativa L.) Using SSR Markers

Aim: Identification of polymorphic markers is prerequisite for conducting any QTL mapping experiment because if the parents are polymorphic for the traits of interest, then further selection of plants in the progenies becomes easy. Hence, the objective of the present study was to identify polymorphic markers for grain quality and yield traits among the parental lines Improved Samba Mahsuri and Badshabhog. Place and Duration of Study: It was carried out at Molecular Breeding Lab, Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi - 221 005, India, during 2019. Methodology: Two parents Improved Samba Mahsuri and Badshabhog were used for the present study. The DNA extraction was done as per the CTAB method suggested by Murray and Thompson. Standard PCR protocol was followed. Results: For parental polymorphism survey, a total of 576 randomly selected SSR markers including 26 gene specific markers related to aroma, cooking and eating quality, grain dimension and yield related traits distributed across the 12 chromosomes of rice were used. Overall, 96 markers including 4 gene specific markers were found to be polymorphic between the two genotypes indicating a total polymorphism percentage of 16.67%. The highest polymorphism percentage was recorded on chromosome 6 (26.67%) followed by chromosome 4 (21.43%) and the lowest polymorphism percentage was observed on chromosome 10 (8.93%). The gene specific markers nksbad2, ARO7, BADEX7_5 and SSI were found to be polymorphic. Conclusion: Based on the present study it may be concluded that the polymorphic markers identified will further be utilized in genotyping of F2:3 population, linkage analysis and mapping QTL’s for grain quality and yield traits.