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Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design in a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80-90% of the target genes, depending on the genome and gene family. We validated the designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis, soybean, and maize. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Despite current advances in next-generation sequencing data analysis procedures, de novo assembly of a reference sequence required for SNP discovery and expression analysis is still a major challenge in genetically uncharacterized, highly heterozygous species. High levels of polymorphism inherent to outbreeding crop species hamper De Bruijn Graph-based de novo assembly algorithms, causing transcript fragmentation and the redundant assembly of allelic contigs. If multiple genotypes are sequenced to study genetic diversity, primary de novo assembly is best performed per genotype to limit the level of polymorphism and avoid transcript fragmentation. Here, we propose an Orthology Guided Assembly procedure that first uses sequence similarity (tBLASTn) to proteins of a model species to select allelic and fragmented contigs from all genotypes and then performs CAP3 clustering on a gene-by-gene basis. Thus, we simultaneously annotate putative orthologues for each protein of the model species, resolve allelic redundancy and fragmentation and create a de novo transcript sequence representing the consensus of all alleles present in the sequenced genotypes. We demonstrate the procedure using RNA-seq data from 14 genotypes of Lolium perenne to generate a reference transcriptome for gene discovery and translational research, to reveal the transcriptome-wide distribution and density of SNPs in an outbreeding crop and to illustrate the effect of polymorphisms on the assembly procedure. The results presented here illustrate that constructing a non-redundant reference sequence is essential for comparative genomics, orthology-based annotation and candidate gene selection but also for read mapping and subsequent polymorphism discovery and/or read count-based gene expression analysis., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Introduction: The genetic diversity of Robusta coffee (Coffea canephora), a cornerstone in the global coffee industry, remains not fully explored, leading to a significant gap in our understanding of its sensory intricacies. Our study evaluated the sensory quality potential of the Robusta cultivars from the INERA Coffee Collection in Yangambi (the Democratic Republic of the Congo), the local wild diversity, and their hybrids. Methods: We evaluated the sensory attributes of 70 genotypes representing the genetic structure of the coffee collection. Of those 70, 22 genotypes were evaluated for two consecutive years to assess the consistency of the sensory quality. Standard coffee cupping with the Fine Robusta Standards and Protocols was enhanced through sensory descriptors from the Coffee Taster's Flavor Wheel. Each genotype's sensory profile was constructed based on the Total cupping score and the frequency of reported sensory descriptors. The Total cupping score ranged from 75.75 to 84.75, with a substantial variation in sensory profiles, even within a genetic cluster. Results and discussion: Nutty/Cocoa was the most frequently reported descriptor class. The sensory profile ideotype exhibits a high frequency of Fruity, Sweet, and Sour/Fermented descriptors and a low frequency of Green/Vegetative, Other, and Roasted descriptors. Evidence suggests that the sensory profile of a genotype is consistent over two harvest years. Genotypes with promising and unique sensory profiles were discovered within the cultivars and the wild - cultivar hybrids. The genetic diversity of wild and cultivated Robusta in the Democratic Republic of the Congo could play an essential role in understanding and improving its sensory quality. [ABSTRACT FROM AUTHOR]

Though facing significant challenges, coffee (Coffea arabica) grown in Haitian agroforestry systems are important contributors to rural livelihoods and provide several ecosystem services. However, little is known about their genetic diversity and the variety mixtures used. In light of this, there is a need to characterize Haitian coffee diversity to help inform revitalization of this sector. We sampled 28 diverse farms in historically important coffee growing regions of northern and southern Haiti. We performed KASP-genotyping of SNP markers and HiPlex multiplex amplicon sequencing for haplotype calling on our samples, as well as several Ethiopian and commercial accessions from international collections. This allowed us to assign Haitian samples to varietal groups. Our analyses revealed considerable genetic diversity in Haitian farms, higher in fact than many farmers realized. Notably, genetic structure analyses revealed the presence of clusters related to Typica, Bourbon, and Catimor groups, another group that was not represented in our reference accession panel, and several admixed individuals. Across the study areas, we found both mixed-variety farms and monovarietal farms with the historical and traditional Typica variety. This study is, to our knowledge, the first to genetically characterize Haitian C. arabica variety mixtures, and report the limited cultivation of C. canephora (Robusta coffee) in the study area. Our results show that some coffee farms are repositories of historical, widely-abandoned varieties while others are generators of new diversity through genetic mixing. [ABSTRACT FROM AUTHOR]

China's and Europe's dependence on imported protein is a threat to the food self-sufficiency of these regions. It could be solved by growing more legumes, including alfalfa that is the highest protein producer under temperate climate. To create productive and high-value varieties, the use of large genetic diversity combined with genomic evaluation could improve current breeding programs. To study alfalfa diversity, we have used a set of 395 alfalfa accessions (i.e. populations), mainly from Europe, North and South America and China, with fall dormancy ranging from 3 to 7 on a scale of 11. Five breeders provided materials (617 accessions) that were compared to the 400 accessions. All accessions were genotyped using Genotyping-by-Sequencing (GBS) to obtain SNP allele frequency. These genomic data were used to describe genetic diversity and identify genetic groups. The accessions were phenotyped for phenology traits (fall dormancy and flowering date) at two locations (Lusignan in France, Novi Sad in Serbia) from 2018 to 2021. The QTL were detected by a Multi-Locus Mixed Model (mlmm). Subsequently, the quality of the genomic prediction for each trait was assessed. Cross-validation was used to assess the quality of prediction by testing GBLUP, Bayesian Ridge Regression (BRR), and Bayesian Lasso methods. A genetic structure with seven groups was found. Most of these groups were related to the geographical origin of the accessions and showed that European and American material is genetically distinct from Chinese material. Several QTL associated with fall dormancy were found and most of these were linked to genes. In our study, the infinitesimal methods showed a higher prediction quality than the Bayesian Lasso, and the genomic prediction achieved high (>0.75) predicting abilities in some cases. Our results are encouraging for alfalfa breeding by showing that it is possible to achieve high genomic prediction quality. [ABSTRACT FROM AUTHOR]

Conventional wisdom states that genetic variation reduces disease levels in plant populations. Nevertheless, crop species have been subject to a gradual loss of genetic variation through selection for specific traits during breeding, thereby increasing their vulnerability to biotic stresses such as pathogens. We explored how genetic variation in Arabica coffee sites in southwestern Ethiopia was related to the incidence of four major fungal diseases. Sixty sites were selected along a gradient of management intensity, ranging from nearly wild to intensively managed coffee stands. We used genotyping‐by‐sequencing of pooled leaf samples (pool‐GBS) derived from 16 individual coffee shrubs in each of the 60 sites to assess the variation in genetic composition (multivariate: reference allele frequency) and genetic diversity (univariate: mean expected heterozygosity) between sites. We found that genetic composition had a clear spatial pattern and that genetic diversity was higher in less managed sites. The incidence of the four fungal diseases was related to the genetic composition of the coffee stands, but in a specific way for each disease. In contrast, genetic diversity was only related to the within‐site variation of coffee berry disease, but not to the mean incidence of any of the four diseases across sites. Given that fungal diseases are major challenges of Arabica coffee in its native range, our findings that genetic composition of coffee sites impacted the major fungal diseases may serve as baseline information to study the molecular basis of disease resistance in coffee. Overall, our study illustrates the need to consider both host genetic composition and genetic diversity when investigating the genetic basis for variation in disease levels. [ABSTRACT FROM AUTHOR]

Knowledge of genetic identity, genetic relationships, ploidy level, and chromosome numbers can enhance the efficiency of ornamental plant breeding programs. In the present study, genome sizes, chromosome numbers, and genetic fingerprints were determined for a collection of 94 Ilex accessions, including 69 I. crenata. The genome size of the entire collection ranged from 1.50 ± 0.03 to 8.01 ± 0.18. Within the species of I. crenata, genome sizes varied (mean ± sd) between 1.98 ± 0.08 and 2.30 ± 0.04, with three outliers: 3.06 ± 0.04, 4.04 ± 0.09, and 4.19 ± 0.08. The chromosome counting results showed 2n = 40 for I. crenata accessions and confirmed the outliers as one triploid and two tetraploids. A high intra-specific genetic diversity in Ilex crenata was found, after genetic fingerprinting using genotyping-by-sequencing (GBS). The species I. crenata was separated into three clades, which coincided with intraspecific differences in genome sizes (mean ± sd) of 2.09 ± 0.006, 2.07 ± 0.05, and 2.19 ± 0.06 pg/2C per clade as mean values for the diploids. Applying a principal coordinate analysis (PCoA) to the genetic fingerprinting data of all species in the collection revealed a wide genetic variation, which has not yet been commercially exploited. These findings could form the basis for selectively breeding parents, in order to create more genetic diversity via intra- and interspecific crosses. [ABSTRACT FROM AUTHOR]

Key message: High variability for and candidate loci associated with resistance to southern anthracnose and clover rot in a worldwide collection of red clover provide a first basis for genomics-assisted breeding. Red clover (Trifolium pratense L.) is an important forage legume of temperate regions, particularly valued for its high yield potential and its high forage quality. Despite substantial breeding progress during the last decades, continuous improvement of cultivars is crucial to ensure yield stability in view of newly emerging diseases or changing climatic conditions. The high amount of genetic diversity present in red clover ecotypes, landraces, and cultivars provides an invaluable, but often unexploited resource for the improvement of key traits such as yield, quality, and resistance to biotic and abiotic stresses. A collection of 397 red clover accessions was genotyped using a pooled genotyping-by-sequencing approach with 200 plants per accession. Resistance to the two most pertinent diseases in red clover production, southern anthracnose caused by Colletotrichum trifolii, and clover rot caused by Sclerotinia trifoliorum, was assessed using spray inoculation. The mean survival rate for southern anthracnose was 22.9% and the mean resistance index for clover rot was 34.0%. Genome-wide association analysis revealed several loci significantly associated with resistance to southern anthracnose and clover rot. Most of these loci are in coding regions. One quantitative trait locus (QTL) on chromosome 1 explained 16.8% of the variation in resistance to southern anthracnose. For clover rot resistance we found eight QTL, explaining together 80.2% of the total phenotypic variation. The SNPs associated with these QTL provide a promising resource for marker-assisted selection in existing breeding programs, facilitating the development of novel cultivars with increased resistance against two devastating fungal diseases of red clover. [ABSTRACT FROM AUTHOR]

We have developed PotatoMASH (Potato Multi-Allele Scanning Haplotags), a novel low-cost, genome-scanning marker platform. We designed a panel of 339 multi-allelic regions placed at 1 Mb intervals throughout the euchromatic portion of the genome. These regions were assayed using a multiplex amplicon sequencing approach, which allows for genotyping hundreds of plants at a cost of 5 EUR/sample. We applied PotatoMASH to a population of over 700 potato lines. We obtained tetraploid dosage calls for 2012 short multi-allelic haplotypes in 334 loci, which ranged from 2 to 14 different haplotypes per locus. The system was able to diagnose the presence of targeted pest-resistance markers, to detect quantitative trait loci (QTLs) by genome-wide association studies (GWAS) in a tetraploid population, and to track variation in a diploid segregating population. PotatoMASH efficiently surveys genetic variation throughout the potato genome, and can be implemented as a single low-cost genotyping platform that will allow the routine and simultaneous application of marker-assisted selection (MAS) and other genotyping applications in commercial potato breeding programmes. [ABSTRACT FROM AUTHOR]