WormBase has been the major repository and knowledgebase of information about the genome and genetics of Caenorhabditis elegans and other nematodes of experimental interest for over 2 decades. We have 3 goals: to keep current with the fast-paced C. elegans research, to provide better integration with other resources, and to be sustainable. Here, we discuss the current state of WormBase as well as progress and plans for moving core WormBase infrastructure to the Alliance of Genome Resources (the Alliance). As an Alliance member, WormBase will continue to interact with the C. elegans community, develop new features as needed, and curate key information from the literature and large-scale projects.

WormBase (www.wormbase.org) is the central repository for the genetics and genomics of the nematode Caenorhabditis elegans. We provide the research community with data and tools to facilitate the use of C. elegans and related nematodes as model organisms for studying human health, development, and many aspects of fundamental biology. Throughout our 22-year history, we have continued to evolve to reflect progress and innovation in the science and technologies involved in the study of C. elegans. We strive to incorporate new data types and richer data sets, and to provide integrated displays and services that avail the knowledge generated by the published nematode genetics literature. Here, we provide a broad overview of the current state of WormBase in terms of data type, curation workflows, analysis, and tools, including exciting new advances for analysis of single-cell data, text mining and visualization, and the new community collaboration forum. Concurrently, we continue the integration and harmonization of infrastructure, processes, and tools with the Alliance of Genome Resources, of which WormBase is a founding member.

Few public sector rice breeders have the capacity to use NGS-derived markers in their breeding programmes despite rapidly expanding repositories of rice genome sequence data. They rely on > 18,000 mapped microsatellites (SSRs) for marker-assisted selection (MAS) using gel analysis. Lack of knowledge about target SNP and InDel variant loci has hampered the uptake by many breeders of Kompetitive allele-specific PCR (KASP), a proprietary technology of LGC genomics that can distinguish alleles at variant loci. KASP is a cost-effective single-step genotyping technology, cheaper than SSRs and more flexible than genotyping by sequencing (GBS) or array-based genotyping when used in selection programmes. Before this study, there were 2015 rice KASP marker loci in the public domain, mainly identified by array-based screening, leaving large proportions of the rice genome with no KASP coverage. Here we have addressed the urgent need for a wide choice of appropriate rice KASP assays and demonstrated that NGS can detect many more KASP to give full genome coverage. Through re-sequencing of nine indica rice breeding lines or released varieties, this study has identified 2.5 million variant sites. Stringent filtering of variants generated 1.3 million potential KASP assay designs, including 92,500 potential functional markers. This strategy delivers a 650-fold increase in potential selectable KASP markers at a density of 3.1 per 1 kb in the indica crosses analysed and 377,178 polymorphic KASP design sites on average per cross. This knowledge is available to breeders and has been utilised to improve the efficiency of public sector breeding in Nepal, enabling identification of polymorphic KASP at any region or quantitative trait loci in relevant crosses. Validation of 39 new KASP was carried out by genotyping progeny from a range of crosses to show that they detected segregating alleles. The new KASP have replaced SSRs to aid trait selection during marker-assisted backcrossing in these crosses, where target traits include rice blast and BLB resistance loci. Furthermore, we provide the software for plant breeders to generate KASP designs from their own datasets.

DNA methylation is an important mechanism of epigenetic gene expression control that can be passed between generations. Here, we use sodium bisulfite treatment and targeted gene enrichment to study genome-wide methylation across the three sub-genomes of allohexaploid wheat. While the majority of methylation is conserved across all three genomes we demonstrate that differential methylation exists between the sub-genomes in approximately equal proportions. We correlate sub-genome-specific promoter methylation with decreased expression levels and show that altered growing temperature has a small effect on methylation state, identifying a small but functionally relevant set of methylated genes. Finally, we demonstrate long-term methylation maintenance using a comparison between the D sub-genome of hexaploid wheat and its progenitor Aegilops tauschii. We show that tri-genome methylation is highly conserved with the diploid wheat progenitor while sub-genome-specific methylation shows more variation.

This study aimed to broaden applicability of KASP for Oryza sativa across diverse genotypes through incorporation of ambiguous (degenerate) bases into their primer designs and to validate 4000 of them for genotyping applications. A bioinformatics pipeline was used to compare 129 rice genomes from 89 countries with the indica reference genome R498 and generate ∼1.6 million KASP designs for the more common variants between R498 and the other genomes. Of the designs, 98,238 were for predicted functional markers. Up to five KASP each for 1024 breeder-selected loci were assayed in a panel of 178 diverse rice varieties, generating 3366 validated KASP. The 84% success rate was within the normal range for KASP demonstrating that the ambiguous bases do not compromise efficacy. The 3366-trait-specific marker panel was applied for population structure analysis in the diversity panel and resolved them into four expected groups. Target variations in thirteen of the genome sequences used for designs were compared with the corresponding KASP genotypes of other accessions of the same thirteen varieties in the diversity panel. There was agreement across 12 varieties for 79% of markers. Ten varieties had high agreement (>88%) but a variety selected from a landrace had only 46.5% agreement. Breeders can now search for the validated KASP and >1 million so-far untested designs across three alternative reference genomes (including Niponbare MSU7), search for designs proximal to previously published SSR markers and retrieve the target variations in 129 rice genomes plus their genomic locations with +/-25 bp flanking sequences.

Authentication of Basmati rice has relied on microsatellite markers since 2004, but microsatellites cannot distinguish between all of the forty-one Basmati varieties approved in 2017. This study investigated whether single nucleotide polymorphisms (SNP) and insertion/deletion (InDel) variations developed into KASP™ (Kompetitive Allele Specific PCR; LGC Biosearch Technologies) could be used to distinguish between commercial Basmati varieties. Suitable loci were identified by comparing whole genome sequences of 120 diverse rice accessions. Sequences flanking these loci were standardized across a wide range of rice genomes to produce optimal KASP designs. We selected 364 KASP designs to use for genotyping; they were either near to informative microsatellite markers, within the Badh2 and Waxy genes, or distributed throughout the rice genome. Genotypes for 327 KASP were obtained with 255 loci revealing polymorphism in up to 41 samples of approved Basmati varieties and 20 non-Basmati varieties. The varieties genotyped had not been used in the KASP design process. KASP were able to distinguish between commercial Basmati varieties that could not be distinguished with currently available microsatellites. Thirty-seven Basmati varieties could be distinguished from all others with between 3 and 8 KASP markers out of a pool of 98 informative markers. A reduced set of 24 KASP markers could determine whether a sample belongs to one of eight family groups. All of the KASP markers used in this study can be purchased from LGC Biosearch Technologies. These markers have potential to be used by industry for routine testing and regulation.