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PubMed2026

CamK-DB: A k-mer MinHash fingerprint database for reference-free genotyping of Camellia accessions.

Tea (Camellia sinensis L.), a major global economic crop in Asia, poses challenges for genetic identification because its highly heterozygous, repetitive genome reduces the efficacy of conventional single-nucleotide polymorphism (SNP) and microsatellite markers, and interspecific hybridization further complicates the situation. To address these issues, CamK-DB was developed as a reference-free Camellia fingerprinting database built on MIKE MinHash sketches. We curated 418 candidate resequencing datasets, and built a database using standardized 5× genome-coverage fingerprints. Each accession is stored as a MIKE. jac fingerprint generated with k = 21 and recommended sketch/pre_cnt = 2000. CamK-DB provides a command-line interface for data management and a custom C++ query engine that computes top-10 matches using Jaccard similarity, complemented by a QT-based graphical interface for interactive analysis. This resource offers a robust and scalable framework for precise and routine germplasm identification, genomic phylogenetic inference, and strategic breeding program design. CamK-DB (database and code) is publicly available at https://github.com/sc-zhang/CamK-DB. CamK-DB binaries are provided for Windows 10/11 and Linux (x86_64, glibc ≥ 2.27).

Databases, Genetic↗
PubMed2003

Creating a saturated reference map for the apple (Malus x domestica Borkh.) genome.

The availability of a high quality linkage map is essential for the detection and the analysis of quantitative traits. Such a map should cover a significant part of the genome, should be densely populated with markers, and in order to gain the maximum advantage should be transferable to populations or cultivars other than the ones on which it has been constructed. An apple genetic linkage map has been constructed on the basis of a segregating population of the cross between the cultivars Fiesta and Discovery. A total of 840 molecular markers, 475 AFLPs, 235 RAPDs, 129 SSRs and 1 SCAR, were used for the two parental maps constructed with JoinMap and spanning 1,140 cM and 1,450 cM, respectively. Large numbers of codominant markers, like SSRs, enable a rapid transfer of the map to other populations or cultivars, allowing the investigation of any chosen trait in another genetic background. This map is currently the most advanced linkage map in apple with regard to genome coverage and marker density. It represents an ideal starting point for future mapping projects in Malus since the stable and transferable SSR frame of the map can be saturated quickly with dominant AFLP markers.

Automation↗
PubMed1991

Optimized strategies for sequence-tagged-site selection in genome mapping.

The physical mapping of complex genomes is based on the construction of a genomic library and the determination of the overlaps between the inserts of the mapping clones in order to generate an ordered, cloned representation of nearly all the sequences present in the target genome. Evaluation of the relative efficiency of experimental procedures used to accomplish this goal must minimally include a comparison of the fraction of the genome covered by the ordered arrays (or "contigs"), the average size of the contigs, and the cost, in terms of time and resources, required to generate the map. Sequence-tagged-site (STS) content mapping is one strategy that has been proposed and is being utilized for this type of experiment. This paper describes three STS selection schemes and presents computer simulations of contig-building experiments based on these procedures. The results of these simulations suggest that a nonrandom STS strategy that uses paired probes requires one-third to one-fourth as many STS assays as are required in random and nonpaired approaches, and also results in a map that has both greater genome coverage and a larger average contig size. This strategy promises to reduce the time and cost required to build a high-quality physical map.

Base Sequence↗
PubMed2006

DMRT gene cluster analysis in the platypus: new insights into genomic organization and regulatory regions.

We isolated and characterized a cluster of platypus DMRT genes and compared their arrangement, location, and sequence across vertebrates. The DMRT gene cluster on human 9p24.3 harbors, in order, DMRT1, DMRT3, and DMRT2, which share a DM domain. DMRT1 is highly conserved and involved in sexual development in vertebrates, and deletions in this region cause sex reversal in humans. Sequence comparisons of DMRT genes between species have been valuable in identifying exons, control regions, and conserved nongenic regions (CNGs). The addition of platypus sequences is expected to be particularly valuable, since monotremes fill a gap in the vertebrate genome coverage. We therefore isolated and fully sequenced platypus BAC clones containing DMRT3 and DMRT2 as well as DMRT1 and then generated multispecies alignments and ran prediction programs followed by experimental verification to annotate this gene cluster. We found that the three genes have 58-66% identity to their human orthologues, lie in the same order as in other vertebrates, and colocate on 1 of the 10 platypus sex chromosomes, X5. We also predict that optimal annotation of the newly sequenced platypus genome will be challenging. The analysis of platypus sequence revealed differences in structure and sequence of the DMRT gene cluster. Multispecies comparison was particularly effective for detecting CNGs, revealing several novel potential regulatory regions within DMRT3 and DMRT2 as well as DMRT1. RT-PCR indicated that platypus DMRT1 and DMRT3 are expressed specifically in the adult testis (and not ovary), but DMRT2 has a wider expression profile, as it does for other mammals. The platypus DMRT1 expression pattern, and its location on an X chromosome, suggests an involvement in monotreme sexual development.

Amino Acid Sequence↗
PubMed2006

A genetic linkage map of Silene vulgaris based on AFLP markers.

A genetic linkage map of an intraspecific cross between 2 Silene vulgaris s.l. ecotypes is presented. Three-hundred AFLP markers from 2 different restriction enzyme combinations were used to genotype an F2 mapping population. Maternal and paternal pure-coupling phase maps with 114 and 186 markers on 12 and 13 linkage groups, respectively, were constructed. Total map length of the paternal and maternal maps are 547 and 446 Kosambi cM, respectively. Nearly half of the markers (49%) exhibited significant transmission ratio distortion. Genome coverage and potential causes of the observed segregation ratio distortions are discussed. The maps represent a first step towards the identification of quantitative trait loci associated with habitat adaptation in the non-model species Silene vulgaris.

Adaptation, Biological↗
PubMed2026

Genome-targeted enrichment and sequencing of human-infecting Cryptosporidium spp.

Cryptosporidium spp. are parasites that cause severe illness in vulnerable human populations. Obtaining pure and sufficient Cryptosporidium DNA from clinical and environmental samples is a challenging task. Oocysts shed in available fecal samples can be limited in quantity, require purification (biased towards dominant strains), and yield limited DNA (&#x2009;<&#x2009;40 fg/oocyst). Here, we use updated genomic sequences from a broad diversity of Cryptosporidium species that have been found to infect humans (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to develop and validate a set of 100,000 RNA baits (CryptoCap_100k) with the aim of enriching Cryptosporidium DNA from varied samples. Compared to unenriched libraries, CryptoCap_100k increases the percentage of reads mapping to target genome sequences, increases the depth and breadth of genome coverage, and facilitates analyses of genetic variants in many samples, while decreasing overall costs.

Cryptosporidium↗
PubMed2004

Strain-specific genomic regions of Ruminococcus flavefaciens FD-1 as revealed by combinatorial random-phase genome sequencing and suppressive subtractive hybridization.

Two closely related strains of the Gram-positive, cellulolytic ruminal bacterium Ruminococcus flavefaciens were compared at the genomic level by suppressive subtractive hybridization. The two strains investigated in this study differ by 1.94% in their respective 16S rDNA genes. Three hundred and eighty-four PCR-amplified products were cloned and then screened for their strain identity by dot blot hybridization. Based on redundancy percentages of the clones sequenced, 9.5% of the genome of the R. flavefaciens FD-1 strain is not present in the JM1 strain. The majority of identities of individual cloned subtracted products (642 bp average length) bore no relation to deposited sequences in GenBank (42% of the subtracted library), whereas of those with putative assigned functions 7% are loosely associated with fibre-degradation, 6% with insertion elements, transposons and phage-like ORFs, 5% with cell membrane associated proteins and 3% with signal transduction. Subtracted sequences were then supplemented with the draft (2 x coverage) genome sequence of R. flavefaciens FD-1 to indicate potential regions of rearrangement within the genome, including a novel insertion sequence.

Base Sequence↗
PubMed2005

A highly redundant BAC library of Atlantic salmon (Salmo salar): an important tool for salmon projects.

BACKGROUND: As farming of Atlantic salmon is growing as an aquaculture enterprise, the need to identify the genomic mechanisms for specific traits is becoming more important in breeding and management of the animal. Traits of importance might be related to growth, disease resistance, food conversion efficiency, color or taste. To identify genomic regions responsible for specific traits, genomic large insert libraries have previously proven to be of crucial importance. These large insert libraries can be screened using gene or genetic markers in order to identify and map regions of interest. Furthermore, large-scale mapping can utilize highly redundant libraries in genome projects, and hence provide valuable data on the genome structure. RESULTS: Here we report the construction and characterization of a highly redundant bacterial artificial chromosome (BAC) library constructed from a Norwegian aquaculture strain male of Atlantic salmon (Salmo salar). The library consists of a total number of 305,557 clones, in which approximately 299,000 are recombinants. The average insert size of the library is 188 kbp, representing 18-fold genome coverage. High-density filters each consisting of 18,432 clones spotted in duplicates have been produced for hybridization screening, and are publicly available 1. To characterize the library, 15 expressed sequence tags (ESTs) derived overgos and 12 oligo sequences derived from microsatellite markers were used in hybridization screening of the complete BAC library. Secondary hybridizations with individual probes were performed for the clones detected. The BACs positive for the EST probes were fingerprinted and mapped into contigs, yielding an average of 3 contigs for each probe. Clones identified using genomic probes were PCR verified using microsatellite specific primers. CONCLUSION: Identification of genes and genomic regions of interest is greatly aided by the availability of the CHORI-214 Atlantic salmon BAC library. We have demonstrated the library's ability to identify specific genes and genetic markers using hybridization, PCR and fingerprinting experiments. In addition, multiple fingerprinting contigs indicated a pseudo-tetraploidity of the Atlantic salmon genome. The highly redundant CHORI-214 BAC library is expected to be an important resource for mapping and sequencing of the Atlantic salmon genome.

Animals↗
PubMed2000

Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library.

A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.

Animals↗
PubMed1997

Microsatellite marker development, mapping and applications in rice genetics and breeding.

Microsatellites are simple, tandemly repeated di- to tetra-nucleotide sequence motifs flanked by unique sequences. They are valuable as genetic markers because they are co-dominant, detect high levels of allelic diversity, and are easily and economically assayed by the polymerase chain reaction (PCR). Results from screening a rice genomic library suggest that there are an estimated 5700-10,000 microsatellites in rice, with the relative frequency of different repeats decreasing with increasing size of the motif. A map consisting of 120 microsatellite markers demonstrates that they are well distributed throughout the 12 chromosomes of rice. Five multiple copy primer sequences have been identified that could be mapped to independent chromosomal locations. The current level of genome coverage provided by these simple sequence length polymorphisms (SSLPs) in rice is sufficient to be useful for genotype identification, gene and quantitative trail locus (QTL) analysis, screening of large insert libraries, and marker-assisted selection in breeding. Studies of allelic diversity have documented up to 25 alleles at a single locus in cultivated rice germplasm and provide evidence that amplification in wild relatives of Oryza sativa is generally reliable. The availability of increasing numbers of mapped SSLP markers can be expected to complement existing RFLP and AFLP maps, increasing the power and resolution of genome analysis in rice.

Breeding↗
PubMed2002

Mapping quantitative trait loci in inbred backcross lines of Lycopersicon pimpinellifolium (LA1589).

Although tomato has been the subject of extensive quantitative trait loci (QTLs) mapping experiments, most of this work has been conducted on transient populations (e.g., F2 or backcross) and few homozygous, permanent mapping populations are available. To help remedy this situation, we have developed a set of inbred backcross lines (IBLs) from the interspecific cross between Lycopersicon esculentum cv. E6203 and L. pimpinellifolium (LA1589). A total of 170 BC2F1 plants were selfed for five generations to create a set of homozygous BC2F6 lines by single-seed descent. These lines were then genotyped for 127 marker loci covering the entire tomato genome. These IBLs were evaluated for 22 quantitative traits. In all, 71 significant QTLs were identified, 15% (11/71) of which mapped to the same chromosomal positions as QTLs identified in earlier studies using the same cross. For 48% (34/71) of the detected QTLs, the wild allele was associated with improved agronomic performance. A number of new QTLs were identified including several of significant agronomic importance for tomato production: fruit shape, firmness, fruit color, scar size, seed and flower number, leaf curliness, plant growth, fertility, and flowering time. To improve the utility of the IBL population, a subset of 100 lines giving the most uniform genome coverage and map resolution was selected using a randomized greedy algorithm as implemented in the software package MapPop (http://www.bio.unc.edu/faculty/vision/lab/ mappop/). The map, phenotypic data, and seeds for the IBL population are publicly available (http://soldb.cit.cornell.edu) and will provide tomato geneticists and breeders with a genetic resource for mapping, gene discovery, and breeding.

Genome, Plant↗
PubMed2026

Molecular Epidemiology of Human Metapneumovirus in Kilifi, Coastal Kenya, 2016-2017 and 2021-2024.

BACKGROUND: Human metapneumovirus (hMPV) is a major contributor of acute respiratory infections (ARI) in childhood and vulnerable adults. It comprises two antigenically distinct lineages (A and B), with multiple sub-lineages. Genomic analyses of hMPV strains enable monitoring of viral evolution and transmission to inform future interventions but remain underutilized in Africa. METHODS: We generated 52 near-complete hMPV genomes from respiratory samples collected in Kilifi, Coastal Kenya, using a tiled-amplicon approach and Oxford Nanopore Technologies sequencing. These samples had been identified as hMPV positive by quantitative PCR during (a) a multi-facility outpatient ARI surveillance in nine health facilities in Kilifi between 2016 and 2017, and 2021 to 2023 and (b) a community-based respiratory infection cohort surveillance study between 2023-2024 that sampled enrolled participants irrespective of symptom status. RESULTS: Of the 192 positive samples analyzed from the two studies, children under 5 years accounted for most hMPV cases (134/186, 72%). 52 samples were sequenced (>70% genome coverage), and hMPV-A (27/52, 53.8%) and hMPV-B (25/52, 46.2%) lineages were identified. The recovered sequences mapped into sub-lineages A2c (27/52, 53.8%), B1 (12/52, 21.2%), and B2b (13/52, 25%). A shift in the predominant sub-lineage was observed from B2b (2016) to B1 (2021), and finally to A2c-wild type (2023). In February 2021, for the first time, we detected a single A2c strain with a 111-nucleotide duplication in the G gene among Kenyan samples. CONCLUSION: Our study expands the global nucleotide sequence database for hMPV by adding new whole-genome sequences from Kenya collected over the last decade. It highlights the ongoing replacement of locally predominant hMPV lineages and the importation and local transmission of globally circulating strains. These findings underscore the importance of sustained hMPV genomic surveillance to detect emerging variants and monitor lineage circulation patterns that may impact viral transmission, molecular detection, and future control measures.

A2c-111nt-dup↗
PubMed2006

Diversity arrays technology (DArT) for high-throughput profiling of the hexaploid wheat genome.

Despite a substantial investment in the development of panels of single nucleotide polymorphism (SNP) markers, the simple sequence repeat (SSR) technology with a limited multiplexing capability remains a standard, even for applications requiring whole-genome information. Diversity arrays technology (DArT) types hundreds to thousands of genomic loci in parallel, as previously demonstrated in a number diploid plant species. Here we show that DArT performs similarly well for the hexaploid genome of bread wheat (Triticum aestivum L.). The methodology previously used to generate DArT fingerprints of barley also generated a large number of high-quality markers in wheat (99.8% allele-calling concordance and approximately 95% call rate). The genetic relationships among bread wheat cultivars revealed by DArT coincided with knowledge generated with other methods, and even closely related cultivars could be distinguished. To verify the Mendelian behaviour of DArT markers, we typed a set of 90 Cranbrook x Halberd doubled haploid lines for which a framework (FW) map comprising a total of 339 SSR, restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) markers was available. We added an equal number of DArT markers to this data set and also incorporated 71 sequence tagged microsatellite (STM) markers. A comparison of logarithm of the odds (LOD) scores, call rates and the degree of genome coverage indicated that the quality and information content of the DArT data set was comparable to that of the combined SSR/RFLP/AFLP data set of the FW map.

Chromosomes, Plant↗
PubMed2005

Detection of quantitative trait loci influencing conformation traits and calving ease in Holstein-Friesian cattle.

An extension of our previous genome scan of a North American Holstein-Friesian population was conducted to identify quantitative trait loci (QTL) affecting conformation traits. Resource families consisted of 1404 sons of 10 elite sires. Genome coverage was estimated to be 2713.5 cM (90%) for 406 markers using a granddaughter design. Regression interval mapping was used to detect QTL affecting 22 conformation traits, including body, udder, feet and legs, and dairy conformation as well as calving ease. Analysis of the families jointly identified 41 chromosome-wise significant QTL influencing conformation traits and 3 significant QTL influencing calving ease on 20 chromosomes. The false discovery rate method was used to account for multiple testing and 3/4 of the suggestive and 5/6 of significant QTL should be real effects. Fourteen of the 44 QTL were significant at the genome-wise level. Comparison of these results with other published reports identifies common QTL affecting conformation traits. Regions on 10 chromosomes appear to affect multiple traits, including conformation, milk production, and somatic cell score, within these particular US Holstein families. Additional work is needed to determine the precise locations of the QTL and select positional candidate genes influencing these traits.

Animals↗
PubMed1995

A small-insert bovine genomic library highly enriched for microsatellite repeat sequences.

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA.GT) > 12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of > 100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers.

Animals↗
PubMed2006

A framework linkage map of perennial ryegrass based on SSR markers.

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third (124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci were generated from either SSR-enriched genomic libraries (247) or ESTs (5). Forty-five percent of the GeneThresher SSRs were associated with an expressed gene. Unlike EST-derived SSR markers, GeneThresher SSRs were often associated with genes expressed at a low level, such as transcription factors. The map constructed here fulfills 2 definitions of a "framework map". Firstly, it is composed of codominant markers to ensure map transferability either within or among species. Secondly, it was constructed to achieve a level of statistical confidence in the support-for-order of marker loci. The map consists of 81 framework SSR markers spread over 7 linkage groups, the same as the haploid chromosome number. Most of the remaining 295 SSR markers have been placed into their most likely interval on the framework map. Nine RFLP markers and 1 SSR marker from another map constructed using the same pedigree were also incorporated to extend genome coverage at the terminal ends of 5 linkage groups. The final map provides a robust framework with which to conduct investigations into the genetic architecture of trait variation in this commercially important grass species.

Chromosome Mapping↗
PubMed2004

Creation of a BAC resource to study the structure and evolution of the banana (Musa balbisiana) genome.

The first bacterial artificial chromosome (BAC) library of the banana species Musa balbisiana 'Pisang Klutuk Wulung' (PKW BAC library) was constructed and characterized. One improved and one novel protocol for nuclei isolation were employed to overcome problems caused by high levels of polyphenols and polysaccharides present in leaf tissues. The use of flow cytometry to purify cell nuclei eliminated contamination with secondary metabolites and plastid DNA. Furthermore, the usefulness of the inducible pCC1BAC vector to obtain a higher amount of BAC DNA was demonstrated. The PKW BAC library represents nine haploid genome equivalents of M. balbisiana and its mean insert size is 135 kb. It consists of two sublibraries, of which the first one (SN sublibrary with 24,960 clones) was prepared according to an improved standard nuclei isolation protocol, whereas the second (FN sublibrary with 11,904 clones) was obtained from flow-sorted nuclei. Screening with 12 RFLP probes, which were genetically anchored to 8 genetic linkage groups of the banana species Musa acuminata, revealed an average of 11 BAC clones per probe, thus confirming the genome coverage estimated based on the insert size, as well as a high level of conservation between the two species of Musa. Localization of selected BAC clones to mitotic chromosomes using FISH indicated that the BAC library represented a useful resource for cytogenetic mapping. As the first step in map-based cloning of a genetic factor that is involved in the activation of integrated pararetroviral sequences of Banana streak virus (BSV), the BSV expressed locus (BEL) was physically delimited. The PKW BAC library represents a publicly available tool, and is currently used to reveal the integration and activation mechanisms of BSV sequences and to study banana genome structure and evolution.

Cell Nucleus↗