Informative Single Nucleotide Polymorphism Markers Research Articles

Genetic diversity and population structure in banana (Musa spp.) breeding germplasm.

Bananas (Musa spp.) are one of the most highly consumed fruits globally, grown in the tropical and sub-tropical regions. We evaluated 856 Musa accessions from the breeding programs of the International Institute of Tropical Agriculture of Nigeria, Tanzania, and Uganda; the National Agricultural Research Organization of Uganda; the Brazilian Agricultural Research Corporation (Embrapa); and the National Research Centre for Banana of India. Accessions from the in vitro gene bank at the International Transit Centre in Belgium were included to provide a baseline of available global diversity. A total of 16,903 informative single nucleotide polymorphism markers were used to estimate and characterize the genetic diversity and population structure and identify overlaps and unique material among the breeding programs. Analysis of molecular variance displayed low genetic variation among accessions and diploids and a higher variation among tetraploids (p<0.001). Structure analysis revealed two major clusters corresponding to genomic composition. The results indicate that there is potential for the banana breeding programs to increase the diversity in their breeding materials and should exploit this potential for parental improvement and to enhance genetic gains in future breeding efforts.

Tag SNP selection for prediction of adaptation traits in Braford and Hereford cattle using Bayesian methods

AbstractThis study utilized Bayesian inference in a genome‐wide association study (GWAS) to identify genetic markers associated with traits relevant to the adaptation of Hereford and Braford cattle breeds. We focused on eye pigmentation (EP), weaning hair coat (WHC), yearling hair coat (YHC), and breeding standard (BS). Our dataset comprised 126,290 animals in the pedigree. Out of these, 233 sires were genotyped using high‐density (HD) chips, and 3750 animals with medium‐density (50 K) single‐nucleotide polymorphism (SNP) chips. Employing the Bayes B method with a prior probability of π = 0.99, we identified and tagged single nucleotide polymorphisms (Tag SNPs), ranging from 18 to 117 SNPs depending on the trait. These Tag SNPs facilitated the construction of reduced SNP panels. We then evaluated the predictive accuracy of these panels in comparison to traditional medium‐density SNP chips. The accuracy of genomic predictions using these reduced panels varied significantly depending on the clustering method, ranging from 0.13 to 0.65. Additionally, we conducted functional enrichment analysis that found genes associated with the most informative SNP markers in the current study, thereby providing biological insights into the genomic basis of these traits.

The use of SNP markers for cattle breed identification.

A potential application of single nucleotide polymorphisms (SNPs) in animal husbandry and production is identification of the animal breed. In this study, using chosen marker selection methods and genotypic data obtained with the use of Illumina Bovine SNP50 BeadChip for individuals belonging to ten cattle breeds, the reduced panels containing the most informative SNP markers were developed. The suitability of selected SNP panels for the effective and reliable assignment of the studied individuals to the breed of origin was checked by three allocation algorithms implemented in GeneClass 2. The studied breeds set included both Polish-native breeds under the genetic resources conservation programs and highly productive breeds with a global range. For all of the tested marker selection methods ("delta" and two FST-based variants), two separate methodological approaches of marker assortment were used and three marker panels were created with 96, 192, and 288 SNPs respectively, to determine the minimum number of markers required for effective differentiation of the studied breeds. Moreover, the usefulness of the most effective panels of markers to assess the population structure and genetic diversity of the analyzed breeds was examined. The conducted analyses showed the possibility of using SNP subsets from medium-density genotypic microarrays to distinguish breeds of cattle kept in Poland and to analyze their genetic structure.

Genotyping by sequencing for the construction of oil palm (Elaeis guineensis Jacq.) genetic linkage map and mapping of yield related quantitative trait loci.

Oil palm (Elaeis guineensis Jacq.) is one of the major oil-producing crops. Improving the quality and increasing the production yield of oil palm have been the primary focuses of both conventional and modern breeding approaches. However, the conventional breeding approach for oil palm is very challenging due to its longevity, which results in a long breeding cycle. Thus, the establishment of marker assisted selection (MAS) for oil palm breeding programs would speed up the breeding pipeline by generating new oil palm varieties that possess high commercial traits. With the decreasing cost of sequencing, Genotyping-by-sequencing (GBS) is currently feasible to many researchers and it provides a platform to accelerate the discovery of single nucleotide polymorphism (SNP) as well as insertion and deletion (InDel) markers for the construction of a genetic linkage map. A genetic linkage map facilitates the identification of significant DNA regions associated with the trait of interest via quantitative trait loci (QTL) analysis. A mapping population of 112 F1 individuals from a cross of Deli dura and Serdang pisifera was used in this study. GBS libraries were constructed using the double digestion method with HindIII and TaqI enzymes. Reduced representation libraries (RRL) of 112 F1 progeny and their parents were sequenced and the reads were mapped against the E. guineensis reference genome. To construct the oil palm genetic linkage map, informative SNP and InDel markers were used to discover significant DNA regions associated with the traits of interest. The nine traits of interest in this study were fresh fruit bunch (FFB) yield, oil yield (OY), oil to bunch ratio (O/B), oil to dry mesocarp ratio (O/DM) ratio, oil to wet mesocarp ratio (O/WM), mesocarp to fruit ratio (M/F), kernel to fruit ratio (K/F), shell to fruit ratio (S/F), and fruit to bunch ratio (F/B). A total of 2.5 million SNP and 153,547 InDel markers were identified. However, only a subset of 5,278 markers comprising of 4,838 SNPs and 440 InDels were informative for the construction of a genetic linkage map. Sixteen linkage groups were produced, spanning 2,737.6 cM for the maternal map and 4,571.6 cM for the paternal map, with average marker densities of one marker per 2.9 cM and one per 2.0 cM respectively, were produced. A QTL analysis was performed on nine traits; however, only QTL regions linked to M/F, K/F and S/F were declared to be significant. Of those QTLs were detected: two for M/F, four for K/F and one for S/F. These QTLs explained 18.1-25.6% of the phenotypic variance and were located near putative genes, such as casein kinase II and the zinc finger CCCH domain, which are involved in seed germination and growth. The identified QTL regions for M/F, K/F and S/F from this study could be applied in an oil palm breeding program and used to screen palms with desired traits via marker assisted selection (MAS).

Genetic Diversity and Population Structure in Ethiopian Mustard (Brassica carinata A. Braun) as Revealed by Single Nucleotide Polymorphism Markers.

Ethiopian mustard (Brassica carinata A. Braun) is currently one of the potential oilseeds dedicated to the production for biofuel and other bio-industrial applications. The crop is assumed to be native to Ethiopia where a number of diversified B. carinata germplasms are found and conserved ex situ. However, there is very limited information on the genetic diversity and population structure of the species. This study aimed to investigate the genetic diversity and population structure of B. carinata genotypes of different origins using high-throughput single nucleotide polymorphism (SNP) markers. We used Brassica 90K Illumina InfiniumTM SNP array for genotyping 90 B. carinata genotypes, and a total of 11,499 informative SNP markers were used for investigating the population structure and genetic diversity. The structure analysis, principal coordinate analysis (PcoA) and neighbor-joining tree analysis clustered the 90 B. carinata genotypes into two distinct subpopulations (Pop1 and Pop2). The majority of accessions (65%) were clustered in Pop1, mainly obtained from Oromia and South West Ethiopian People (SWEP) regions. Pop2 constituted dominantly of breeding lines and varieties, implying target selection contributed to the formation of distinct populations. Analysis of molecular variance (AMOVA) revealed a higher genetic variation (93%) within populations than between populations (7%), with low genetic differentiation (PhiPT = 0.07) and poor correlation between genetic and geographical distance (R = 0.02). This implies the presence of gene flow (Nm > 1) and weak geographical structure of accessions. Genetic diversity indices showed the presence of moderate genetic diversity in B. carinata populations with an average genetic diversity value (HE = 0.31) and polymorphism information content (PIC = 0.26). The findings of this study provide important and relevant information for future breeding and conservation efforts of B. carinata.

The genetic diversity of Ethiopian barley genotypes in relation to their geographical origin.

Ethiopia is recognized as a center of diversity for barley, and its landraces are known for the distinct genetic features compared to other barley collections. The genetic diversity of Ethiopian barley likely results from the highly diverse topography, altitude, climate conditions, soil types, and farming systems. To get detailed information on the genetic diversity a panel of 260 accessions, comprising 239 landraces and 21 barley breeding lines, obtained from the Ethiopian biodiversity institute (EBI) and the national barley improvement program, respectively were studied for their genetic diversity using the 50k iSelect single nucleotide polymorphism (SNP) array. A total of 983 highly informative SNP markers were used for structure and diversity analysis. Three genetically distinct clusters were obtained from the structure analysis comprising 80, 71, and 109 accessions, respectively. Analysis of molecular variance (AMOVA) revealed the presence of higher genetic variation (89%) within the clusters than between the clusters (11%), with moderate genetic differentiation (PhiPT = 0.11) and five accessions were detected as first-generation migrants using Monte Carlo resampling methods. The Mantel test revealed that the genetic distance between accessions is poorly associated with their geographical distance. Despite the observed weak correlation between geographic distance and genetic differentiation, for some regions like Gonder, Jimma, Gamo-Gofa, Shewa, and Welo, more than 50% of the landraces derived from these regions are assigned to one of the three clusters.

Species identification in Haliotis genus from the northeastern Pacific Ocean using genome-wide RAD-SNPs

Abalone is a marine gastropod mollusk that belongs to the genus Haliotis high-priced and with commercial importance worldwide. Because of its high demand, its products are susceptible to retail fraud by substituting lower-value products or protected species instead of higher-value ones. Therefore, species identification is a crucial traceability element, and restriction-site-associated DNA sequencing (RAD-seq) is a genomic tool that permits achieving this goal in non-model species with few developments in genetic data. This study analyzed five ecological and socio-economically important abalone species that inhabit the west coast and islands of the Baja California Peninsula (BCP), Mexico: green H. fulgens, pink H. corrugata, red H. rufescens, black H. cracherodii and threaded/pinto H. kamtschatkana abalones. The number of putative bi-allelic single nucleotide polymorphisms (SNPs) found across the five species was 150 326 loci. After filtering data, 1 127 SNPs (Total panel) were retained from 223 sequenced abalones. Subsequently, two criteria were used to identify the most informative SNPs markers: private alleles (species-specific) and FST outliers (adaptive markers). The reduced 66 Private SNPs allele panel performs similarly to the Total panel with a 100% correct assignment among the five species, whereas the 24 SNPs FST outlier panel could only correctly assign four out of five species, confusing the threaded/pinto and red abalones. Furthermore, both reduced panels successfully identify black abalone individuals, which has a commercial and conservation importance for seafood trade in Mexico. Therefore, all these markers can be used in seafood traceability and specimen identification in enforcing fishery management measures among these five abalone species.

Genome-wide association study identifies SNP markers and putative candidate genes for terpene traits important for Leptocybe invasa resistance in Eucalyptus grandis.

Terpenes are an important group of plant specialized metabolites influencing, amongst other functions, defence mechanisms against pests. We used a genome-wide association study to identify single nucleotide polymorphism (SNP) markers and putative candidate genes for terpene traits. We tested 15,387 informative SNP markers derived from genotyping 416 Eucalyptus grandis individuals for association with 3 terpene traits, 1,8-cineole, γ-terpinene, and p-cymene. A multilocus mixed model analysis identified 21 SNP markers for 1,8-cineole on chromosomes 2, 4, 6, 7, 8, 9, 10, and 11, that individually explained 3.0%–8.4% and jointly 42.7% of the phenotypic variation. Association analysis of γ-terpinene found 32 significant SNP markers on chromosomes 1, 2, 4, 5, 6, 9, and 11, explaining 3.4–15.5% and jointly 54.5% of phenotypic variation. For p-cymene, 28 significant SNP markers were identified on chromosomes 1, 2, 3, 5, 6, 7, 10, and 11, explaining 3.4–16.1% of the phenotypic variation and jointly 46.9%. Our results show that variation underlying the 3 terpene traits is influenced by a few minor loci in combination with a few major effect loci, suggesting an oligogenic nature of the traits.

Affected-embryo-based SNP haplotyping with NGS for the preimplantation genetic testing of Marfan syndrome

ABSTRACT Marfan syndrome (MFS), an autosomal dominant heritable disease of the connective tissue, is characterized by broad clinical manifestations in the musculoskeletal, cardiovascular, pulmonary, and ocular systems. In this study, a male patient with MFS caused by a heterozygous mutation NM_000138.5(FBN1):c.6037 + 2 T > C in the fibrillin 1 gene (FBN1) underwent preimplantation genetic testing (PGT) by using affected-embryo-based single nucleotide polymorphism (SNP) haplotyping. Multiple displacement amplification was used for whole genome amplification of biopsied trophectoderm cells after controlled ovarian stimulation. Sanger sequencing and next-generation sequencing (NGS) were used to detect the state of FBN1 mutation. A total of 14 blastocysts formed after intracytoplasmic sperm injection were biopsied. After NGS, 60 informative polymorphic SNP markers located upstream and downstream of the FBN1 gene and its pathogenic mutation site were linked to individual alleles. Sanger sequencing further confirmed that 8 blastocysts carried the mutation NM_000138.5(FBN1):c.6037 + 2 T > C, while 6 did not. Four of the non-carriers were euploid verified by copy number variation results. A female infant without MFS was born at 37 weeks gestation after a subsequent frozen embryo transfer. In conclusion, the successful case indicates that SNP haplotyping using sibling embryos as a reference is applicable to PGT in monogenetic diseases. Abbreviations MFS: Marfan syndrome; PGT: preimplantation genetic testing; FBN1: fibrillin 1 gene; NGS: next-generation sequencing; SNP: single nucleotide polymorphism.

A single nucleotide polymorphism assay sheds light on the extent and distribution of genetic diversity, population structure and functional basis of key traits in cultivated north American cannabis

BackgroundThe taxonomic classification of Cannabis genus has been delineated through three main types: sativa (tall and less branched plant with long and narrow leaves), indica (short and highly branched plant with broader leaves) and ruderalis (heirloom type with short stature, less branching and small thick leaves). While still under discussion, particularly whether the genus is polytypic or monotypic, this broad classification reflects putative geographical origins of each group and putative chemotype and pharmacologic effect.MethodsHere we describe a thorough investigation of cannabis accessions using a set of 23 highly informative and polymorphic SNP (Single Nucleotide Polymorphism) markers associated with important traits such as cannabinoid and terpenoid expression as well as fibre and resin production. The assay offers insight into cannabis population structure, phylogenetic relationship, population genetics and correlation to secondary metabolite concentrations. We demonstrate the utility of the assay for rapid, repeatable and cost-efficient genotyping of commercial and industrial cannabis accessions for use in product traceability, breeding programs, regulatory compliance and consumer education.ResultsWe identified 5 clusters in the sample set, including industrial hemp (K5) and resin hemp, which likely underwent a bottleneck to stabilize cannabidiolic acid (CBDA) accumulation (K2, Type II & III). Tetrahydrocannabinolic acid (THCA) resin (Type I) makes up the other three clusters with terpinolene (K4 - colloquial “sativa” or “Narrow Leaflet Drug” (NLD), myrcene/pinene (K1) and myrcene/limonene/linalool (K3 - colloquial “indica”, “Broad Leaflet Drug” (BLD), which also putatively harbour an active version of the cannabichrometic acid Synthase gene (CBCAS).ConclusionThe final chemical compositions of cannabis products have key traits related to their genetic identities. Our analyses in the context of the NCBI Cannabis sativa Annotation Release 100 allows for hypothesis testing with regards to secondary metabolite production. Genetic markers related to secondary metabolite production will be important in many sectors of the cannabis marketplace. For example, markers related to THC production will be important for adaptable and compliant large-scale seed production under the new US Domestic Hemp Production Program.

Genome-wide association mapping for resistance to leaf, stem, and yellow rusts of common wheat under field conditions of South Kazakhstan.

Common or bread wheat (Triticum aestivum L.) is the most important cereal crop in the world, including Kazakhstan, where it is a major agricultural commodity. Fungal pathogens producing leaf, stem, and yellow (stripe) rusts of wheat may cause yield losses of up to 50–60%. One of the most effective methods for preventing these losses is to develop resistant cultivars with high yield potential. This goal can be achieved using complex breeding studies, including the identification of key genetic factors controlling rust disease resistance. In this study, a panel consisting of 215 common wheat cultivars and breeding lines from Kazakhstan, Russia, Europe, USA, Canada, Mexico, and Australia, with a wide range of resistance to leaf rust (LR), stem rust (SR), and yellow rust (YR) diseases, was analyzed under field conditions in Southern Kazakhstan. The collection was genotyped using the 20K Illumina iSelect DNA array, where 11,510 informative single-nucleotide polymorphism markers were selected for further genome-wide association study (GWAS). Evaluation of the phenotypic diversity over 2 years showed a mostly mixed reaction to LR, mixed reaction/moderate susceptibility to SR, and moderate resistance to YR among wheat accessions from Kazakhstan. GWAS revealed 45 marker–trait associations (MTAs), including 23 for LR, 14 for SR, and eight for YR resistances. Three MTAs for LR resistance and one for SR resistance appeared to be novel. The MTAs identified in this work can be used for marker-assisted selection of common wheat in Kazakhstan in breeding new cultivars resistant to LR, SR, and YR diseases. These findings can be helpful for pyramiding genes with favorable alleles in promising cultivars and lines.

An improved 7K SNP array, the C7AIR, provides a wealth of validated SNP markers for rice breeding and genetics studies.

Single nucleotide polymorphisms (SNPs) are highly abundant, amendable to high-throughput genotyping, and useful for a number of breeding and genetics applications in crops. SNP frequencies vary depending on the species and populations under study, and therefore target SNPs need to be carefully selected to be informative for each application. While multiple SNP genotyping systems are available for rice (Oryza sativa L. and its relatives), they vary in their informativeness, cost, marker density, speed, flexibility, and data quality. In this study, we report the development and performance of the Cornell-IR LD Rice Array (C7AIR), a second-generation SNP array containing 7,098 markers that improves upon the previously released C6AIR. The C7AIR is designed to detect genome-wide polymorphisms within and between subpopulations of O. sativa, as well as O. glaberrima, O. rufipogon and O. nivara. The C7AIR combines top-performing SNPs from several previous rice arrays, including 4,007 SNPs from the C6AIR, 2,056 SNPs from the High Density Rice Array (HDRA), 910 SNPs from the 384-SNP GoldenGate sets, 189 SNPs from the 44K array selected to add information content for elite U.S. tropical japonica rice varieties, and 8 trait-specific SNPs. To demonstrate its utility, we carried out a genome-wide association analysis for plant height, employing the C7AIR across a diversity panel of 189 rice accessions and identified 20 QTLs contributing to plant height. The C7AIR SNP chip has so far been used for genotyping >10,000 rice samples. It successfully differentiates the five subpopulations of Oryza sativa, identifies introgressions from wild and exotic relatives, and is useful for quantitative trait loci (QTL) and association mapping in diverse materials. Moreover, data from the C7AIR provides valuable information that can be used to select informative and reliable SNP markers for conversion to lower-cost genotyping platforms for genomic selection and other downstream applications in breeding.

A Genome-Wide Association Study for Resistance to the Insect Pest Leptocybe invasa in Eucalyptus grandis Reveals Genomic Regions and Positional Candidate Defense Genes.

The galling insect, Leptocybe invasa, causes significant losses in plantations of various Eucalyptus species and hybrids, threatening its economic viability. We applied a genome-wide association study (GWAS) to identify single-nucleotide polymorphism (SNP) markers associated with resistance to L. invasa. A total of 563 insect-challenged Eucalyptus grandis trees, from 61 half-sib families, were genotyped using the EUChip60K SNP chip, and we identified 15,445 informative SNP markers in the test population. Multi-locus mixed-model (MLMM) analysis identified 35 SNP markers putatively associated with resistance to L. invasa based on four discreet classes of insect damage scores: (0) not infested, (1) infested showing evidence of oviposition but no gall development, (2) infested with galls on leaves, midribs or petioles and (3) stunting and lethal gall formation. MLMM analysis identified three associated genomic regions on chromosomes 3, 7 and 8 jointly explaining 17.6% of the total phenotypic variation. SNP analysis of a validation population of 494 E. grandis trees confirmed seven SNP markers that were also detected in the initial association analysis. Based on transcriptome profiles of resistant and susceptible genotypes from an independent experiment, we identified several putative candidate genes in associated genomic loci including Nucleotide-binding ARC- domain (NB-ARC) and toll-interleukin-1-receptor-Nucleotide binding signal- Leucine rich repeat (TIR-NBS-LRR) genes. Our results suggest that Leptocybe resistance in E. grandis may be influenced by a few large-effect loci in combination with minor effect loci segregating in our test and validation populations.

Genotyping-By-Sequencing Reveals Population Structure and Genetic Diversity of a Buffelgrass (Cenchrus ciliaris L.) Collection

Buffelgrass (Cenchrus ciliaris L.) is an important forage grass widely grown across the world with many good characteristics including high biomass yield, drought tolerance, and adaptability to a wide range of soil conditions and agro-ecologies. Two hundred and five buffelgrass accessions from diverse origins, conserved as part of the in-trust collection in the ILRI genebank, were analyzed by genotyping-by-sequencing using the DArTseq platform. The genotyping generated 234,581 single nucleotide polymorphism (SNP) markers, with polymorphic information content (PIC) values ranging from 0.005 to 0.5, and the short sequences of the markers were aligned with foxtail millet (Setaria italica) as a reference genome to generate genomic map positions of the markers. One thousand informative SNP markers, representing a broad coverage of the reference genome and with an average PIC value of 0.35, were selected for population structure and diversity analyses. The population structure analysis suggested two main groups, while the hierarchical clustering showed up to eight clusters in the collection. A representative core collection containing 20% of the accessions in the collection, with germplasm from 10 African countries and Oman, was developed. In general, the study revealed the presence of considerable genetic diversity and richness in the collection and a core collection that could be used for further analysis for specific traits of interest.

Population Structure and Genetic Diversity of Nile Tilapia (Oreochromis niloticus) Strains Cultured in Tanzania.

Understanding population structure and genetic diversity within and between local Nile tilapia lines cultured in Tanzania is important for sustainable aquaculture production. This study investigated the genetic structure and diversity among seven Nile tilapia populations in Tanzania (Karanga, Igunga, Ruhila, Fisheries Education and Training Agency, Tanzania Fisheries Research Institute, Kunduchi, and Lake Victoria). Double-digest restriction site-associated DNA (ddRAD) libraries were prepared from 140 individual fish (20 per population) and sequenced using an Illumina HiSeq 4000 resulting in the identification of 2,180 informative single nucleotide polymorphisms (SNPs). Pairwise Fst values revealed strong genetic differentiation between the closely related populations; FETA, Lake Victoria, and Igunga and those from TAFIRI and Karanga with values ranging between 0.45 and 0.55. Population structure was further evaluated using Bayesian model-based clustering (STRUCTURE) and discriminant analysis of principal components (DAPC). Admixture was detected among Karanga, Kunduchi, and Ruhila populations. A cross-validation approach (25% of individual fish from each population was considered of unknown origin) was conducted in order to test the efficiency of the SNP markers to correctly assign individual fish to the population of origin. The cross-validation procedure was repeated 10 times resulting in 77% of the tested individual fish being allocated to the correct population. Overall our results provide a new database of informative SNP markers for both conservation management and aquaculture activities of Nile tilapia strains in Tanzania.

Biyocoğrafik Soy Tahmini ve Adli Bilimlerde Kullanımı

Adli bilimlerde SNP (Single nucleotide polymorphism, tek nükleotid polimorfizm) markırları kimliklendirmede, fenotip belirlemede, nesep ve soy tayininde kullanılmaktadır. Biyocoğrafik soy tahmininde farklı coğrafik bölgelerde bulunan popülasyonlar arasındaki farklılıkları gösteren soy SNP markırları (AISNPs, Ancestry Informative SNP Markers) kullanılmaktadır. AISNP markırları ile bir kişinin biyocoğrafik orijini ve her biyocoğrafik bölgeden alınan genetik soy yüzdesi tespit edilebilir. Bu bilgiler adli bilimlerde bilinmeyen bir şüphelinin, felaket kurbanlarının veya kayıp kişilerin kimliklendirilmesinde kullanılabilir. Bu makalede biyocoğrafik soy kavramı, biyocoğrafik soy tahmininde kullanılan AISNPs panelleri, uygulama yöntemleri, kullanılan istatistiksel yöntemler ve güncel adli uygulamalar tartışılmıştır.

Identification and development of a core set of informative genic SNP markers for assaying genetic diversity in Chinese cabbage

Rapid, economical, and reliable genotyping is an important requirement for germplasm analysis and cultivar identification in crop species. Chinese cabbage (Brassica rapa L. subsp. pekinensis (Lour.) Hanelt) originated in China and is now an economically important vegetable crop worldwide, especially in East Asia. In this study, we evaluated 1167 single nucleotide polymorphisms (SNPs) among 166 representative Chinese cabbage inbred lines using a KASP genotyping assay. On the basis of polymorphisms and principal component analysis, we selected 60 core SNPs distributed on all Brassica rapa chromosomes with allele frequencies sufficiently balanced so as to provide adequate information for genetic identification. The core set of SNPs was used for construction of a neighbor-joining dendrogram, in which the 166 inbred lines were clustered into spring, summer, and autumn ecotype groups. Clustering of the ecotype groups was better resolved than that achieved with 1167 and 360 polymorphic SNP datasets. Stability and resolution of the core SNP markers were tested using 178 commercial hybrid Chinese cabbage cultivars to confirm their utility in genetic identification. The set of 60 informative and stable SNP markers showed high discriminatory power and relatively uniform genomic distribution (4–9 markers per chromosome). The SNPs represent a cost-efficient and accurate marker set for germplasm analysis and cultivar identification and are suitable for molecular marker-assisted breeding in Chinese cabbage.

CropSNPdb: a database of SNP array data for Brassica crops and hexaploid bread wheat.

Advances in sequencing technology have led to a rapid rise in the genomic data available for plants, driving new insights into the evolution, domestication and improvement of crops. Single nucleotide polymorphisms (SNPs) are a major component of crop genomic diversity, and are invaluable as genetic markers in research and breeding programs. High-throughput SNP arrays, or 'SNP chips', can generate reproducible sets of informative SNP markers and have been broadly adopted. Although there are many public repositories for sequencing data, which are routinely uploaded, there are no formal repositories for crop SNP array data. To make SNP array data more easily accessible, we have developed CropSNPdb (http://snpdb.appliedbioinformatics.com.au), a database for SNP array data produced by the Illumina Infinium™ hexaploid bread wheat (Triticum aestivum) 90K and Brassica 60K arrays. We currently host SNPs from datasets covering 526 Brassica lines and 309 bread wheat lines, and provide search, download and upload utilities for users. CropSNPdb provides a useful repository for these data, which can be applied for a range of genomics and molecular crop-breeding activities.

P113 Developing an assay to detect and quantify donor-derived dna in the blood and urine of solid organ transplant patients

Aim Detection of circulating donor-derived cell-free DNA (cfDNA) as a non-invasive biomarker of graft injury shows tremendous potential for transplant monitoring. Increases in the amount of cfDNA in blood or urine indicate injury to the transplanted organ, including rejection. Also known as a liquid biopsy, this method is less invasive and more sensitive than an actual biopsy to detect early rejection. We proposed to develop a clinical test based on the detection and quantification of donor single-nucleotide polymorphisms (SNPs) using cfDNA extracted from plasma and urine of solid organ transplant patients. Methods Using genomic DNA, informative SNP markers from donor and recipient (ThermoFisher) will be identified using real-time PCR (Roche). cfDNA from transplant recipients will be extracted from plasma and urine (Qiagen), and informative SNPs will be quantified using digital-droplet PCR (ddPCR), (Bio-Rad). For development of the assay blood and urine samples were collected from 25 healthy individuals, genomic and cfDNA were extracted, and analytical parameters determined including limits of detection (LOD) and limits of quantification (LOQ) using ddPCR. Results cfDNA extraction was successfully performed from blood and urine. The average cfDNA concentration was 1.36 ± 0.54 ng/ul from plasma, and 1.15 ± 2.1 ng/ul from urine. Determined from genomic DNA, an average of 4.9 informative markers was present between any two of the 25 individuals, although one pairing had no informative markers present, indicating the need for a larger panel using more than 36 SNPs. LOD studies determined the false positivity rate to be less than 0.0025%. Preliminary LOQ testing indicates that 0.5–0.75% donor-derived DNA can be detected. Conclusions We have established a laboratory developed test to detect donor-derived cfDNA from multiple sample types such as blood and urine at a sensitivity that appears acceptable for clinical testing. In collaboration with the local solid organ transplant program, the clinical utility of this assay will be evaluated in kidney transplant recipients.

Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing

BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or “orphan” crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6–$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1–13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384–576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.