snpqc – an R pipeline for quality control of Illumina SNP genotyping array data

In genome‐wide association studies, quality control (QC) of genotypes is important to avoid spurious results. It is also important to maintain long‐term data integrity, particularly in settings with ongoing genotyping (e.g. estimation of genomic breeding values). Here we discuss snpqc , a fully automated pipeline to perform QC analyses of Illumina SNP array data. It applies a wide range of common quality metrics with user‐defined filtering thresholds to generate a comprehensive QC report and a filtered dataset, including a genomic relationship matrix, ready for further downstream analyses which make it amenable for integration in high‐throughput environments. snpqc also builds a database to store genotypic, phenotypic and quality metrics to ensure data integrity and the option of integrating more samples from subsequent runs. The program is generic across species and array designs, providing a convenient interface between the genotyping laboratory and downstream genome‐wide association study or genomic prediction.     

High‐throughput genomics in sorghum: from whole‐genome resequencing to a SNP screening array

With its small, diploid and completely sequenced genome, sorghum (Sorghum bicolor L. Moench) is highly amenable to genomics‐based breeding approaches. Here, we describe the development and testing of a robust single‐nucleotide polymorphism (SNP) array platform that enables polymorphism screening for genome‐wide and trait‐linked polymorphisms in genetically diverse S. bicolor populations. Whole‐genome sequences with 6× to 12× coverage from five genetically diverse S. bicolor genotypes, including three sweet sorghums and two grain sorghums, were aligned to the sorghum reference genome. From over 1 million high‐quality SNPs, we selected 2124 Infinium Type II SNPs that were informative in all six source genomes, gave an optimal Assay Design Tool (ADT) score, had allele frequencies of 50% in the six genotypes and were evenly spaced throughout the S. bicolor genome. Furthermore, by phenotype‐based pool sequencing, we selected an additional 876 SNPs with a phenotypic association to early‐stage chilling tolerance, a key trait for European sorghum breeding. The 3000 attempted bead types were used to populate half of a dual‐species Illumina iSelect SNP array. The array was tested using 564 Sorghum spp. genotypes, including offspring from four unrelated recombinant inbred line (RIL) and F₂ populations and a genetic diversity collection. A high call rate of over 80% enabled validation of 2620 robust and polymorphic sorghum SNPs, underlining the efficiency of the array development scheme for whole‐genome SNP selection and screening, with diverse applications including genetic mapping, genome‐wide association studies and genomic selection.     

SNP markers‐based map construction and genome‐wide linkage analysis in Brassica napus

An Illumina Infinium array comprising 5306 single nucleotide polymorphism (SNP) markers was used to genotype 175 individuals of a doubled haploid population derived from a cross between Skipton and Ag‐Spectrum, two Australian cultivars of rapeseed (Brassica napus L.). A genetic linkage map based on 613 SNP and 228 non‐SNP (DArT, SSR, SRAP and candidate gene markers) covering 2514.8 cM was constructed and further utilized to identify loci associated with flowering time and resistance to blackleg, a disease caused by the fungus Leptosphaeria maculans. Comparison between genetic map positions of SNP markers and the sequenced Brassica rapa (A) and Brassica oleracea (C) genome scaffolds showed several genomic rearrangements in the B. napus genome. A major locus controlling resistance to L. maculans was identified at both seedling and adult plant stages on chromosome A07. QTL analyses revealed that up to 40.2% of genetic variation for flowering time was accounted for by loci having quantitative effects. Comparative mapping showed Arabidopsis and Brassica flowering genes such as Phytochrome A/D, Flowering Locus C and agamous‐Like MADS box gene AGL1 map within marker intervals associated with flowering time in a DH population from Skipton/Ag‐Spectrum. Genomic regions associated with flowering time and resistance to L. maculans had several SNP markers mapped within 10 cM. Our results suggest that SNP markers will be suitable for various applications such as trait introgression, comparative mapping and high‐resolution mapping of loci in B. napus.     

A genome‐wide scan for copy number variations using high‐density single nucleotide polymorphism array in Simmental cattle

Copy number variations (CNVs) have recently been identified as promising sources of genetic variation, complementary to single nucleotide polymorphisms (SNPs). As a result, detection of CNVs has attracted a great deal of attention. In this study, we performed genome‐wide CNV detection using Illumina Bovine HD BeadChip (770k) data on 792 Simmental cattle. A total of 263 CNV regions (CNVRs) were identified, which included 137 losses, 102 gains and 24 regions classified as both loss and gain, covering 35.48 Mb (1.41%) of the bovine genome. The length of these CNVRs ranged from 10.18 kb to 1.76 Mb, with an average length of 134.78 kb and a median length of 61.95 kb. In 136 of these regions, a total of 313 genes were identified related to biological functions such as transmembrane activity and olfactory transduction activity. To validate the results, we performed quantitative PCR to detect nine randomly selected CNVRs and successfully confirmed seven (77.6%) of them. Our results present a map of cattle CNVs derived from high‐density SNP data, which expands the current CNV map of the cattle genome and provides useful information for investigation of genomic structural variation in cattle.     

Identification of a large SNP dataset in Larimichthys crocea using specific‐locus amplified fragment sequencing

The large yellow croaker, Larimichthys crocea, is a commercially important drum fish (Family: Sciaenidae) native to the East and South China Sea. Habitat deterioration and overfishing have led to significant population decline and the collapse of its fishery over the past decades. Today, the market supply of L. crocea depends solely on stocks produced in hatcheries and farms. Common issues that occur in the culture of L. crocea include germplasm degradation, precocious puberty, elevated disease susceptibility and growth retardation. In this study, we employed SLAF‐seq (specific‐locus amplified fragment sequencing) technology to identify single nucleotide polymorphism (SNP) loci across the L. crocea genome. Sixty samples were selected for SLAF analysis out of 1000 progeny in the same cohort of a cultured stock. Our analysis obtained a total of 151 253 SLAFs, of which 65.88% (99 652) were identified to be polymorphic, scoring a total of 710 567 putative SNPs. Further filtration resulted in a final panel of 1782 SNP loci. The data derived from this work could be beneficial for understanding the genetics of complex phenotypic traits as well as for developing marker‐selection‐assisted breeding programs in L. crocea.     

Estimation of linkage disequilibrium and interspecific gene flow in Ficedula flycatchers by a newly developed 50k single‐nucleotide polymorphism array

With the access to draft genome sequence assemblies and whole‐genome resequencing data from population samples, molecular ecology studies will be able to take truly genome‐wide approaches. This now applies to an avian model system in ecological and evolutionary research: Old World flycatchers of the genus Ficedula, for which we recently obtained a 1.1 Gb collared flycatcher genome assembly and identified 13 million single‐nucleotide polymorphism (SNP)s in population resequencing of this species and its sister species, pied flycatcher. Here, we developed a custom 50K Illumina iSelect flycatcher SNP array with markers covering 30 autosomes and the Z chromosome. Using a number of selection criteria for inclusion in the array, both genotyping success rate and polymorphism information content (mean marker heterozygosity = 0.41) were high. We used the array to assess linkage disequilibrium (LD) and hybridization in flycatchers. Linkage disequilibrium declined quickly to the background level at an average distance of 17 kb, but the extent of LD varied markedly within the genome and was more than 10‐fold higher in ‘genomic islands’ of differentiation than in the rest of the genome. Genetic ancestry analysis identified 33 F₁ hybrids but no later‐generation hybrids from sympatric populations of collared flycatchers and pied flycatchers, contradicting earlier reports of backcrosses identified from much fewer number of markers. With an estimated divergence time as recently as <1 Ma, this suggests strong selection against F₁ hybrids and unusually rapid evolution of reproductive incompatibility in an avian system.