Abstract
Genebanks harbor original landraces carrying many original favorable alleles for mitigating biotic and abiotic stresses. Their genetic diversity remains, however, poorly characterized due to their large within genetic diversity. We developed a high-throughput, cheap and labor saving DNA bulk approach based on single-nucleotide polymorphism (SNP) Illumina Infinium HD array to genotype landraces. Samples were gathered for each landrace by mixing equal weights from young leaves, from which DNA was extracted. We then estimated allelic frequencies in each DNA bulk based on fluorescent intensity ratio (FIR) between two alleles at each SNP using a two step-approach. We first tested either whether the DNA bulk was monomorphic or polymorphic according to the two FIR distributions of individuals homozygous for allele A or B, respectively. If the DNA bulk was polymorphic, we estimated its allelic frequency by using a predictive equation calibrated on FIR from DNA bulks with known allelic frequencies. Our approach: (i) gives accurate allelic frequency estimations that are highly reproducible across laboratories, (ii) protects against false detection of allele fixation within landraces. We estimated allelic frequencies of 23,412 SNPs in 156 landraces representing American and European maize diversity. Modified Roger’s genetic Distance between 156 landraces estimated from 23,412 SNPs and 17 simple sequence repeats using the same DNA bulks were highly correlated, suggesting that the ascertainment bias is low. Our approach is affordable, easy to implement and does not require specific bioinformatics support and laboratory equipment, and therefore should be highly relevant for large-scale characterization of genebanks for a wide range of species.
Highlights
Genetic resources maintained in situ or ex situ in genebanks represent a vast reservoir of traits/alleles for future genetic progress and an insurance against unforeseen threats to agricultural production (Tanksley, 1997; Hoisington et al, 1999; Kilian and Graner, 2012; McCouch et al, 2012)
We developed a new method to estimate allelic frequencies of single-nucleotide polymorphism (SNP) within pools of individuals using the fluorescent intensity ratio (FIR) between A and B alleles from Illumina MaizeSNP50 array
Allelic frequencies at SNPs belonging to MaizeSNP50 array were estimated within 156 maize landraces by pooling randomly 15 individuals per population and by calibrating a predictive two-step model (Figure 1)
Summary
Genetic resources maintained in situ or ex situ in genebanks represent a vast reservoir of traits/alleles for future genetic progress and an insurance against unforeseen threats to agricultural production (Tanksley, 1997; Hoisington et al, 1999; Kilian and Graner, 2012; McCouch et al, 2012). Single nucleotide polymorphisms (SNPs) have become the marker of choice for various crop species such as maize (Ganal et al, 2011), rice (McCouch et al, 2010), barley (Moragues et al, 2010), and soybean (Lam et al, 2010) They are the most abundant class of sequence variation in the genome, co-dominantly inherited, genetically stable and appropriate to high-throughput automated analysis (Rafalski, 2002). For maize 50K Infinium SNP array, only “PZE” prefixed SNPs (so called later PZE SNPs in this study) give consistent results for diversity analysis as compared with previous studies based on SSR markers and are suitable for assessing genetic variability (Ganal et al, 2011; Inghelandt et al, 2011; Bouchet et al, 2013; Frascaroli et al, 2013). 50K Infinium SNP array has been used successfully to decipher genetic diversity of inbred lines (van Heerwaarden et al, 2011; Bouchet et al, 2013; Frascaroli et al, 2013; Rincent et al, 2014), landraces using either doubled haploids (Strigens et al, 2013) or a single individual per accession (van Heerwaarden et al, 2011; Arteaga et al, 2016), or teosinte with few individuals per accession (Aguirre-Liguori et al, 2017)
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