Next-generation sequencing (NGS) library construction often requires high-quality DNA extraction, precise adjustment of DNA concentration, and restriction enzyme digestion to reduce genome complexity, which results in increased time and cost in sample preparation and processing. To address these challenges, a PCR-based method for rapid NGS library preparation, named dpMIG-seq, has been developed and proven effective for high-throughput genotyping. However, the application of dpMIG-seq has been limited to diploid and polyploid species with disomic inheritance. In this study, we obtained genome-wide single nucleotide polymorphism (SNP) markers for tetraploid blueberry to evaluate genotyping and downstream analysis outcomes. Comparison of genotyping qualities inferred across samples with different DNA concentrations and multiple bioinformatics approaches revealed high accuracy and reproducibility of dpMIG-seq-based genotyping, with Pearson's correlation coefficients between replicates in the range of 0.91 to 0.98. Furthermore, we demonstrated that dpMIG-seq enables accurate genotyping of samples with low DNA concentrations. Subsequently, we applied dpMIG-seq to a tetraploid F1 population to examine the inheritance probability of parental alleles. Pairing configuration analysis supported the random meiotic pairing of homologous chromosomes on a genome-wide level. On the other hand, preferential pairing was observed on chr-11, suggesting that there may be an exception to the random pairing. Genotypic data suggested quadrivalent formation within the population, although the frequency of quadrivalent formation varied by chromosome and cultivar. Collectively, the results confirmed applicability of dpMIG-seq for allele dosage genotyping and are expected to catalyze the adoption of this cost-effective and rapid genotyping technology in polyploid studies.

中文翻译：

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一种用于阐明多体作物复杂遗传的低成本 dpMIG-seq 方法：四倍体蓝莓的案例研究


二代测序 （NGS） 文库构建通常需要高质量的 DNA 提取、精确调整 DNA 浓度和限制性内切酶消化，以降低基因组复杂性，从而导致样品制备和处理的时间和成本增加。为了应对这些挑战，已经开发了一种基于 PCR 的快速 NGS 文库制备方法，名为 dpMIG-seq，并被证明对高通量基因分型有效。然而，dpMIG-seq 的应用仅限于具有二倍体遗传的二倍体和多倍体物种。在这项研究中，我们获得了四倍体蓝莓的全基因组单核苷酸多态性 （SNP） 标记，以评估基因分型和下游分析结果。对具有不同 DNA 浓度和多种生物信息学方法的样品中推断的基因分型质量的比较表明，基于 dpMIG-seq 的基因分型具有很高的准确性和可重复性，重复之间的 Pearson 相关系数在 0.91 至 0.98 的范围内。此外，我们证明 dpMIG-seq 能够对低 DNA 浓度的样品进行准确的基因分型。随后，我们将 dpMIG-seq 应用于四倍体 F1 群体，以检查亲本等位基因的遗传概率。配对配置分析支持全基因组水平上同源染色体的随机减数分裂配对。另一方面，在 chr-11 上观察到优先配对，表明随机配对可能存在例外。基因型数据表明种群内形成四价，尽管四价形成的频率因染色体和品种而异。 总的来说，结果证实了 dpMIG-seq 用于等位基因剂量基因分型的适用性，并有望促进这种经济高效且快速的基因分型技术在多倍体研究中采用。