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Targeted genotyping is an extremely powerful approach for the detection of known genetic variations that are biologically or clinically important. However, for non-model organisms, large-scale target genotyping in a cost-effective manner remains a major challenge. To address this issue, we present an ultrahigh-multiplex, in-solution probe array-based high-throughput diverse marker genotyping (HD-Marker) approach that is capable of targeted genotyping of up to 86 000 loci, with coverage of the whole gene repertoire, in what is a 27-fold and six-fold multiplex increase in comparison with the conventional Illumina GoldenGate and original HD-Marker assays, respectively. We perform extensive analyses of various ultrahigh-multiplex levels of HD-Marker (30 k-plex, 56 k-plex, and 86 k-plex) and show the power and excellent performance of the proposed method with an extremely high capture rate (about 96%) and genotyping accuracy (about 96%). With great advantages in terms of cost (as low as 0.0006 USD per genotype) and high technical flexibility, HD-Marker is a highly efficient and powerful tool with broad application potential for genetic, ecological, and evolutionary studies of non-model organisms.

Transcriptome-wide targeted genotyping is highly attractive for evolutionary and ecological studies but, until recently, accomplishing this goal presented a major technical barrier for the study of non-model organisms. Our group has recently developed a high-throughput targeted genotyping approach (called HD-Marker) based on the high specificity and accuracy of oligo extension-ligation assays that facilitates the design of assays tailored to meet specific genotyping needs. HD-Marker allows for targeted genotyping of over 10 000 genes in a single tube, with strikingly high capture rate (98%-99%) and genotyping accuracy (97%-99%). With the remarkable advantages of cost-effectiveness and flexibility, we envision that HD-Marker has broad application potential in evolutionary and ecological studies. [ABSTRACT FROM AUTHOR]