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The identification of informative molecular markers is useful for linkage mapping and can benefit genome-wide association studies by providing fine-scale information about sequence variations. However, high-throughput genotyping approaches are not cost-effective for labs that require frequent use, such as breeding programs that need to perform genotyping on large populations with hundreds of individuals. The number of single nucleotide polymorphism markers generated by those approaches can be far more than needed for most breeding programs; instead, breeders focus on the use of at most hundreds of polymorphic molecular markers for analysis. To help make use of molecular markers a routine tool for breeding programs, we aim to develop a cost-effective genotyping system by using the Tecan Allegro Targeted Resequencing V2 kit. This provides a customized probe design, which indicates that all the DNA fragments synthesized are known targets. SNPs obtained from previous peanut next-generation sequencing data were pre-filtered and selected as targets. These SNP targets were polymorphic among different tetraploid accessions and were selected to be distinguishable from paralogs. A total of 5154 probes were designed to detect 2770 SNP targets and were tested on 48 accessions, which include some closely related sister lines from a breeding population. The results indicated that genotyping by a targeted resequencing approach reduced the cost from around USD 28 (SNP chip and GBS) to USD 18 per sample, while providing polymorphic markers with accurate SNP calls. With this cost-effective genotyping platform, pre-selected SNP markers can be used effectively and routinely for more breeding programs. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ) is a deadly infectious disease having a major impact on global health. Using the CMV vector for development of novel vaccines is a promising new strategy that elicits strong and durable, high frequency memory T cell responses against heterologous immunogens. We conducted functional transcriptomic analysis of whole blood samples collected from cohorts of rhesus (Rh) macaques that were administered RhCMV/TB vector using a prime-boost strategy. Two modified CMV vectors were used in this study, including 68-1 RhCMV/TB-6Ag (encoding 6 Mtb protein immunogens, including Ag85A, ESAT-6, Rv3407, Rv2626, Rpf A, and Rpf D) and its attenuated variant, 68-1 RhCMV/Δpp71-TB-6Ag (a cell-to-cell spread-deficient vaccine vector lacking the Rh110 gene encoding the pp71 tegument protein). Bulk mRNA sequencing, differential gene expression, and functional enrichment analyses showed that these RhCMV/TB vaccines induce the innate and adaptive immune responses with specific transcriptomic signatures, including the IL-15-induced protective gene signature previously defined to be linked with protection against simian immunodeficiency virus (SIV) by the 68-1 RhCMV/SIV vaccine. While both vectors exhibited a transcriptomic response of the IL-15 protective signature in whole blood, we show that lack of pp71 does not maintain induction of the protective signature for the full duration of the study compared to the parental non-attenuated vector. Our observations indicate that RhCMV vector vaccines induce a transcriptomic response in whole blood that include a conserved IL-15 signature of which vector-encoded pp71 is an important component of response durability that upon future Mtb challenge may define specific vaccine protection outcomes against Mtb infection., Competing Interests: OHSU and LP and SH have a significant financial interest in Vir Biotechnology, Inc., a company that may have a financial interest in the results of CMV-based vector research and technology. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sung, Whitmore, Smith, Chang, Tisoncik-Go, Barber-Axthelm, Selseth, Feltham, Ojha, Hansen, Picker and Gale.)