Nuclear insertions of mitochondrial DNA sequences (numts) can introduce errors in population genetic studies using mitochondrial DNA markers (mtDNA markers). Despite their prevalence in mammalian genomes and the potential risk of contamination with mtDNA markers, adequate precautions are often overlooked. When investigating the genetic diversity of Vietnam sheep, we found numt contamination in D-loop amplification. Consequentially, to continue, we conducted a comprehensive in silico survey of numts using the reference genomes from the NCBI database. We identified four so-called “mega-numts” that are not only highly similar in sequence to the mitogenome but also span a large part of it, thus posing a serious problem for amplifying authentic mtDNA markers. We demonstrated the existence of two numts in three randomly selected Vietnam sheep individuals. Based on the assumed mutation rate of the D-loop region, we proposed a sequence identity threshold to help distinguish authentic D-loop sequences from their corresponding numts when studying contemporary sheep populations. We hope this study can serve as a guideline for future research into Vietnam sheep using mtDNA markers.

The Phan Rang sheep, considered the sole indigenous breed of Vietnam, are primarily concentrated in the two central provinces of Ninh Thuan and Binh Thuan, with Ninh Thuan accounting for more than 90% of the country’s sheep population. These provinces are known for their high temperatures and frequent droughts. The long-standing presence of the Phan Rang sheep in these regions suggests their potential resilience to heat stress—a trait of increasing interest in the face of global climate change. Despite the breed’s significance, a critical knowledge gap hinders conservation and breeding programs. To address this, our study employed a two-pronged approach. First, we collected body conformational data to aid in breed identification. Second, we analyzed mitochondrial DNA (D-loop) and Y chromosome markers (SRY and SRYM18) to elucidate the maternal and paternal lineages. Among the 68 Phan Rang sheep analyzed for their D-loop, 19 belonged to mitochondrial haplogroup A, while 49 belonged to haplogroup B. The haplogroups can be subdivided into 16 unique haplotypes. All 19 rams surveyed for their paternal lineages belonged to haplotypes H5 and H6. These findings strongly support the hypothesis of dual origins for the Phan Rang sheep. This study presents the first genetic data for the Phan Rang breed, providing crucial insights for future research and conservation efforts.

Codon optimization is a widely adopted strategy to improve heterologous expression in Saccharomyces cerevisiae, while other approaches, such as optimizing regulatory elements and cultivation conditions, are less common. Here, using enhanced green fluorescent protein (eGFP) as a heterologous reporter gene, we aimed to compare the effects of terminator selection, cultivation temperature, and coding sequence optimization in the S. cerevisiae strain 2805. Flow cytometry and immunoblotting were employed to assess changes in expression levels. While codon optimization still produced the largest improvement in expression, terminator choice and cultivation temperature also produced significant improvements in the expression of an already codon-optimized sequence. In particular, codon optimization was responsible for a 5-9-fold increase in the mean fluorescence intensity (MFI) compared with an increase of only 1.3-2.6-fold through terminator selection and an approximately 1.2-fold increase obtained by lowering the cultivation temperature from 30 oC to 20 oC. Among the 5 terminators tested, CPS1 produced the strongest effect on expression, increasing MFI by 2.6-fold compared with that obtained using the original Gal7 terminator. Taken together, the findings demonstrate that, within the tested strain and episomal expression system, terminator selection and reduced cultivation temperature can provide additional improvements after codon optimization and therefore serve as complementary steps in yeast expression optimization.