Sat-BSA: an NGS-based method using local de novo assembly of long reads for rapid identification of genomic structural variations associated with agronomic traits.

Advances in next generation sequencing (NGS)-based methodologies have accelerated the identifications of simple genetic variants such as point mutations and small insertions/deletions (InDels). Structural variants (SVs) including large InDels and rearrangements provide vital sources of genetic diversity for plant breeding. However, their analysis remains a challenge due to their complex nature. Consequently, novel NGS-based approaches are needed to rapidly and accurately identify SVs. Here, we present an NGS-based bulked-segregant analysis (BSA) technique called Sat-BSA (SVs associated with traits) for identifying SVs controlling traits of interest in crops. Sat-BSA targets allele frequencies at all SNP positions to first identify candidate genomic regions associated with a trait, which is then reconstructed by long reads-based local de novo assembly. Finally, the association between SVs, RNA-seq-based gene expression patterns and trait is evaluated for multiple cultivars to narrow down the candidate genes. We applied Sat-BSA to segregating F2 progeny obtained from crosses between turnip cultivars with different tuber colors and successfully isolated two genes harboring SVs that are responsible for tuber phenotypes. The current study demonstrates the utility of Sat-BSA for the identification of SVs associated with traits of interest in species with large and heterozygous genomes.

Isolation of the high polyamine-producing bacterium Staphylococcus epidermidis FB146 from fermented foods and identification of polyamine-related genes.

It has been reported that the intake of polyamines contributes to the extension of healthy life span in animals. Fermented foods contain high concentrations of polyamines thought to be derived from fermentation bacteria. This suggests that bacteria that produce high levels of polyamines could be isolated from fermented foods and utilized as a source of polyamines for human nutrition. In this study, Staphylococcus epidermidis FB146 was isolated from miso, a Japanese fermented bean paste, and found to have a high concentration of putrescine in its culture supernatant (452 µM). We analyzed the presence of polyamines in the culture supernatants and cells of the type strains of 21 representative Staphylococcus species in addition to S. epidermidis FB146, and only S. epidermidis FB146 showed high putrescine productivity. Furthermore, whole-genome sequencing of S. epidermidis FB146 was performed, and the ornithine decarboxylase gene (odc), which is involved in putrescine synthesis, and the putrescine:ornithine antiporter gene (potE), which is thought to contribute to the release of putrescine into the culture supernatant, were present on plasmid DNA harbored by S. epidermidis FB146.

Phylogenomic analysis in Latilactobacillus sakei by using polymorphisms detected by next-generation sequencing.

Latilactobacillus sakei is a lactic acid bacterium used to produce a wide range of fermented food products. To understand their characteristics and adaptability to various nutrient sources, we applied strain-specific, nucleotide-concatenated (SSC) sequences to the phylogenetic analysis of 32 L. sakei strains isolated from various locations and products. SSC sequences were developed by concatenating the polymorphisms detected by whole-genome sequencing. This enabled us to use sufficient polymorphisms and avoid the bias caused by selecting partial sequences, such as that in core genome and multi-locus sequence typing. SSC sequence-based analysis revealed that the phylogenetic relations for L. sakei are based on the different nutrition sources rather than geographical distance.

Putrescine Production by Latilactobacillus curvatus KP 3-4 Isolated from Fermented Foods.

Polyamines are aliphatic hydrocarbons with terminal amino groups and are essential for biological activities. It has been reported that polyamines have health-promoting effects in animals, such as the extension of lifespan by polyamine intake. The identification of a high polyamine-producing bacterium from foods could lead to the development of a novel probiotic candidate. We aimed to identify high polyamine-producing bacteria from food, and isolated and collected bacteria from vegetables and fermented foods produced in Japan. We successfully acquired Latilactobacillus curvatus KP 3-4 isolated from Kabura-zushi as a putrescine producing lactic acid bacteria. Comparing the polyamine synthesis capability of L. curvatus KP 3-4 with that of typical probiotic lactic acid bacteria and L. curvatus strains available from the Japan Collection of Microorganisms, it was found that only L. curvatus KP 3-4 was capable of exporting high levels of putrescine into the culture supernatant. The enhancement of putrescine production by the addition of ornithine, and whole-genome analysis of L. curvatus KP 3-4, suggest that putrescine is synthesized via ornithine decarboxylase. The administration of L. curvatus KP 3-4 to germ-free mice increased the concentration of putrescine in the feces.