Background: Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. Results: A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2× to 5× plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions. Conclusions: MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

Gut microbiome contributes to host health by maintaining homeostasis, increasing digestive efficiency, and facilitating the development of immune system. Wild greylag geese ( Anser anser ) and ruddy shelducks ( Tadorna ferruginea ), migrating along the central Asian flyway, appear to be one of the most popular species in the rare birds rearing industries of China. However, the structure and function of the gut microbial communities associated with these two bird species remain poorly understood. Here, for the first time, we compared gut metagenomes from greylag geese to ruddy shelducks and investigated the similarities and differences between these two bird species in detail. Taxonomic classifications revealed the top three bacterial phyla, Firmicutes , Proteobacteria, and Fusobacteria , in both greylag geese and ruddy shelducks. Furthermore, between the two species, 12 bacterial genera were found to be more abundant in ruddy shelducks and 41 genera were significantly higher in greylag geese. A total of 613 genera (approximately 70%) were found to be present in both groups. Metabolic categories related to carbohydrate metabolism, metabolism of cofactors and vitamins, lipid metabolism, amino acid metabolism, and glycan biosynthesis and metabolism were significantly more abundant in ruddy shelducks, while greylag geese were enriched in nucleotide metabolism and energy metabolism. The herbivorous greylag geese gut microbiota harbored more carbohydrate‐active enzymes than omnivorous ruddy shelducks. In our study, a range of antibiotic resistance categories were also identified in the gut microbiota of greylag geese and ruddy shelducks. In addition to providing a better understanding of the composition and function of wild birds gut microbiome, this comparative study provides reference values of the artificial domestication of these birds.

As the most widely distributed scavenger birds on the Qinghai-Tibetan Plateau, Himalayan vultures (Gyps himalayensis) feed on the carcasses of various wild and domestic animals, facing the dual selection pressure of pathogens and antibiotics and are suitable biological sentinel species for monitoring antibiotic resistance genes (ARGs). This study used metagenomic sequencing to comparatively investigate the ARGs and mobile genetic elements (MGEs) of wild and captive Himalayan vultures. Overall, the resistome of Himalayan vultures contained 414 ARG subtypes resistant to 20 ARG types, with abundances ranging from 0.01 to 1,493.60 ppm. The most abundant resistance type was beta-lactam (175 subtypes), followed by multidrug resistance genes with 68 subtypes. Decreases in the abundance of macrolide-lincosamide-streptogramin (MLS) resistance genes were observed in the wild group compared with the zoo group. A total of 75 genera (five phyla) of bacteria were predicted to be the hosts of ARGs in Himalayan vultures, and the clinical (102 ARGs) and high-risk ARGs (35 Rank I and 56 Rank II ARGs) were also analyzed. Among these ARGs, twenty-two clinical ARGs, nine Rank I ARG subtypes, sixteen Rank II ARG subtypes were found to differ significantly between the two groups. Five types of MGEs (128 subtypes) were found in Himalayan vultures. Plasmids (62 subtypes) and transposases (44 subtypes) were found to be the main MGE types. Efflux pump and antibiotic deactivation were the main resistance mechanisms of ARGs in Himalayan vultures. Decreases in the abundance of cellular protection were identified in wild Himalayan vultures compared with the captive Himalayan vultures. Procrustes analysis and the co-occurrence networks analysis revealed different patterns of correlations among gut microbes, ARGs, and MGEs in wild and captive Himalayan vultures. This study is the first step in describing the characterization of the ARGs in the gut of Himalayan vultures and highlights the need to pay more attention to scavenging birds.

Land use change affects soil aggregate composition and stability, which impacts soil structure and health. To reveal how land use change impacted soil aggregates of alpine grassland in a high-altitude watershed, soil samples from 161 sites including alpine grassland, cropland and abandoned land were selected to measure and analyze the distribution of aggregate fractions (macro-aggregates, micro-aggregates, silt+clay), soil aggregate stability (mean weight diameter, geometric mean diameter, fractal dimension, etc.) and related soil properties (soil organic carbon content, soil particle composition, etc.) in the Huangshui River watershed of the Qinghai–Tibet Plateau. The results showed: (1) As alpine grasslands were converted to croplands and croplands to abandoned lands, the proportion of macro-aggregates and the aggregate stability index showed a trend of first decreasing and then increasing (p < 0.05), indicating that tillage and abandonment have significant influences on soil aggregate structure. (2) Compared with temperate grassland, alpine grassland had richer soil organic carbon, and a higher ratio of macro-aggregates and aggregate stability. (3) Soil organic carbon and sand content had distinct influences on the fractions and stability of aggregates during land use change. These results suggested that cultivation can substantially reduce the soil aggregate stability in alpine grassland, whereas abandonment can effectively improve soil aggregate structure.