Chloroplast Genome of Native Silene latifolia subsp. alba from Fennoscandia Shows High Level of Differences from Invasive White Campion

Silene latifolia is an herbaceous plant with great invasive potential. Spread along trade routes from Europe to almost all continents, white campion became particularly widespread in North America. We sequenced the chloroplast genome of S. latifolia subsp. alba from a native range in southeast Fennoscandia. The chloroplast genome of native S. latifolia subsp. alba forms a 151,747-bp circle, has two inverted repeat regions (25,993 bp each), large single copy (82,708 bp), and small single copy (17,106 bp) regions. It contains 77 protein-coding genes, 30 tRNA genes, and four rRNA genes. SSRs and long DNA repeats were identified. Comparison of a newly sequenced plastome of S. latifolia subsp. alba with plastomes of invasive specimens of species from North America and Japan revealed a high level of single nucleotide polymorphisms (SNPs) among them. A total of 214 SNPs were found, among which 110 were identified in intergenic spacers, 74 in exons, and 30 in introns. Intraspecific shifts in inverted repeat boundaries were identified. Our research suggests that high polymorphic regions may be potential molecular markers for population studies and that high intraspecific genetic polymorphism may contribute to a species’ invasive success.

     

Characterisation of the genetic effects of the ADFP gene and its association with production traits in dairy goats

Adipose differentiation-related protein (ADFP) is important for regulation of lipid metabolism and insulin secretion in beta-cells. In this study, we investigated polymorphisms within the caprine ADFP gene and determined its relationship with production traits. As there was no sequence information available for the caprine ADFP gene, we generated DNA sequence data and examined the genomic organisation. The caprine ADFP gene is organised into 7 exons and 6 introns that span approximately 8.7 kbp and is transcribed into mRNA containing 1353 bp of sequence coding for a protein of 450 amino acids. The protein sequences showed substantial similarity (71–99%) to orthologues from cattle, human and mouse. We identified polymorphisms in the sequences using DNA sequencing, PCR-RFLP and forced PCR-RFLP methods. Seven single nucleotide polymorphisms (SNPs) were identified using samples from 4 different goat populations consisting of 1408 healthy and unrelated individuals. Six haplotypes involving the 7 SNPs from the caprine ADFP gene were identified and their effects on production traits were analysed. Haplotype 6 had the highest haplotype frequency and was highly significantly associated with chest circumference and milk yield in the analysed populations. The results of this study suggest that the ADFP gene is a strong candidate gene affecting production traits and may be used for marker-assisted selection and management in Chinese dairy goat breeding programmes.     

Cloning and expression of arginine kinase from a swimming crab, <Emphasis Type="Italic">Portunus trituberculatus</Emphasis>

Arginine kinase (AK) is an important phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the cDNA of AK (designated as PtAK) was identified from the eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA was 1,479 bp, containing an open reading frame of 1,074 bp that coded for 357 amino acids. The estimated molecular mass of mature PtAK was 40.30 kDa and theoretical isoelectric point was 6.18. Amino acid sequence alignment showed that PtAK had very high similarity with other shrimp and crab AKs ranging from 0.876 to 0.983. The genomic DNA fragments of about 1,434 bp consisted of two exons interrupted by an intron. Totally 24 SNPs, including 17 in the coding region and seven in the non-coding region, were detected by direct sequencing of 19 genomic samples. In exon 1, the coding SNPs (cSNPs) were only found in the disease-resistant specimens. The fluorescent real-time PCR analysis revealed that the expression of PtAK was detected in all the examined tissues with the highest expression in the muscle and the lowest in the eyestalk. The expression of PtAK after Vibrio alginolyticus injection was tested in haemocytes, showing that two peak values were 5.01-fold (at 3 h) and 3.60-fold (at 24 h) compared with the control values, respectively. The results suggested that AK might play an important role in the immune response in crabs.     

Identification, characterisation and application of single nucleotide polymorphisms for diversity assessment in cassava (Manihot esculenta Crantz)

To monitor genetic diversity in the field it is important that it is measured accurately. Here, we elucidate the potential of single nucleotide polymorphisms (SNPs) for measuring genetic diversity in cassava. The nature and frequency of SNPs was characterised and their utility in genetic diversity assessment compared to that of simple sequence repeats (SSRs). This was achieved by direct sequencing of amplicons in diverse cassava varieties. A total of 26 SNPs were identified from quality sequences of nine genes, giving an estimated frequency of one SNP every 121 nucleotides. Nucleotide diversity ranged from 7.8 × 10⁻⁴ to 5.6 × 10⁻³. Average haplotype-based polymorphic information content (PIC = 0.414) was higher than for individual SNPs (PIC = 0.228). The Mantel test indicated interdependence (r = 0.219; P < 0.001) between SNP and SSR genotypic data. Individual SNPs had lower PIC values than SSRs. For this reason larger numbers of SNPs may be necessary to achieve the same level of discrimination among genotypes provided by SSRs.     

Genome-wide discovery of DNA polymorphism in Brassica rapa

Single nucleotide polymorphisms (SNPs) and/or insertion/deletions (InDels) are frequent sequence variations in the plant genome, which can be developed as molecular markers for genetic studies on crop improvement. The ongoing Brassica rapa genome sequencing project has generated vast amounts of sequence data useful in genetic research. Here, we report a genome-wide survey of DNA polymorphisms in the B. rapa genome based on the 557 bacterial artificial clone sequences of B. rapa ssp. pekinensis cv. Chiifu. We identified and characterized 21,311 SNPs and 6,753 InDels in the gene space of the B. rapa genome by re-sequencing 1,398 sequence-tagged sites (STSs) in eight genotypes. Comparison of our findings with a B. rapa genetic linkage map confirmed that STS loci were distributed randomly over the B. rapa whole genome. In the 1.4 Mb of aligned sequences, mean nucleotide polymorphism and diversity were θ = 0.00890 and π = 0.00917, respectively. Additionally, the nucleotide diversity in introns was almost three times greater than that in exons, and the frequency of observed InDel was almost 17 times higher in introns than in exons. Information regarding SNPs/InDels obtained here will provide an important resource for genetic studies and breeding programs of B. rapa.     

TLR4 single nucleotide polymorphisms (SNPs) associated with Salmonella shedding in pigs

Toll-like receptor 4 (TLR4) is a key factor in the innate immune recognition of lipopolysaccharide (LPS) from Gram-negative bacteria. Previous studies from our group identified differences in the expression profile of TLR4 and genes affected by the TLR4 signaling pathway among pigs that shed varying levels of Salmonella, a Gram-negative bacterium. Therefore, genetic variation in this gene may be involved with the host’s immune response to bacterial infections. The current study screened for single nucleotide polymorphisms (SNPs) in the TLR4 gene and tested their association with Salmonella fecal shedding. Pigs (n?=?117) were intranasally challenged at 7 weeks of age with 1?×?10⁹ CFU of S. Typhimurium χ4232 and were classified as low or persistent Salmonella shedders based on the levels of Salmonella being excreted in fecal material. Salmonella fecal shedding was determined by quantitative bacteriology on days 2, 7, 14, and 20/21 post exposure, and the cumulative levels of Salmonella were calculated to identify the low (n?=?20) and persistent (n?=?20) Salmonella shedder pigs. From those 40 animals, the TLR4 region was sequenced, and 18 single nucleotide polymorphisms (SNPs) in TLR4 were identified. Twelve SNPs have been previously described and six are novel SNPs of which five are in the 5′ untranslated region and one is in intron 2. Single marker association test identified 13 SNPs associated with the qualitative trait of Salmonella fecal shedding, and seven of those SNPs were also associated with a quantitative measurement of fecal shedding (P?<?0.05). Using a stepwise regression process, a haplotype composed of SNPs rs80787918 and rs80907449 (P?≤?4.0?×?10^?3) spanning a region of 4.9 Kb was identified, thereby providing additional information of the influence of those SNPs on Salmonella fecal shedding in pigs.     

Identification of genetic associations of ECHS1 gene with milk fatty acid traits in dairy cattle

Our previous genome‐wide association study identified 83 genome‐wide significant SNPs and 20 novel promising candidate genes for milk fatty acids in Chinese Holstein. Among them, the enoyl‐CoA hydratase, short chain 1 (ECHS1) and enoyl‐CoA hydratase and 3‐hydroxyacyl CoA dehydrogenase (EHHADH) genes were located near two SNPs and one SNP respectively, and they play important roles in fatty acid metabolism pathways. We herein validated whether the two genes have genetic effects on milk fatty acid traits in dairy cattle. By re‐sequencing the full‐length coding region, partially adjacent introns and 3000 bp up/downstream flanking sequences, we identified 12 SNPs in ECHS1: two in exons, four in the 3′ flanking region and six in introns. The g.25858322C>T SNP results in an amino acid replacement from leucine to phenylalanine and changes the secondary structure of the ECHS1 protein, and single‐locus association analysis showed that it was significantly associated with three milk fatty acids (P = 0.0002–0.0013). The remaining 11 SNPs were found to be significantly associated with at least one milk fatty acid (P = <0.0001–0.0040). Also, we found that two haplotype blocks, consisting of nine and two SNPs respectively, were significantly associated with eight milk fatty acids (P = <0.0001–0.0125). However, none of polymorphisms was observed in the EHHADH gene. In conclusion, our findings are the first to indicate that the ECHS1 gene has a significant genetic impact on long‐chain unsaturated and medium‐chain saturated fatty acid traits in dairy cattle, although the biological mechanism is still undetermined and requires further in‐depth validation.     

Genetic structure,polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella

Laboratory of genetics and physiology 2 (LGP2) is an actual detector and regulator during RNA viral infection in innate immunity. In this study, 5′-flanking region and all introns of LGP2 in grass carp (Ctenopharyngodon idella) were excavated. The genomic CiLGP2 (C. idella LGP2) was 8062 bp in length, with a 364 bp 5′-flanking region, twelve exons and eleven introns. Besides, the promoter activity of the upstream region before initiator codon was identified. By sequencing, six single nucleotide polymorphisms (SNPs) and one 20-bp insertion/deletion polymorphism were detected in CiLGP2. With a challenge experiment, the genotype and allele distributions of these seven polymorphisms were examined. Analytic result revealed only the − 1392 C/G, 494 A/T and 4403 C/T loci were significantly associated with the resistance/susceptibility to grass carp reovirus (GCRV) (P < 0.05). To further identify these correlations, another independent challenge test was performed. The analytic result based on the cumulative mortality demonstrated that the stock in − 1392 GG genotype was more susceptible to GCRV than that in CC genotype, while the stocks in 494 TT genotype and 4403 TT genotype were more resistant to GCRV than that in AA and CC genotype stocks, respectively (P < 0.05). Those significant SNPs might be potential gene markers for the future molecular selection of C. idella strains that are resistant to GCRV.