Analysis on DNA sequence of <Emphasis Type="Italic">GPR54</Emphasis> gene and its association with litter size in goats

The kisspeptin/GPR54 pathway is crucial in the process of puberty onset. Six pairs of primers were designed to clone goat GPR54 and scan polymorphisms and one pair of primers to detect polymorphisms of GPR54 in sexual precocious and sexual late-maturing goat breeds. A DNA fragment of 4258 bp of goat GPR54 was obtained, which contains an open reading frame (ORF) of 1137 bp and encodes 378 amino acids, having a high homology with other mammals. The protein was predicted to have seven transmembrane regions. There were no base pair variation in exons 1–4 and three base changes (G4014A, G4136A and C4152T) in exon 5 by sequencing and the three mutations may have some correlation with sexual precocity in goats. For the 4152 locus, the Jining Grey goat does with genotype TT and CT had 1.02 and 0.84 (P < 0.01) kids more than those with genotype CC, respectively. No significant difference (P > 0.05) was found in litter size between TT and CT genotypes in Jining Grey goat. For the other two loci, no significant difference (P > 0.05) was found in litter size between different genotypes in Jining Grey goats. The present study preliminarily indicated an association between allele T of the 4152 locus in GPR54 and high litter size in Jining Grey goats.     

Restriction analysis and partial sequencing of the 16S rRNA gene as index for rapid identification of <Emphasis Type="Italic">Bacillus</Emphasis> species

Restriction fragment length analysis of PCR amplified 16S rDNA with AluI revealed the presence of a 265 bp fragment in all species of Bacillus with the exception of B. cereus and B. thuringiensis, which contains two restriction sites within this fragment which results in three smaller fragments totalling to 265 bp. Some distant species of Bacillus with no evidence of this fragment could be delineated into other genera based on phenotypic and genotypic parameters. BLAST search for homologous sequences of individual species revealed that it is a highly conserved region. Multiple alignment of the fragment suggests that a region between 160 and 265 bp of the 265 bp fragment was a hypervariable region and were highly species-specific. A set of primers was designed for amplification of this hypervariable region. Partial sequencing of the hypervariable region within the 265 bp fragment seems an index for identification of Bacillus species.     

Molecular detection of the entomopathogenic bacterium Pseudomonas entomophila using PCR

Aims: To develop a specific, fast and simple molecular method useful to detect the entomopathogenic bacterium Pseudomonas entomophila. Methods and Results: The use of bioinformatics tools allowed the identification of unique genes present in P. entomophila genome. Using such genes, we designed primers aiming to detect specifically P. entomophila by PCR. Furthermore, a pair of primers specifically designed to amplify the 16S rRNA gene in Pseudomonas species was used. Primer specificity was checked using environmental pseudomonad and nonpseudomonad species. A 618 ‐bp fragment was amplified only in Pseudomonas using the 16S rDNA primers. Primers (PSEEN1497) designed to detect P. entomophila amplified a 570 ‐bp fragment only in P. entomophila. A duplex PCR was developed combining 16S rDNA and PSEEN1497 primers that allowed the detection of P. entomophila present in experimentally infected Drosophila melanogaster. Conclusions: We developed a molecular method useful to detect P. entomophila present in bacterial cultures or directly from infected insects. Significance of the Study: To the best of our knowledge, this is the first molecular method aiming to detect P. entomophila in environmental samples. The use of our method will facilitate studies related to ecology and insect host range of this entomopathogenic bacterium.     

Application of loop-mediated isothermal amplification (LAMP)-based technology for authentication of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

In this study, loop-mediated isothermal amplification (LAMP)-based molecular marker was developed for authentication of Catharanthus roseus, a medicinal plant. Samples of this plant were collected from different geographical locations in India. Random amplified polymorphic deoxyribonucleic acid (DNA) analysis of collected samples was carried out with 25 random primers. A 610-bp DNA fragment, common to all accessions, was eluted, cloned, and sequenced. Four LAMP primers were designed on the basis of sequence of 610 bp DNA fragment. LAMP reaction, containing 10× Bst DNA polymerase reaction buffer, Bst DNA polymerase, four in-house designed primers, dNTPs, MgSO₄, and betaine, was incubated at 65°C for 1 h. The resulting amplicon was visualized by adding SYBR Green I to the reaction tube. The data showed confirmatory results. Since the assay method is simple, sensitive, and cost-effective, it is a feasible method for identifying and authentication of C. roseus.     

Molecular Characterization of Acanthamoeba spp. Occurring in Water Bodies and Patients in Poland and Redefinition of Polish T16 Genotype

Acanthamoeba genus is divided into 20 genotypes (T1–T20) on the basis of the gene encoding 18S rRNA sequence. Using of at least 2 kbp gene fragments is strongly recommended to identify new genotypes and 5% difference is commonly used as a criterion of new genotypes, however, this value is questionable. In this paper, Polish Acanthamoeba strains described earlier on the basis of ~850 bp Ami fragment of 18S rRNA gene as T4, T11 and a new T16 genotype, have been analyzed using near‐complete sequence of the gene. This analysis was needed because the Ami fragment does not reveal full variability within 18S rRNA gene. Phylogenetic analysis based on Ami fragment is biased by artifacts in the construction of the tree, so the fragment should not be used for identification of new putative Acanthamoeba genotypes. The analysis confirmed that the Polish sequences represent T4 and T11 genotypes and that the strains described earlier as T16 genotype are in fact a new subgroup of the T20 genotype and that this genotype should be divided into two subgroups: T20a (two strains described by [J. Eukaryot. Microbiol. 62 (2015) 69]) and T20b (11 Polish strains described in this study). The T20b subgroup was isolated from both clinical samples and water bodies used by people as bathing places and there is a risk of infection for humans during contact with water.     

Development and application of a triplex-PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus from pigs

A triplex-PCR assay was developed and evaluated for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) recovered from various biological samples of pig. Three sets of primers were designed to target mecA, 16S rRNA and nuc genes of MRSA. The specific amplification generated three bands on agarose gel, with sizes 280 bp for mecA, 654 bp for 16S rRNA and 481 bp for nuc, respectively. A potential advantage of the PCR assay is its sensitivity with a detection limit of 10² CFU per ml of bacteria. In all, 79 MRSA isolates recovered from various samples of pigs were subjected to the amplification by the triplex-PCR assay and all the isolates yielded three bands corresponding to the three genes under this study. No false-positive amplification was observed, indicating the high specificity of the developed triplex-PCR assay. This assay will be a useful and powerful method for differentiation of MRSA from methicillin-sensitive S. aureus, coagulase-negative methicillin-resistant staphylococci and coagulase-negative methicillin-sensitive staphylococci.     

耐盐绒毛白蜡特异性SCAR分子标记的鉴定

该研究以耐盐型和盐敏感型绒毛白蜡及其F1代为材料,采用混合品系分析法进行RAPD分析。结果显示:在随机选取的150个10碱基随机引物中,仅有引物S20在耐盐基因池和盐敏感基因池间扩增出特异而可重复的592bp的多态性片段,命名为S20-592。获得的RAPD标记S20-592经克隆、测序、重新设计一对特异性引物转化成更稳定的SCAR标记。通过F1代个体验证,耐盐型个体均能扩增出此差异条带而盐敏感型个体中不能扩增出此差异条带,证明该SCAR标记的特异引物可用于耐盐绒毛白蜡物种的快速分子鉴定。     

Development of a sequence‐characterized amplified region marker using inter‐simple sequence repeats for detection of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Early and accurate detection of the pathogens would facilitate effective control of the diseases. DNA‐based methods now provide essential tools for accurate plant disease diagnosis. In this study, inter‐simple sequence repeats (ISSR) technique has been successfully applied to develop a sequence‐characterized amplified region (SCAR) marker for diagnosis of stripe rust of wheat and detection of Pst. In this study, one fragment unique to Pst was identified by ISSR and then sequenced. Based on the specific fragment, a pair of SCAR primers (616AF/616AR) was designed to amplify a 299‐bp DNA fragment within the sequenced region. The primers can amplify a unique DNA fragment for all tested isolates of Pst but not for the other pathogens of wheat leaves and the uninfected leaves. The polymerase chain reaction (PCR) assay could detect as low as 0.1 ng of genomic DNA in a 25.0 μl PCR reaction mixture and detect the pathogen from asymptomatic wheat leaves inoculated with Pst under glasshouse conditions.     

An EST-SSR marker linked with yellow rust resistance in wheat

Expressed sequenced tags containing simple sequence repeats (EST-SSRs) were used to identify molecular markers associated with yellow rust resistance in wheat (Triticum aestivum L.). A cross between yellow rust resistant (PI178383) and susceptible (Harmankaya99) wheat genotypes was performed and respective DNA pools from the resistant and susceptible F₂ seedlings were constructed. 78 EST-SSR primers were used for bulked segregant analysis and one EST-SSR marker (Pk54), identified as 200 bp fragment, was present in the resistant parent and resistant F₂ hybrids but not in the susceptible ones. 108 wheat genotypes differing in yellow rust resistance were screened with Pk54 and 68 % of the wheat genotypes, known to be yellow rust resistant, had the Pk54 marker, further suggesting that the presence of this marker correlates with yellow rust resistance.     

Isolation and characterization of microsatellite markers in Perilla frutescens Brit

We isolated and characterized 13 polymorphic microsatellite primers from Perilla frutescens Brit. var. frutescens by using a modified method that involves one‐way PCR amplification with single primer prior to enrichment with an ‘oligo hook’. The efficiency of this procedure for isolating unique microsatellite sequences was approximately 77%. The number of alleles per microsatellite locus ranged from three to 10 with an average of 6.5 alleles per locus while fragment size varied from 156 to 298 bp. The observed and expected heterozygosities ranged from 0.52 to 0.86 and 0.52 to 0.89, respectively. These newly isolated microsatellite markers are expected to provide valuable resources for different genetic studies currently underway in our Perilla genome research program.     

Use of random amplified polymorphic DNA to identify several novel markers for sex identification in the crested serpent eagle and crested goshawk

The crested serpent eagle (Spilornis cheela hoya) has no distinct sexual dimorphic traits. In the current study, we report the results of an EE0.6 (EcoRI 0.6-kb fragment) sequence applied to S. cheela hoya and a novel random amplified polymorphic DNA (RAPD) marker that can be used to sex individuals within the species S. cheela hoya and Accipiter trivigatus formosae (crested goshawk). We used sex-specific primers for the avian CHD1 (chromo-helicase-DNA-binding 1) gene and the EE0.6 sequence in PCR assays to determine sex. In addition, 120 random primers were used for RAPD fingerprinting to search for novel sex-specific fragments of S. cheela hoya. The OPBB08 random primer generated a 1241-bp sex-specific fragment in all female S. cheela hoya. From the nucleotide sequence, PCR primers were designed to amplify 553-, 895-, and 194-bp sex-specific fragments present in all female S. cheela hoya. One of these primer pairs (ScBB08-7F/R) also amplified a male/female common fragment that can be used as an internal control (543 bp). Moreover, one of the primer pairs (ScBB08-5aF/5bR) could be used to identify genders of A. trivigatus formosae. In conclusion, we identified novel sex-specific DNA markers of S. cheela hoya and A. trivigatus formosae that can be used for rapid and accurate sex identification.     

Specific PCR to identify the heavy-metal-resistant bacterium <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>

The aim of this study is to develop a polymerase chain reaction (PCR) assay for rapid detection of Cupriavidus metallidurans. PCR primers targeting the Signal transduction histidine kinase gene were designed and designated Cm-F1/Cm-R1. Strains of C. metallidurans were positively identified. The size of the PCR products was 437 bp, as expected. This PCR method enables monitoring of industrial, environmental and clinical sources for presence of C. metallidurans.     

Molecular characterization of atrazine resistance in common ragweed (Ambrosia artemisiifolia L.)

Common ragweed (Ambrosia artemisiifolia L.) is the most frequent weed in the Carpathian Basin and is spreading fast in other parts of Europe. In recent years, besides the wild type, a mutant genotype resistant to atrazine herbicides has evolved and is now widespread in many areas. The present study demonstrates that the atrazine resistance of ragweed is maternally inherited, and is caused by a point mutation inthe psb A chloroplast gene. The promoter 5′-untranslated region and the open reading frame regions of the gene were analysed, and a homology search was performed. Both the atrazine-resistant and susceptible types of cpDNA were present in atrazine-resistant plants, while the mixed presence of both genotypes in the same plant, known as heteroplasmy, was not unequivocally detectable in susceptible plants.     

An ISSR‐based Approach for the Molecular Detection and Diagnosis of Dwarf Bunt of Wheat,Caused by Tilletia controversa Kühn

Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is an important international quarantine disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from intersimple sequence repeat (ISSR) for rapid identification of T. controversa. A total of 60 primers were tested by ISSR to detect DNA polymorphisms between T. controversa and related species. The primer ISSR818 generated a polymorphic pattern displaying a 952‐ bp DNA fragment specific for T. controversa. The marker was converted into a sequence characterized amplified region (SCAR), and specific primers (TCKSF2/TCKSR2) were designed for use in a PCR detection assay. Its detection limit was 1 ng of DNA, which could be yielded by 1.1 μg of teliospores in a 25‐ μl PCR. Conclusively, a method to distinguish T. controversa from similar pathogenic fungi has been successfully developed based on the use of a SCAR marker.     

Design of Mini‐barcode for Catfishes for assessment of archival biodiversity

Recovery of DNA barcode sequences is often challenging from the archived specimens. However, short fragments of DNA may be recovered, which would significantly improve many unresolved taxonomic conflicts. Here, we designed a mini‐barcode for catfishes comprising several species and many cryptic taxa. We analysed a data set of 3048 publicly available COI barcode sequences representing 547 worldwide catfish species and performed 152 628 interspecies comparisons. A significantly more positively correlated interspecies distance was detected with transversion (0.78, P < 0.001) than with transition (0.70, P < 0.001). This suggested that transversions were better diagnostics for species identification. In the aligned data set, two transversion‐rich fragments (53 bp and 119 bp) were identified. Transition/transversion bias value was 1.04 in 53‐bp fragment, 1.23 in 119‐bp fragment and 1.50 in full‐length barcode. The interspecies distance with full‐length barcode was 0.212 ± 0.037, while that with 53‐bp and 119‐bp fragments was 0.325 ± 0.039 and 0.218 ± 0.045, respectively. Survey of 53‐bp fragment showed a possibility of only 1144 barcodes, while that of 119‐bp fragment showed >4 million barcodes. Thus, the 119‐bp fragment is a viable mini‐barcode for catfishes comprising >3000 extant species. Experiment with 82 archived catfishes showed successful recovery of this mini‐barcode using the designed primer. The mini‐barcode sequences showed species‐specific similarity in the range of 98‐100% with the global database. Therefore, survey of a transversion‐rich fragment within the full‐length barcode would be an ideal approach of mini‐barcode design for biodiversity assessment.     

Mutations in the exon 10 of prolactin receptor gene change the egg production performance in Wanjiang white goose

To select the molecular genetic markers related to egg performance of Wanjiang white goose, prolactin receptor gene (PRLR) was adopted to be a candidate gene in our study. Five pairs of primers (P1–P5) were designed to detect the SNPs of PRLR gene by PCR-SSCP method. The results revealed that polymorphisms were discovered in the PCR products amplified with P4 primers in PRLR exon 10, three genotypes were found: AA, AB and AC. The sequence of AB genotype is the same as original sequence (DQ660982) in NCBI. There are five mutations in AA genotype: C → A at 840 bp, C → T at 862 bp, T → C at 875 bp, T → A at 963 bp, A → T at 989 bp, resulting in amino acid mutations: His → Asn, Thr → Ile, Asn → Lys, Thr → Ser, and synonymous mutation at 875 bp. Sequencing revealed five mutations in AC genotype: G → T at 816 bp, A → T at 861 bp, C → T at 862 bp, T → C at 875 bp, A → G at 948 bp, causing amino acid mutations of Val → Phe, Thr → Phe, synonymous mutations at 875 and 963 bp. Besides, there are an N-glycosylation site (NQSR), three casein kinase II phosphorylation sites including SIIE, SKTE, and SLMD in AA genotype; three casein kinase II phosphorylation sites including SIIE, SKTE, and TLMD in AB genotype; three casein kinase II phosphorylation sites including SIFE, SKTE, and TLMD in AC genotype. The annual egg yielding of AB genotype geese are significantly more than those of AA and AC genotype geese on the average (P < 0.05). It is suggested for the first time that PRLR is a promising candidate gene that can affect egg performance in Wanjiang white goose.     

Differences in parasitism of Meloidogyne incognita and two genotypes of M. arenaria on Solanum torvum in Japan

Two genotypes of root‐knot nematode, Meloidogyne arenaria (A2‐O and A2‐J), are found in Japan. They were distinguished from each other based on mitochondrial DNA sequences. The primer set (C2F3/1108) amplified a 1.7‐kb fragment from A2‐J, whereas a 1.1‐kb fragment was amplified from A2‐O. M. arenaria (A2‐O) was detected in local regions of southern Japan, whereas M. arenaria (A2‐J) was widespread from the Kyushu region to the Tohoku region. The distribution of M. arenaria (A2‐J) overlaps with the cultivation area of eggplant. Solanum torvum is used worldwide as a rootstock for eggplant cultivation, and it is resistant to Meloidogyne spp. In particular, it is reported that S. torvum is resistant to M. arenaria outside Japan. In this study, we inoculated S. torvum rootstock cultivars with M. arenaria (A2‐J), M. arenaria (A2‐O) and Meloidogyne incognita populations. Although M. incognita and M. arenaria (A2‐O) produced only a few egg masses on S. torvum, thereby confirming its resistance, the four geographical populations of M. arenaria (A2‐J) produced large numbers of egg masses on S. torvum. This study confirmed that S. torvum is resistant to M. incognita and M. arenaria (A2‐O) populations, but susceptible to populations of M. arenaria (A2‐J) in the eggplant production area of Japan.     

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.