A Novel Real-time Fluorescence Mutant-allele-specific Amplification Method for Rapid Single Nucleotide Polymorphism Analysis

Current methods for single nucleotide polymorphism （SNP） analysis are timeconsuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification （MASA） method for rapid SNP analysis. The method is a marriage of two technologies： MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.

Polymorphism Analysis of RXFP2 in Different Sheep Breeds from Qinghai-Tibet Plateau of China

Relaxin/insulin-like family peptide receptor 2 （RXFP2） is a robust candidate gene related to horn types in sheep. A series of independent genome-wide association studies have reported that RXFP2 underlies the existence and lack of horns. In this study, High-Resolution Melting （HRM） analysis and DNA sequencing were employed to detect the polymorphism of RXFP2 gene in three sheep breeds from Qinghai-Tibet Plateau of China （ Tibetan sheep, Qinghai fine wool sheep and Alpine Merino sheep） and to determine the impacts of genotypes of RXFP2 on expression of horn phenotypes. The results showed that one single nucleotide pol- ymorphism （SNP） was identified as RXFP 2SNP c. 29389966 A 〉 G. The frequency of genotype AA in Alpine Merino ram （polled） was significantly higher than that in Tibetan ram （horned） and Qinghai fine wool ram （horned） Ix2（1, N= 421） = 72.25, P〈 0.001; xZ（1, N= 402） = 4.28, P〈 0.005）]; the fre- quency of genotype AA in Qinghai fine wool ewe （polled） was also much higher than that in Qinghai fine wool ewe （horned） and Tibetan ewe （horned） [x2（1, N = 196） = 42.04, P 〈 0. 001 ; x2 （ 1, N = 192） = 24. 69, P 〈 0. 005 ） ]. This mutation could potentially be exploited in marker-assisted selection （MAS） pro- grams within sheep industry to breed horned or polled animals.

Evaluation of the Method Based on Restriction Fragment Length Polymorphism Analysis as Simple Analysis Method of Lactic Acid Bacteria in Foods

Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g-1) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as to amplify Bifidobacterium spp., before the method will be used as an inspection method for lactic acid bacteria.

Diversity Analysis in Selected Non-basmati Scented Rice Collection

Diversity analysis among 23 rice varieties including 16 non-basmati scented accessions, 5 basmati accessions and 2 non-scented accessions was performed by random amplified polymorphic DNA （RAPD） and inter-simple sequence repeat （ISSR） marker systems. The varieties analyzed by 11 RAPD and 8 ISSR primers yielded an average of 65% and 80% polymorphism, respectively. The average number of polymorphic bands generated per RAPD primer was 6 and per ISSR primer was 5.87. RAPD and ISSR data analysis individually could not segregate basmati and non-basmati scented rice accessions. However, the analysis using a combined data could group basmati and non-basmati scented rice accessions separately. The bands present specifically among three accessions of non-basmati scented rice were also identified. The study revealed a high genetic diversity among non-basmati scented rice accessions.

Genetic diversity analysis of Lepidium sativum(Chandrasur) using inter simple sequence repeat(ISSR) markers

Lepidium sativum（commonly known as garden cress） belongs to the family Brassicaceae. It is a fastgrowing erect, annual herbaceous plant. Its seeds possess significant fracture healing, anti-asthmatic, anti-diabetic,hypoglycemic, nephrocurative and nephroprotective activities. In the present study, we assessed the genetic diversity of various genotypes of L. sativum using inter-simple sequence repeat（ISSR） markers. Out of 41 ISSR primers screened, 32 primers showed significant, clear and reproducible bands. A total of 510 amplified bands were obtained using 32 ISSR primers, out of which 422 bands were polymorphic and 88 bands were monomorphic. The percentage of polymorphism was found to be 82. A total of 35 unique alleles ranging insize from 200 to 2,900 bp were observed.Cluster analysis based on unweighted pair-group method,arithmetic mean divided the 18 genotypes into two main clusters, with the first having only HCS-08 genotype of L.sativum and other having all of the other 17 genotypes. The Jaccard similarity coefficient revealed a broad range32–72 % genetic relatedness among the 18 genotypes.

Large-scale genome-wide SNP analysis reveals the rugged(and ragged)landscape of global ancestry,phylogeny,and demographic history in chicken breeds

The worldwide chicken gene pool encompasses a remarkable,but shrinking,number of divergently selected breeds of diverse origin.This study was a large-scale genome-wide analysis of the landscape of the complex molecular architecture,genetic variability,and detailed structure among 49 populations.These populations represent a significant sample of the world's chicken breeds from Europe(Russia,Czech Republic,France,Spain,UK,etc.),Asia(China),North America(USA),and Oceania(Australia).Based on the results of breed genotyping using the Illumina 60K single nucleotide polymorphism(SNP)chip,a bioinformatic analysis was carried out.This included the calculation of heterozygosity/homozygosity statistics,inbreeding coefficients,and effective population size.It also included assessment of linkage disequilibrium and construction of phylogenetic trees.Using multidimensional scaling,principal component analysis,and ADMIXTURE-assisted global ancestry analysis,we explored the genetic structure of populations and subpopulations in each breed.An overall 49-population phylogeny analysis was also performed,and a refined evolutionary model of chicken breed formation was proposed,which included egg,meat,dual-purpose types,and ambiguous breeds.Such a large-scale survey of genetic resources in poultry farming using modern genomic methods is of great interest both from the viewpoint of a general understanding of the genetics of the domestic chicken and for the further development of genomic technologies and approaches in poultry breeding.In general,whole genome SNP genotyping of promising chicken breeds from the worldwide gene pool will promote the further development of modern genomic science as applied to poultry.

Development of EST-SSR primers and their practicability test for Laminaria

Laminaria （Saccharina） is one of the economic important seaweeds. Through sequence analysis of 4 099 ESTs from Laminaria （971 were generated from our own cDNA laboratories and 3 128 were downloaded from updated EST databases） with SSRIT software, two hundred and fifty-four SSRs in 206 ESTs were found. From the 254 SSRs, sixty-three SSR primer-pairs were designed. In order to test their practicability, the 63 primer-pairs were tested in commonly used SSR reaction conditions using 12 Laminara DNA samples as templates. The results show that 23 SSR primer-pairs gave good amplification patterns on most （more than 80%） of the 12 Laminara DNA templates. Genetic diversity study of 12 Laminaria lines, which were widely used in breeding and economic cultivation in China, was performed based on the obtained SSR data.

Identification and Enumeration Method of Both Eukaryotic and Prokaryotic Microorganisms in Food Sample

The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g-1, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g-1. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g-1. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g-1) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g-1), and the other Firmicutes (3.7 × 106 MPN g-1). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g-1), Burkhokderia sp. (1.5 × 106 MPN g-1), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g-1). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.

A Simple Evaluation System for Microbial Property in Soil and Manure

Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 106 MPN g-1 dry soil), followed by γ-Proteobacteria (1.32 × 106 MPN), δ-Proteobacteria (0.006 × 106 MPN), and the other gram negative bacteria (0.006 × 106 MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 106 MPN), followed by α-Proteobacteria (26.0 × 106 MPN), β-Proteobacteria (17.1 × 106 MPN), δ-Proteobacteria (11.2 × 106 MPN), the other Firmicutes (1.71 × 106 MPN), γ-Proteobacteria (0.5 × 106 MPN), and the other gram negative bacteria (0.05 × 106 MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 106 MPN), followed by Actinobacteria (2.14 × 106 MPN), Bacillus sp. (2.14 × 106 MPN), and γ-Proteobacteria (0.35 × 106 MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was found suitable as a candidate of a new evaluation system of soil and manure.

A New Evaluation Method for Antibiotic-Resistant Bacterial Groups in Environment

In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg·L-1) were included in row cattle feces (1.78 × 104 MPN g-1) and cattle feces manure (>3.84 × 104 MPN g-1). Compost originated from leftover food (>44.8 × 104 MPN g-1) and shochu lee (>320 × 104 MPN g-1) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg·L-1), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 104 MPN g-1), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg·L-1 of ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 104 MPN g-1), followed by cattle feces manure (4.19 × 104 MPN g-1), and shochu lee (0.36 × 104 MPN g-1), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.

Novel ACTG1 mutation causing autosomal dominant non-syndromic hearing impairment in a Chinese family

γ -actin （ACTG1） gene is a cytoplasmic nonmuscle actin gene, which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea. Mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing loss linked to the DFNA 20/26 locus on chromosome 17q25.3 in European and American families, respectively. In this study, a novel missense mutation （c.364A〉G; p.I122V） co-segregated with the affected individuals in the family and did not exist in the unaffected family members and 150 unrelated normal controls. The alteration of residue Ile122 was predicted to damage its interaction with actin-binding proteins, which may cause disruption of hair cell organization and function. These findings strongly suggested that the I122V mutation in ACTG1 caused autosomal dominant non-syndromic hearing impairment in a Chinese family and expanded the spectrum of ACTG1 mutations causing hearing loss.

Sequence Information on Simple Sequence Repeats and Single Nucleotide Polymorphisms through Transcriptome Analysis of Mungbean

Mungbean （Vigna radiata （L.） Wilczek） is a unique species in its ability to fix atmospheric nitrogen, with early maturity, and relatively good drought resistance. We used 454 sequencing technology for transcriptome sequencing. A total of 150 159 and 142 993 reads produced 5 254 and 6 374 large contigs （〉_500 bp） with an average length of 833 and 853 for Sunhwa and Jangan, respectively. Functional annotation to known sequences yielded 41.34% and 41.74% unigenes for Jangan and Sunhwa. A higher number of simple sequence repeat （SSR） motifs was identified in Jangan （1 630） compared with that of Sunhwa （1 334）. A similar SSR distribution pattern was observed in both varieties. A total of 8 249 single nucleotide polymorphisms （SNPs） and indels with 2 098 high-confidence candidates were identified in the two mungbean varieties. The average distance between individual SNPs was -860 bp. Our report demonstrates the utility of transcriptomic data for implementing a functional annotation and development of genetic markers. We also provide large resource sequence data for mungbean improvement programs.

A dCAPS marker developed from a stress associated protein gene TaSAP7-B governing grain size and plant height in wheat

Stress associated proteins（SAPs） are the A20/AN1 zinc-finger proteins which confer to abiotic stresses in plants. In this study, TaSAP7-B, including two AN1 domains, was isolated from B genome of wheat（Triticum aestivum L.）. Sequencing analysis on TaSAP7-B illustrated one In Del（insertion-deletion） and one SNP（single nucleotide polymorphism） in the promoter region while no diversity was observed in the coding region. On the basis of SNP in the promoter region（–260 bp）, a dCAPS（derived cleaved amplified polymorphic sequences） marker SNP-260 was developed for TaSAP7-B. Using a natural population consisting of 262 wheat accessions, significant associations were detected between the marker SNP-260 and agronomic traits, such as plant height（PH）, peduncle length（PL）, length of penultimate internode（LPI）, number of spike per plant（NSP）, and 1 000-grain weight（TGW）. Two genotypes were identified using marker SNP-260 in the natural population. Among them, the genotypes possessing C allele exhibited a higher TGW and shorter PH than the T genotypes. Hence, base C was considered as the superior allele. The dCAPS marker of TaSAP7-B can be instrumental for marker-assisted selection for high grain size and short plant height.

A genetic diversity assessment of starch quality traits in rice landraces from the Taihu basin,China

There are nearly 1 000 rice landrace varieties in the Taihu basin, China. To assess the genetic diversity of the rice, 24 intragenic molecular markers（representing 17 starch synthesis-related genes） were investigated in 115 Taihu basin rice landraces and 87 improved cultivars simultaneously. The results show that the average genetic diversity and polymorphism information content values of the landraces were higher than those of improved cultivars. In total, 41 and 39 allele combinations（of the 17 genes） were derived from the landraces and improved cultivars, respectively; only two identical allele combinations were found bet ween the two rice variety sources. Cluster analysis, based on the molecular markers, revealed that the rice varieties could be subdivided into five groups and, within these, the japonica improved rice and japonica landrace rice varieties were in two separate groups. According to the quality reference criteria to classify the rice into grades, some of the landraces were found to perform we ll, in terms of starch quality. For example, according to NY /T595-2002 criteria from the Ministry of Agriculture of China, 25 and 33 landraces reached grade 1, in terms of their apparent amylose content and gel consistency. Th e varieties that had outstanding quality could be used as breeding materials for rice quality breeding programs in the future. Our study is useful for future applications, such as genetic diversity studies, the protection of rice variety and improvment of rice quality in breeding programs.

Association Analysis of Four Single Nucleotide Polymorphisms with Leukocyte Telomere Length in Two Chinese Populations

Telomeres are protein--DNA complex structure at the ends of chromosomes, which are involved in genomic stability （Blackburn, 2010）. In most human cells, telomere erosion with each round of cell division eventually limits cell proliferation and tissue renewal, thereby impacting age-dependent pathol- ogies （Lundblad, 2012）. Leukocyte telomere length （LTL） undertakes a slow loss throughout life across human pop- ulations in general （Blackburn, 2010）. Telomerase is a ribo- nucleoprotein that adds telomeric DNA to chromosomal ends and contains two essential components：

Analysis of the Genetic Structure of Sclerotinia sclerotiorum （Lib.） de Bary Populations from Different Regions and Host Plants by Random Amplified Polymorphic DNA Markers

The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA (RAPD) method with 20 random decamer primer pairs in order to provide some information on the phylogenetic taxa and breeding for resistance to sclerotinia stem rot. A minimum of three and a maximum of 15 unambiguously amplified bands were generated, furnishing a total of 170 bands ranging in size from 100 to 3 200 bp, corresponding to an average of 8.5 bands per primer pair. One hundred and four of these 170 bands (61.2%) were polymorphic, the percentage of polymorphic bands for each primer pair ranging from 0.0% to 86.7%. The genetic relationships among the isolates, based on the results of RAPD analysis, were examined. The genetic similarity of all selected isolates was quite high. At the species level, the genetic diversity estimated by Nei's gene diversity (h) was 0.197 and Shannon's index of diversity (I) was 0.300. The unweighted pair-group mean analysis (UPGMA) cluster analysis showed that most isolates from the same regions were grouped in the same cluster or a close cluster. The population of isolates from Hefei (Anhui Province, China) was more uniform and relatively distant to other populations. The Canadian population collected from carrot (Daucus carota var. sativa DC.) was relatively close to the Polish population collected from oilseed rape (Brassica napus L.) plants. There was no relationship between isolates from the same host plants. An analysis of molecular variance (AMOVA) revealed that the percentage of variance attributable to variation among and within populations was 50.62% and 49.38%, respectively. When accessions from China, Europe, and Canada were treated as three separate groups, the variance components among groups, among populations within groups, and within populations were ?0.96%, 51.48%, and 49.47%, respectively. The genetic differentiations among and within populations were highly significant (P < 0.001). Similarly, the coefficient of gene differentiation (Gst) in total populations calculated by population genetic analysis was 0.229 4, which indicated that the genetic variation among populations was 22.94%. The gene flow (Nm) was 1.68, which indicated that the gene permutation and interaction among populations was relatively high.

Mining Functional Gene Modules Linked with Rheumatoid Arthritis Using a SNP-SNP Network

The identification of functional gene modules that are derived from integration of information from different types of networks is a powerful strategy for interpreting the etiology of complex diseases such as rheumatoid arthritis （RA）. Genetic variants are known to increase the risk of developing RA. Here, a novel method, the construction of a genetic network, was used to mine functional gene modules linked with RA. A polymorphism interaction analy- sis （PIA） algorithm was used to obtain cooperating single nucleotide polymorphisms （SNPs） that contribute to RA disease. The acquired SNP pairs were used to construct a SNP-SNP network. Sub-networks defined by hub SNPs were then extracted and turned into gene modules by mapping SNPs to genes using dbSNP database. We per- formed Gene Ontology （GO） analysis on each gene module, and some GO terms enriched in the gene modules can be used to investigate clustered gene function for better understanding RA pathogenesis. This method was applied to the Genetic Analysis Workshop 15 （GAW 15） RA dataset. The results show that genes involved in func- tional gene modules, such as CD160 （rs744877） and RUNX1 （rs2051179）, are especially relevant to RA, which is supported by previous reports. Furthermore, the 43 SNPs involved in the identified gene modules were found to be the best classifiers when used as variables for sample classification.

Quantification of flupirtine maleate polymorphs using X-ray powder diffraction

Flupirtine maleate, a pharmaceutical compound for treating psychotic disease in clinics, has seven polymorphs. Form A, with better crystal stability and bioavailability, has been widely used as the pharmaceutical crystal form. Unfortunately, it is usually found in a polymorphic mixture with form B. In this study, pure crystal forms of A and B were prepared and characterized by X-ray powder diffraction （XRPD）, Fourier transform infrared spectroscopy （FT-IR） and thermal analysis. An XRPD-based method for the quantitative determination of the amount of the flupirtine maleate polymorphs form A and form B was also established through a systematic optimization of instrumental parameters. The results of the analytical methodology validation showed that the XPRD method had a broad quantitative range of 0- 100% （w/w）, good linear relationship, with R2= 0.999, excellent repeatability and precision and low limits of detection （LoD） of 0.15% （w/w） and quantification （LoQ） of 0.5% （w/w）. The results also showed that the single-peak method was not as good as the whole pattern in reducing the influence of the preferred orientation, but this can be compensated for by a systematic optimization of instrumental parameters and validating the analytical methodology to reduce errors and obtain a good, repeatable, sensitive, and accurate method. This XRPD method can be used to analyze mixtures of flupirtine maleate polymorphs （forms A and B） quantitatively and control the quality of the bulk drug.