Male-specific band in random amplified microsatellite polymorphism fingerprints of Holstein cattle

A total of 65 blood samples collected from Holstein cattle were employed for DNA extraction. Genomic DNA were amplified by means of random amplified polymorphic DNA (RAPD). One hundred and one random primers (Operon kits OPAA, OPAO, OPAV, OPC, OPE and OPA-06) were used for polymerase chain reactions (PCR). The PCR products were size fractionated by means of electrophoresis in agarose gel, transferred to a nylon membrane, and then hybridized with a alpha-32P-labeled microsatellite probe for random amplified microsatellite polymorphism (RAMPO) analysis. All the primers produced many amplification patterns in RAMPO fingerprints, but only OPE-01 (5'-CCCAAGGTCC-3') yielded a male-specific strong signal. Among the 65 cases, the specific band could be observed in the RAMPO fingerprints of the 24 male samples but not in the 41 female samples. Therefore, this specific band is peculiar to male cattle.     

Cloning of Taiwan water buffalo male-specific DNA sequence for sexing

Random amplified polymorphic DNA (RAPD) fingerprinting was carried out to investigate the sex-specific DNA sequence for sexing in Taiwan water buffalos. One hundred and forty random primers were used for RAPD-PCR (polymerase chain reaction). One of these primers, OPC-16, produced a 321 bp fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing, a novel male-specific sequence was obtained. Two primers (BuSexOPC16-F and -R) were designed according to the cloned male-specific sequence to amplify the male-specific fragment using PCR for sexing. Sex-specific bands in the gel were represented in the males but none were found in the females when the Taiwan water buffalo genomic DNA samples were amplified with these two primers using PCR. The same results were also obtained from Taiwan yellow, Holstein, Angus, and Hereford cattle samples. This showed that the sex of these five breeds could be easily and effectively determined using the PCR technique.     

一个新的泥鳅雄性特异DNA片段（简报）

世界上现存鱼类多达24000余种．是脊椎动物中分布最广,种类最多的类群．具有多种多样的生物学特性和重大的经济价值。与高等脊椎动物相比．其性别决定具有多样性和可塑性。大多数鱼类的性别决定机制很原始。性染色体的分化处于萌芽状态。在已进行细胞遗传学研究的1700多种鱼类中．大约有176种（占10．4％）发现有明显的异型性染色体。     

猪MyoG基因的PCR-RFLP多态性分析

以杜洛克、长白、大约克、南昌白、二花脸、梅山猪、玉山黑猪、乐平花猪、金华两头乌及上高两头乌等中外10个猪种共计561头猪为研究材料,采用3对引物（PCR1、PCR2、PCR3）分别扩增猪肌细胞生成素（MyoG）基因的不同区域,扩增产物经限制性核酸内切酶MspⅠ酶切后发现：（1）在PCR1 MspⅠ-RFLP位点上,外来品种杜洛克、长白、大约克及培育品种南昌白中极大多数个体表现为AA型,个别为BB型;而6个中国地方猪种除乐平花猪外均以BB型居多。（2）在PCR2 MspⅠ-RFLP位点上,6个中国地方猪种除一头玉山黑猪表现为MN型外,其余均为MM型;而外来品种以NN型占大多数,培育品种南昌白更趋向于外来品种。（3）在PCR3 MspⅠ-RFLP位点上,所有猪种均可得到扩增产物,但无MspⅠ酶切位点。（4）在梅山猪及与其亲缘关系较近的二花脸猪中,没有发现Soumillion等(1997)报道的梅山猪特异性MspⅠ多态性酶切位点。     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of porcine leptin receptor polymorphisms and their association with reproduction and production traits

Fatness in pigs is of prime economic importance due to market incentives for production of lean pork and elevated fatness increasing the feed costs. Leptin (LEP) action, mediated through its specific receptors (LEPR), was reported to be involved in the regulation of fatness via feed intake, energy expenditure, and whole-body energy balance in pigs. In this study, we have designed 17 primer sets based on the human and mouse LEPR sequences and successfully amplified coding regions of 15 porcine LEPR exon fragments by polymerase chain reactions (PCR). Four single nucleotide polymorphisms (SNP) of Intron 2, Exons 2, 6, and 18 were found in Landrace, Yorkshire, and Duroc by mutagenetically separated-PCR (MS-PCR) and PCR-restriction fragment length polymorphisms (PCR-RFLP). Chi-square statistics was used to calculate homogeneity of genotypic frequencies of 4 gene polymorphisms for three breeds of animals. Effects of Intron 2, Exon 2, and Exon 18 polymorphisms on the reproduction trait such as litter sizes of sows were evident (p < 0.05) in Duroc and Yorkshire. There was no (p > 0.05) significant influence on the production trait of average daily gain due to four candidate gene polymorphisms in three porcine breeds. However, effects of Exon 6 and 18 polymorphisms on the production trait of backfat thickness were significant (p < 0.05) in Landrace and Yorkshire, respectively. Effects of Exon 18 polymorphisms on feed efficiency were also evident (p < 0.05) in Duroc.     

DNA probe and PCR-specific reaction for Lactobacillus plantarum

A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using a dot-blot DNA hybridization technique for its ability to identify Lact. plantarum strains. This probe hybridized with all Lact. plantarum strains tested and with some strains of Lact. pentosus , albeit more weakly. Two internal primers of this probe were selected (LbPl1 and LbPl2) and polymerase chain reaction (PCR) was carried out. All Lact. plantarum strains tested amplified a 250 bp fragment contrary to the other LAB species tested. This specific PCR for Lact. plantarum was also performed from colonies grown on MRS medium with similar results. These methods enabled the rapid and specific detection and identification of Lact. plantarum .     

Growth hormone gene polymorphisms and growth performance traits in Duroc, Landrace and Tao-Yuan pigs

This study investigated the restriction fragment length polymorphism (RFLP) of the porcine growth hormone (pGH) gene in Duroc, Landrace, and Tao-Yuan pigs and its effects on growth performance and levels of plasma growth hormone in peripheral circulation. Genomic DNA extracted from 81 Tao-Yuan, 60 Landrace and 48 Duroc pigs were subjected to Southern blot hybridization with a pGH cDNA probe. Polymorphism was detected with the restriction enzymes TaqI and DraI. A comparison of these three breeds showed significant differences in allelic frequencies. Blood samples for radioimmunoassay (RIA) of GH were collected biweekly during the experimental period from pigs 12 to 40 weeks of age. Tao-Yuan pigs showed a mean plasma GH level (2.51 +/- 1.23 ng/mL) that was much lower than that of the Landrace (3.80 +/- 1.52 ng/mL) and Duroc (4.20 +/- 1.03 ng/mL) pigs (P < 0.05). Moreover, the Tao-Yuan pigs also showed poorer growth performance than the Landrace and the Duroc pigs both in the daily weight gain (0.37 +/- 0.06 vs. 0.67 +/- 0.05 and 0.70 +/- 0.05 kg/day, P < 0.01) and feed efficiency (3.12 +/- 0.28 vs. 2.60 +/- 0.14 and 2.52 +/- 0.12, P < 0.05). These results suggest that the growth performance trait in these pigs is highly correlated with their growth hormone genotype.     

Use of primers derived from a new sequence of the bovine Y chromosome for sexing Bos taurus and Bos indicus embryos

A bovine male-specific marker was identified in our laboratory through random amplified polymorphic DNA (RAPD) analysis. This fragment of 3216 bp was cloned, sequenced and mapped by fluorescent in situ hybridization (FISH) on the taurine Yq. Primers derived from this sequence were initially screened by polymerase chain reaction (PCR) for their ability to detect Y-specific segments in zebu and taurine genomic DNA. Two of these primers amplified a 655 bp Y-specific sequence present in taurine and zebu male genomic DNA. These primers were then used for detecting the 655 bp male sequence in DNA from 173 zebu and 30 taurine embryos, which had been previously sexed using primers for the sequence BC 1.2. The results revealed an accuracy of 100%.     

Male-specific DNA in the dioecious species Atriplex garrettii (Chenopodiaceae)

The mechanism of sex determination in dioecious species of the genus Atriplex (Chenopodiaceae) has not been determined. This paper reports the discovery of a male-specific DNA fragment in the diploid dioecious species A. garrettii. DNA samples extracted individually from ten male and ten female plants were bulked by sex. Random amplified polymorphic DNA (RAPD) fragments were generated in the two bulks in order to identify markers that were polymorphic between male and female plants. A total of 158 decamer primers were tested. A 2075 base-pair (bp) male-specific DNA fragment generated with the OPAF-14 primer was identified. The fragment was cloned and partially sequenced and 24-mer primers that exclusively amplified this fragment were constructed. When 124 male plants, 126 female plants, and one hermaphroditic plant were tested individually, the male-specific 2075-bp DNA fragment was present in the hermaphrodite and all but one of the male plants, and was absent in all female plants. A smaller DNA fragment (~1800 bp) that was homologous to the 2075-bp fragment was amplified from the single male plant that lacked the 2075-bp fragment. Cytogenetic analysis revealed no apparent heteromorphic sex chromosomes. These observations suggest that sex determination in A. garrettii is genetic, with no evidence of heteromorphic sex chromosomes.     

3个猪品种黑素皮质素受体1(MC1R)基因变异研究

利用测序、PCR-RFLP和PCR-SSCP等技术对杜洛克、长白、大白猪MC1R基因进行研究发现了5个多态位点。其中,668位点G→C突变发生在5′UTR,其余4个多态位点nt894insCC(894位点CC插入),1318C→T,1554G→A和1197G→A发生在编码区。nt894insCC导致编码蛋白过早终止。1318C→T,1554G→A和1197G→A突变分别导致a164Val,Ala243Thr和Asp124Asn氨基酸的改变。所有长白、大白猪个体在894位点均存在CC插入,其余多态位点基因型分别为668GG,1197AA,1318CC,1554GG。所有杜洛克个体在894位点均不存在CC插入,其余多态位点基因型分别为668CC,1197GG,1318TT,1554AA。所有突变位点无杂合子出现。由此可以推测,668G→C,1318C→T和1554G→A可能与杜洛克的红毛色存在相关,导致1197G→A突变无意义的894位点CC插入可能与长白、大白猪白毛色存在相关。     

Male-specific DNA markers from African catfish (Clarias gariepinus)

We searched for sex-specific DNA sequences in the male and female genomes of African catfish, Clarias gariepinus (Burchell, 1822) by comparative random amplified polymorphic DNA (RAPD) assays performed on pooled DNA samples. Two sex-linked RAPD markers were identified from the male DNA pool and confirmed on individual samples, showing good agreement with phenotypic sex. Both markers were isolated, cloned and characterized. The first marker (CgaY1) was nearly 2.6 kb long, while the length of second one (CgaY2) was 458 bp. Southern blot analysis with a CgaY1 probe showed strong hybridizing fragments only in males and not in females under stringent conditions, indicating the presence of multiple copies of CgaY1 in the male genome. When tested by zoo blot on the genomes of two closely related species from the Clariidae family, CgaY1 hybridized to the DNA of Heterobranchus longifilis and generated a faint male-specific band at low stringency. CgaY2 produced similar hybridization pattern in both sexes of C. gariepinus, C. macrocephalus and H. longifilis. Specific primers were designed to the sequences and the markers were amplified in multiplex PCR reactions together with a control band common to all individuals. This allowed for rapid, molecular sexing of the species on the basis of a simple three band (male) versus one band (female) pattern. According to our knowledge these are the first sex-specific DNA markers isolated from a siluroid fish species. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel molecular genetic marker for gender determination of pigeons

The absence of conspicuous sexual dimorphism in pigeons often makes it difficult to determine their sex on the basis of external morphology. We identified a novel female-specific DNA marker in pigeons, presenting the possibility of pigeon gender determination using a PCR-based method. One-hundred and twenty random primers were used for RAPD fingerprinting in order to find any sex-specific fragments in pigeons. One of these primers, OPC-20, produced a female-specific band in the DNA fingerprints. This DNA fragment was isolated from the gel and inserted into a vector for nucleotide sequencing. A novel female-specific 732 bp sequence was obtained. A pair of primers (DoveOPC20F & R) was designed, based on the cloned sequence, for amplifying the female-specific band by PCR for pigeon gender determination. Sex-specific bands in the gel were observed in all females but not in males. The PCR products in the gel were then transferred onto nylon membranes and hybridized with a DIG-labeled probe of the cloned female-specific DNA fragment. Clear hybridization signals were found only in all of the female pigeons; the same result was obtained from dot blot hybridization. This demonstrates that the sex of pigeons can be accurately and rapidly identified by PCR.     

猪Mu阿片受体基因单核苷酸多态性分析

Mu阿片受体（简称MOR）属于G蛋白藕联受体,分布在痛觉传导区,以及与情绪和行为有关的区域,影响动物的神经反应和行为表现。该研究以长白猪、大白猪和杜洛克猪为试验材料, 用8对引物对Mu阿片受体基因的5′ UTR区域、整个编码区和3′ UTR区域用PCR-SSCP方法进行了扫描,发现5处突变基因座（GenBank登录号：AF521309）。统计结果发现基因型频率分布与品种有关,大白猪突变基因型频率显著高于长白和杜洛克,本研究推测分布上的差异可能是由于长期的选择压力造成的。 Abstract:Mu opioid receptor (MOR) is a member of G protein-coupled receptor family,distributed in the pain transduction region in the brain and related to emotion and behaviour.This study was designed to investigate the Single Nucleotide Polymorphism (SNP) of Mu opioid receptor gene in various breeds,including duroc,landrace and Yorkshire.5′ UTR ( untranslate region),coding region and 3′ UTR of Mu opioid receptor gene were amplified by eight pairs of primers,and the Single Nucleotide Polymorphism (SNP) were detected by SSCP.Five polymorphisms were found (Genebank Accession number:AF521309).The results of χ2 test showed that the frequencies of genotypes in different breeds were significantly different (P＜0.01).The frequencies of mutation genotypes in Yorkshire were significantly higher than Duroc and Landrace.According to the above results,we can speculate the difference of the frequencies of genotypes may be the results of long term choice pressure.     

Identification of Y chromosomal PCR marker and production of a selected strain for molecular sexing in the brown planthopper, Nilaparvata lugens

A laboratory colony was established in order to enable molecular sexing in premature stages in the brown planthopper, Nilaparvata lugens. We found four male-specific amplified fragment length polymorphisms (AFLPs) in the planthopper, and sequenced one of the AFLPs along with its 5' flanking region (1,423 bp in total). PCR primers were designed based on the nucleotide sequence information so that the PCR product was present in male planthoppers and absent in female planthoppers. However, we could not completely distinguish males from females, because the PCR amplification product was absent in some of the males screened. We, therefore, established a laboratory colony, in which all males carried this sequence. We can directly sex pre-adult stages in this colony using our PCR primers, making this strain of considerable value for studies that require sex separation in egg and nymphal stages.     

猪MyoG基因3'端PCR-SSCP遗传多态性及其遗传效应

采用PCR—SSCP方法对长白猪、大白猪、杜洛克猪、山西黑猪和马身猪共636头猪的肌细胞生成素（Myogenin,简称MyoG）基因3’端的遗传多态性进行检测,分析MyoG基因对猪的初生体质量、断奶体质量、6月龄体质量和背膘厚的影响。根据已发表的猪MyoG基因3’端侧翼序列设计3对引物,发现F1／R1引物对扩增的片段有多态性。统计结果发现：长白、大白、杜洛克猪种B基因为优势基因,其基因频率分别为0．8807、0．7256和0．8581：山西黑猪种4基因为优势基因,其基因频率为0．9359;马身猪种只检测到4基因。χ^2独立性检验表明,基因型分布在外来猪种（长白猪、大白猪、杜洛克猪）与地方猪种（山西黑猪、马身猪）间存在极显著差异（P〈0．01）。固定效应模型分析结果表明,初生体质量基因型闻差异显著（P〈0．05）,而断奶体质量、6月龄体质量和背膘厚基因型闻差异不显著（P〉0．05）。最小二乘分析结果表明,BB基因型与其它2种基因型比较有较大的初生质量,同似和AB型比较差异极显著（P〈0．01）。因此,推测MyoG基因对个体的初生体质量存在一足的影响,选择带有B等位基因的个体有望提高个体的初生体质量。     

Sex identification of pigs using polymerase chain reaction amplification of the amelogenin gene

The amelogenin (AMEL) gene exists on both sex chromosomes of various mammalian species and the length and sequence of the noncoding regions differ between the two chromosome-specific alleles. Because both forms can be amplified using a single primer set, the use of AMEL in polymerase chain reaction (PCR)-based methods has facilitated sex identification in various mammalian species, including cattle, sheep and humans. In this study, we designed PCR primers to yield different-sized products from the AMEL genes on the X (AMELX) and Y (AMELY) chromosomes of pigs. PCR amplification of genomic DNA samples collected from various breeds of pigs (European breeds: Landrace, Large White, Duroc and Berkshire; Chinese breeds: Meishan and Jinhua and their crossbreeds) yielded the expected products. For all breeds, DNA from male pigs produced two bands (520 and 350 bp; AMELX and AMELY, respectively), whereas samples from female pigs generated only the 520 bp product. We then tested the use of PCR of AMEL for sex identification of in vitro-produced (IVP) porcine embryos sampled at 2 or 5 to 6 days after fertilization; germinal vesicle (GV)-stage oocytes and electroactivated embryos were used as controls. More than 88% of the GV-stage oocytes and electroactivated embryos yielded a single 520 bp single band and about 50% of the IVP embryos tested produced both bands. Our findings show that PCR analysis of the AMEL gene is reliable for sex identification of pigs and porcine embryos.     

猪STCH基因的Bi-PASA遗传标记及多态性研究

微粒体应激 70蛋白三磷酸腺苷酶 (STCH)基因属于应激 70蛋白基因伴侣家族 ,在机体免疫反应和疾病抵抗力等方面起重要作用。根据人和小鼠STCH基因的保守序列设计引物 ,PCR扩增到猪STCH基因第5外显子 4 4 5bp片段。序列测定显示 ,猪STCH基因与人和小鼠STCH基因分别具有 87 13%和 80 4 5 %的同源性。通过测定和比较中国梅山猪、欧洲约克夏猪及PIC商品猪的STCH基因序列 ,发现在猪STCH基因编码区第 5外显子 10 5 0位点上存在一个单碱基突变位点。利用双向特定等位基因PCR扩增法 (Bi PASA)建立了检测猪STCH基因变异的遗传标记 ,并用该标记分析了STCH基因在中国家猪 (梅山猪、荣昌猪和金华猪 )、欧洲家猪 (约克夏猪、大白猪 )、商品猪 (PIC合成系 )以及欧洲野猪的基因频率和多态性。本研究建立的Bi PASA遗传标记和基因变异信息 ,将为进一步分析猪STCH基因变异与经济性状的相关分析提供基础资料。     

Characterization of swine leptin (LEP) polymorphisms and their association with production traits

Four polymorphisms in the swine leptin (LEP) gene were characterized and evaluated for association with economically important production traits in Yorkshire, Landrace and Duroc pigs. Our results show that these polymorphisms are generally of low frequency or are absent in pig populations. Two polymorphisms (A2845T and T3469C) may be associated (P < 0.0078) with feed intake and growth rate traits in Landrace pigs.