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Biochemical Genetics (v.46, #3-4)

Mice Genotyping Using Buccal Swab Samples: An Improved Method by Dinko Mitrečić; Sandra Mavrić; Božica Vrabec Branica; Srećko Gajović (pp. 105-112).

Routine methods used to genotype mice involve isolation of DNA from partially amputated neonate’s tail, toe, or ear. The inevitable drawbacks of such techniques are the animal’s pain response and the increased time and funds required for DNA purification. In order to implement a noninvasive and simple protocol for mouse DNA isolation, we have improved the method based on samples collected by swabbing of the inner cheek. Combining alkaline and temperature lysis, it was possible to isolate a DNA solution ready for PCR in less than an hour. Testing the method on three different mouse lines showed that it is highly efficient, the volume of the PCR samples could be reduced to 25 μl, and fragments up to 800 bp were successfully amplified. This protocol reduces animal discomfort, shortens the time for DNA isolation, and enables amplification of larger DNA fragments with optimal success rate, thus considerably facilitating large-scale genotyping of different mouse lines.

Keywords: DNA isolation; Buccal swabs; Genotyping; PCR; Mouse

Molecular Variability of Hop stunt viroid: Identification of a Unique Variant with a Tandem 15-nucleotide Repeat from Naturally Infected Plum Tree by Yuan-Ai Yang; Hong-Qing Wang; Zu-Jian Wu; Zhuo-Min Cheng; Shi-Fang Li (pp. 113-123).

For this study, 68 plum samples were collected from 12 provinces of China. Low molecular weight RNAs were extracted and used for dot-blot, reverse transcription–polymerase chain reaction (RT-PCR), return–polyacrylamide gel electrophoresis, and biological indexing using cucumber. Results showed that 15 out of the 68 plum samples were positive for Hop stunt viroid (HSVd). Four positive samples were selected for cloning and sequence analysis. Results indicated that most HSVd sequences from the plum in China had 1–3 nucleotide changes from the closest HSVd in GenBank. In addition, the sample PB21 (cv. ‘Friar’) collected from Hebei province had one sequence with a 15-nt duplication (named HSVd D-15) at the 244/245 position in the lower central region. By biological indexing, cucumber seedlings, an indicator plant for HSVd, were inoculated with RNAs directly extracted from the original plum source (PB21, cv. ‘Friar’). Nucleotide sequencing analysis of the progeny showed that HSVd, but not HSVd-D15, was recovered from inoculated cucumbers. Although very unlikely, the possibility that this extra sequence was the result of a PCR artifact cannot be completely ruled out.

Keywords: Hop stunt viroid ; Plum; Recombination; Intramolecular recombination; Sequence repeat

Genetic Diversity and Structure of Two Prominent Zebu Cattle Breeds Adapted to the Arid Region of India Inferred from Microsatellite Polymorphism by M. Sodhi; M. Mukesh; S. P. S. Ahlawat; R. C. Sobti; G. C. Gahlot; S. C. Mehta; B. Prakash; B. P. Mishra (pp. 124-136).

This study aims to assess the genetic diversity and population structure of two major zebu dairy breeds (Tharparkar and Rathi) adapted to the arid region of Rajasthan state of India. Various variability estimates indicate the existence of sufficient within-breed genetic diversity. Mean estimates of F-statistics are significantly different from zero: F _IS = 0.112 ± 0.029, F _IT = 0.169 ± 0.033, F _ST = 0.065 ± 0.017. The overall positive value of F _IS (0.112) and an F _IT value (0.169) that is more than the F _ST (0.065) indicate departure from random mating. The drift-based estimates reflect a moderate yet significant level of breed differentiation between the Tharparkar and Rathi breeds. The evaluation of an exact test, showing that allele frequencies across all the loci differed significantly, supports the population differentiation. This is paralleled by the outcome of neighbor-joining clustering based on allele-sharing distance measures. The allocation of a high percentage of individuals (95.7%) to their population of origin and correspondence analysis further substantiates the existence of a cohesive genetic structure in both the breeds.

Keywords: Indian zebu cattle; Microsatellite markers; Genetic diversity; Population structure

Polymorphism in the Matrix Metalloproteinase-2 Gene Promoter is Associated with Cervical Neoplasm Risk in Mexican Women by Luz M. Baltazar-Rodriguez; Aristoteles Anaya-Ventura; Maricela Andrade-Soto; Elisa A. Monrroy-Guizar; Jose R. Bautista-Lam; Gregorio Jonguitud-Olguin; Friedman R. Cepeda-Lopez; Veronica A. Centeno-Aguilar; Nelida A. Gonzalez-Hernandez; Alejandro D. Soriano-Hernández; Mario Ramirez-Flores; Angel Lugo-Trampe; Iram P. Rodriguez-Sanchez; Oscar A. Newton-Sanchez; Ivan Delgado-Enciso (pp. 137-144).

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity on matrix proteins, and previous studies have revealed a strong association between the MMP-2 −1306C→T polymorphism and the risk of several types of cancer. Our study looked at whether this polymorphism contributed to the development of cervical neoplasia by analyzing 54 patients with invasive squamous cell cervical cancer, 100 patients with cervical intraepithelial neoplasia, and 126 control subjects. The MMP-2 CC genotype was more frequent in the cancer patients when compared with the control group (OR 2.57; 95% CI 1.15–5.86). The association of cervical cancer with the CC genotype was more pronounced in women who had first coitus at an early age (OR 3.96; 95% CI 1.46–11.06). The CC genotype was associated with intraepithelial neoplasia only in women with first coitus at 19 years old or younger. The data suggest that the MMP-2 −1306C→T polymorphism contributes to the development of squamous cell cervical cancer in the population studied, especially in women who had first coitus at an early age.

Keywords: Cervix neoplasms; Gelatinase A; First intercourse; Risk factors; Mexico

Development of Genotyping Methods for Single Nucleotide Polymorphism in the Human Pancreatic Ribonuclease Gene (RNASE1) and Their Application to Population Studies by Misuzu Ueki; Reiko Iida; Junko Fujihara; Haruo Takeshita; Isao Yuasa; Yoshihiko Kominato; Toshihiro Yasuda (pp. 145-153).

Two potential single nucleotide polymorphisms [SNPs; rs1804215 (G979T) and rs11545379 (G1169T)] have been identified in the human pancreatic ribonuclease, RNase 1, gene (RNASE1) that could give rise to an amino acid substitution in the protein, but relevant population data are not available. We have developed genotyping methods for each SNP using the mismatched PCR-restriction fragment length polymorphism technique. These methods are advantageous in comparison with other SNP genotyping methods because they are technically simpler and do not require specialized instruments. We applied these genotyping methods to examine the genotype distribution of each SNP in four populations, including Japanese populations living in two prefectures, an Ovambo population, and a Turkish population. In all the populations studied, however, only a single genotype for each SNP was found. Therefore, irrespective of differences in ethnic groups, RNASE1 might show markedly low heterogeneity in its genetic structure with regard to these SNPs.

Keywords: Pancreatic ribonucleases; Single nucleotide polymorphism; Population data; Genotyping method; PCR

A Rare Y Chromosome Missense Mutation in Exon 25 of Human USP9Y Revealed by Pyrosequencing by Lynn M. Sims; Jack Ballantyne (pp. 154-161).

Ubiquitin-specific protease 9, Y-linked (USP9Y), is a protein encoded by the Y chromosome. Its precise function in the cell is unknown, although a role in the regulation of protein turnover has been postulated. Nonetheless, mutations in this gene could result in the over- or under-abundance of proteins involved in the regulation of spermatogenesis. We have identified a novel mutation, SM1, located in exon 25 of USP9Y (c.3642G→A), which results in an amino acid substitution (p.V1214I). The mutation is in close linkage (four bases distant) from a silent mutation, referred to as M222 (p.E1212E, c.3636G→A). In our male population (n = 374), SM1 was found in one individual (0.3%) who belongs to the recently described haplogroup R1b3h, defined by the U152 SNP. This new mutation is expected to represent a new haplogroup, (R1b1c10a); therefore, within our population of individuals from haplogroup R1b3h (R1b110) (n = 16), it has a frequency of 6.3% (95% CI: 2.7–9.9%).

Keywords: Y-SNP; USP9Y; M222; U152; Infertility

Molecular Characterization of Tree Peony Germplasm Using Sequence-Related Amplified Polymorphism Markers by Xiao Yan Han; Liang Sheng Wang; Qing Yan Shu; Zheng An Liu; Su Xia Xu; Takuya Tetsumura (pp. 162-179).

This study examined 63 tree peony specimens, consisting of 3 wild species and 63 cultivars, using sequence-related amplified polymorphism (SRAP) markers for the purpose of detecting genomic polymorphisms. Bulk DNA samples from each specimen were evaluated with 23 SRAP primer pairs. Among the 296 different amplicons, 262 were polymorphic. The maximum parsimony, neighbor-joining, and unweighted pair-group method using arithmetic average trees were largely in congruence. In the three trees, the wild species Paeonia ludlowii and P. delavayi formed separate clusters with strong bootstrap support, and P. ostii was closely related to all cultivars. The cultivars were divided into groups with various corresponding bootstrap values. The genetic similarity among the genotypes ranged from 0.02 to 0.73. These results demonstrate that SRAP markers are effective in detecting genomic polymorphisms in the tree peony and should be useful for linkage map construction and molecular marker assisted selection breeding.

Keywords: Tree peony; SRAP marker; Genomic polymorphism; MAS breeding

Genetic Diversity and Geographic Differentiation in the Threatened Species Dysosma pleiantha in China as Revealed by ISSR Analysis by Min Zong; Hai-Long Liu; Ying-Xiong Qiu; Shu-Zhen Yang; Ming-Shui Zhao; Cheng-Xin Fu (pp. 180-196).

Dysosma pleiantha, an important threatened medicinal plant species, is restricted in distribution to southeastern China. The species is capable of reproducing both sexually and asexually. In this study, inter-simple sequence repeat marker data were obtained and analyzed with respect to genetic variation and genetic structure. The extent of clonality, together with the clonal and sexual reproductive strategies, varied among sites, and the populations under harsh ecological conditions tended to have large clones with relatively low clonal diversity caused by vegetative reproduction. The ramets sharing the same genotype show a clumped distribution. Across all populations surveyed, average within-population diversity was remarkably low (e.g., 0.111 for Nei’s gene diversity), with populations from the nature reserves maintaining relatively high amounts of genetic diversity. Among all populations, high genetic differentiation (AMOVA: Φ_ST = 0.500; Nei’s genetic diversity: G _ST = 0.465, Bayesian analysis: Φ_B = 0.436) was detected, together with an isolation-by-distance pattern. Low seedling recruitment due to inbreeding, restricted gene flow, and genetic drift are proposed as determinant factors responsible for the low genetic diversity and high genetic differentiation observed.

Keywords: Dysosma pleiantha ; Threatened medicinal plant; Conservation; Clonal diversity; Genetic variation; ISSR

Effect of Strontium Ranelate on Hydrogen Peroxide-Induced Apoptosis of CRL-11372 Cells by Alpaslan Senkoylu; Akin Yilmaz; Mehmet Ali Ergun; Mustafa N. İlhan; Aykin Simsek; Necdet Altun; Selcuk Bolukbasi; Sevda Menevse (pp. 197-205).

In vitro and in vivo studies have proven strontium to be an osteoinductive trace element. The effect of strontium ranelate (SR) on H₂O₂-induced apoptosis of CRL-11372 cells and optimization of its anti-apoptotic dose were the aims of this study. After 1 h of pretreatment with SR 1 μM, 50 μM, 100 μM, 500 μM, and 1,000 μM concentrations, CRL-11372 osteoblasts were exposed to 100 μM H₂O₂ for periods of 6–12 h. The same experiments were repeated without H₂O₂. The apoptotic index and viability of cells were assessed quantitatively with a fluorescent dye and qualitatively with agarose gel electrophoresis. Concentrations of 1–100 μM of SR with a 6-h treatment and only 1 μM concentration with a 12-h treatment inhibited the apoptotic effect of H₂O₂ on cultured osteoblasts significantly (P < 0.05). SR was shown to inhibit H₂O₂-induced apoptosis of CRL-11372 cells in a dose-dependent manner.

Keywords: Strontium ranelate; Osteoblast; Apoptosis; Tissue culture

Dissecting the Matrilineal Components of Tongjiang Cattle from Southwest China by Shi-Yi Chen; Yi-Ping Liu; Wei Wang; Cheng-Zhong Gao; Yong-Gang Yao; Song-Jia Lai (pp. 206-215).

Tongjiang cattle are a local cattle population of Sichuan Province, China, numbering approximately half a million in 2005. They have long been grouped into the Bashan breed, although they have a unique breeding history and phenotypic characteristics, as well as a restricted geographic distribution. Morphologically, they can be divided into two groups based on the basic coat color (black and russet). In order to dissect the matrilineal components of Tongjiang cattle and to compare the body size traits of the two morphological groups, we measured five body size traits among 59 Tongjiang cattle samples and further sequenced the mtDNA D-loop sequence of 54 individuals. Among the 54 mtDNAs, 37 (68.5%) were Bos taurus types and 17 (31.5%) were Bos indicus types. Four known B. taurus haplogroups (T1–T4) and one B. indicus haplogroup (I1) were detected in these samples. Two body size traits differed significantly (P < 0.05) between the black group and the russet group, although the two groups possessed similar matrilineal genetic structure. This is the first report to identify all four B. taurus haplogroups in one local Chinese cattle population. Our results suggest that the contribution of different matrilineal lineages to Chinese cattle might be more complex than we originally thought.

Keywords: Tongjiang cattle; Chinese local breed; mtDNA; D-loop; Matrilineal components; Body size; Genetic diversity

Evidence for Introgressive Hybridization of Captive Markhor (Capra falconeri) with Domestic Goat: Cautions for Reintroduction by Sabine E. Hammer; Harald M. Schwammer; Franz Suchentrunk (pp. 216-226).

Markhors (Capra falconeri) are among the most endangered mammal species, and several conservation measures, including ex situ breeding, are implemented to prevent their extinction. We studied sequence diversity and differentiation of the first hypervariable segment of the mitochondrial DNA control region among C. f. heptneri and C. f. megaceros kept in four zoos in relationship to lineages of other wild and domestic goats, to assess for the first time the level of molecular distinctness and variability among those subspecies, and to check for possible introgression by related Capra taxa, such as domestic goats. Levels of differentiation between some Capra falconeri lineages and modern domestic goats were similar to levels between other wild goat species (i.e., Capra aegagrus, Capra ibex) and domestic goats. Among pure markhor lineages, paraphyly was observed for C. f. heptneri, suggesting occurrence of shared ancestral polymorphism among markhor subspecies and/or ancient or recent gene exchange between subspecies. Interestingly, 35.7% of all studied markhors from three zoos are introgressed by the domestic goat. Furthermore, despite relatively small breeding group sizes, markhors have maintained a relatively high proportion of mtDNA variation within zoo groups. In any case, the existence of markhors introgressed with domestic goat DNA in zoos should be considered when selecting markhors for ex situ breeding programs with the aim of building up a stock for later reintroduction into the wild.

Keywords: Capra falconeri ; Markhor; Mitochondrial control region; Introgression; Conservation genetics

A Study of Genetic Structure of Stephania yunnanensis (Menispermaceae) by DALP by Yun-shu Ma; Hong Yu; Yong-yi Li; Hong Yan; Xin Cheng (pp. 227-240).

Genetic diversity and genetic structure within and among ten populations of Stephania yunnanensis H. S. Lo and three populations of S. epigaea H. S. Lo from Yunnan province were evaluated by direct amplification of length polymorphism (DALP) markers. Five primer groups were screened, and a total of 287 DNA fragments were amplified, among which 266 were polymorphic, averaging 53.2 polymorphic bands per primer group in S. yunnanensis. The percentage of polymorphic bands of S. yunnanensis was 92.68% at the species level and 61.92% within the ten populations sampled. At the species level, the observed number of alleles (N _a) was 1.9268 and the effective number of alleles (N _e) was 1.5933; Nei’s gene diversity (H) was 0.3414; Shannon’s information index (I) was 0.5057. At the population level, N _a = 1.6192, N _e = 1.4001, H = 0.2298, and I = 0.3401. Total gene diversity of S. yunnanensis was 0.3419. Gene diversity within population was 0.2298, coefficient of gene differentiation was 0.3278, and estimated gene flow was 1.0254. The results indicated that the genetic differentiation was relatively higher among populations of S. yunnanensis. DALP markers were an informative and useful method for assaying genetic diversity and authenticating species of Stephania. These data could provide basic molecular evidence for establishing a reasonable strategy for protecting and exploiting the resource of S. yunnanensis.

Keywords: Stephania yunnanensis ; Stephania epigaea ; DALP; Genetic diversity; Genetic structure

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Biochemical Genetics (v.46, #3-4)

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Biochemical Genetics (v.46, #3-4)