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Biochemical Genetics (v.48, #7-8)

AFLP-Based Genetic Diversity Assessment of Commercially Important Tea Germplasm in India by R. K. Sharma; M. S. Negi; S. Sharma; P. Bhardwaj; R. Kumar; E. Bhattachrya; S. B. Tripathi; D. Vijayan; A. R. Baruah; S. C. Das; B. Bera; R. Rajkumar; J. Thomas; R. K. Sud; N. Muraleedharan; M. Hazarika; M. Lakshmikumaran; S. N. Raina; P. S. Ahuja (pp. 549-564).

India has a large repository of important tea accessions and, therefore, plays a major role in improving production and quality of tea across the world. Using seven AFLP primer combinations, we analyzed 123 commercially important tea accessions representing major populations in India. The overall genetic similarity recorded was 51%. No significant differences were recorded in average genetic similarity among tea populations cultivated in various geographic regions (northwest 0.60, northeast and south both 0.59). UPGMA cluster analysis grouped the tea accessions according to geographic locations, with a bias toward China or Assam/Cambod types. Cluster analysis results were congruent with principal component analysis. Further, analysis of molecular variance detected a high level of genetic variation (85%) within and limited genetic variation (15%) among the populations, suggesting their origin from a similar genetic pool.

Keywords: AFLP; Genetic diversity; Tea accessions; Institute of Himalayan Bioresource Technology (IHBT); Tea Research Association; UPASI Tea Research Foundation

High Genetic Differentiation of Hippophae rhamnoides ssp. yunnanensis (Elaeagnaceae), a Plant Endemic to the Qinghai-Tibet Plateau by Wen Chen; Xue Su; Hui Zhang; Kun Sun; Ruijun Ma; Xuelin Chen (pp. 565-576).

RAPD markers were used to detect genetic diversity and population genetic differentiation of Hippophae rhamnoides ssp. yunnanensis, a sea buckthorn endemic to the Qinghai-Tibet plateau. The genetic parameters of percentage of polymorphic bands (92.86%), Nei’s gene diversity (h, 0.255), and Shannon’s index (I, 0.397) indicated high genetic diversity in this subspecies. The subpopulation differentiation suggested that 45.9% of genetic variation was among populations. High genetic differentiation among populations was also detected using AMOVA (47.02%). The main factors responsible for high genetic differentiation are probably related to natural geographic barriers among populations, gene drift, and limited gene flow caused by restricted pollen flow and seed flow. A Mantel test indicated that geographic distances were significantly correlated with genetic distances. The UPGMA phenogram based on Nei’s unbiased genetic distances and the result of three-dimensional model plots performed by principal coordinate analysis also supported the correlation. Altitude, however, did not have any clear effect on genetic differentiation.

Keywords: Hippophae rhamnoides ssp. yunnanensis ; Genetic diversity; Genetic differentiation; RAPD

A Colorimetric Method for Detection of K-ras Codon 12 Point Mutations in DNA Extracted from Tissue and Peripheral Blood in Pancreatic Disorders by Robert A. Ollar; Avram M. Cooperman; Michael E. Wayne; James F. Barrecchia; Niket Sonpal; Sushil Duddempudi; Franklin E. Kasmin (pp. 577-589).

Molecular-based methods to monitor point mutations require special and expensive equipment unavailable in most hospitals. Colorimetric-based analysis is an ideal platform for K-ras codon 12 gene point mutations because it uses commonly found hospital equipment. The colorimetric assay is sensitive and specific, detecting mutated DNA levels as low as 1% in a wild-type background. Paired genomic DNA extracts of fixed tissue and cellular fractions of peripheral blood are more sensitive and accurate than unpaired samplings. This approach has the potential to improve K-ras point mutation scans as well as to detect micrometastases in circulating tumor cells.

Keywords: Colorimetric; Point mutation; Somatic mutation ratio; Fixed tissue; Cellular fraction; K-ras 12; Micrometastases

Rangewide Genetic Diversity in Natural Populations of Chinese Pine (Pinus tabulaeformis) by Meng-Ben Wang; Zhen-Zhen Hao (pp. 590-602).

Thirteen natural populations from throughout the range of the Chinese pine (Pinus tabulaeformis Carr.) were examined using inter-simple sequence repeat markers to characterize the genetic structure at the species level and to compare the extent and distribution of genetic variation among central, intermediate, and marginal populations. Although the total genetic variation in the Chinese pine was mainly maintained within populations, the genetic differentiation among populations was significant (P < 0.001). The genetic divergence was significantly correlated with geographic distance (P < 0.05). Genetic diversity tended to decrease from the central to intermediate and marginal populations. The marginal populations had significantly lower intrapopulation genetic diversity than central populations (P < 0.05). Cluster analysis based on Nei’s unbiased genetic distances confirmed the difference among four central populations and the rest. Both historical and contemporary factors may have played key roles in shaping the spatial genetic structure of this species.

Keywords: Pinus tabulaeformis ; Inter-simple sequence repeat (ISSR); Biodiversity; Genetic structure

Genetic Diversity Within and Among Lepidium draba Populations from Eastern Anatolia Based on RAPD Analysis by Ozkan Aksakal; Serap Sunar; Yusuf Kaya; Guleray Agar (pp. 603-611).

Genetic variation and structure of six natural populations of Lepidium draba L. from Eastern Anatolia were assessed using random amplified polymorphic DNA (RAPD) markers. For RAPD analysis, 12 primers generated 218 reproducible bands across the six populations analyzed, of which 73 bands (33.3%) were polymorphic. The mean Nei’s gene diversity value for all six populations was 0.1771. Shannon’s information index varied with population (0.2278–0.3082), averaging 0.2608. Analysis of molecular variance (AMOVA) showed that genetic diversity was greater within populations (58.66%) than among populations (30.68%). In addition, the variation between groups was 10.33%. The genetic differentiation among populations (G _ST) was 0.3210, indicating that most genetic diversity occurs within populations. Gene flow (Nm) was low, at only 0.5288.

Keywords: AMOVA; Lepidium draba ; Gene flow; Genetic variation; RAPD

Comparative Analysis of SOX3 Protein Orthologs: Expansion of Homopolymeric Amino Acid Tracts During Vertebrate Evolution by Marija Mojsin; Natasa Kovacevic-Grujicic; Aleksandar Krstic; Jelena Popovic; Milena Milivojevic; Milena Stevanovic (pp. 612-623).

To understand more fully the structure and evolution of the SOX3 protein, we comparatively analyzed its orthologs in vertebrates. Since complex disorders are associated with human SOX3 polyalanine expansions, our investigation focused on both compositional and evolutionary analysis of various homopolymeric amino acid tracts observed in SOX3 orthologs. Our analysis revealed that the observed homopolymeric alanine, glycine, and proline tracts are mammal-specific, except for one polyglycine tract present in birds. Since it is likely that the SOX3 protein acquired additional roles in brain development in Eutheria, we might speculate that development of novel brain functions during the course of evolution was affected, at least in part, by such structural–functional changes in the SOX3 protein.

Keywords: SOX3 protein; Homopolymeric amino acid tract; Polyalanine expansion; Comparative protein analysis

A Set of Polymorphic Trinucleotide and Tetranucleotide Microsatellite Markers for Silver Crucian Carp (Carassius auratus gibelio) and Cross-Amplification in Crucian Carp by X. H. Zheng; C. Y. Lu; Y. Y. Zhao; C. Lee; D. C. Cao; Y. M. Chang; L. Q. Liang; X. W. Sun (pp. 624-635).

Silver crucian carp (Carassius auratus gibelio Bloch), as a gynogenetic fish, is a promising model for the study of the evolutionary genetics of vertebrates. We have developed 59 polymorphic trinucleotide and tetranucleotide markers for the silver crucian carp through the biotin capture method and radioactive-labeling hybridization. The number of alleles ranged from 2 to 12 in the population, and the average proportion of heterozygotes (including tri- and diallelic) at polymorphic loci was 76.8%. In addition, these loci were successfully applied to a close relative, the crucian carp (Carassius auratus), by cross-amplification, as shown by the range of alleles (2–19), observed heterozygosity (0.1765–0.9706), expected heterozygosity (0.2392–0.9421), and polymorphism information content (0.2186–0.9236).

Keywords: Carassius auratus gibelio ; Microsatellites; Trinucleotides; Tetranucleotides

Cytochrome b Genetic Diversity and Maternal Origin of Chinese Domestic Donkey by Yun-Sheng Zhang; Xue-Yi Yang; Xiao-Bin Wang; Chun-Mei Zhang; Fang Qin; Zhen-Hui Zhou; Xian-Yong Lan; Hong Chen; Chu-Zhao Lei (pp. 636-646).

To clarify the origin of Chinese domestic donkeys, we investigated the mitochondrial Cytb gene from 244 animals from 13 native breeds. We found 55 variable sites in the Cytb gene sequence and subsequently defined 58 haplotypes. Analysis of haplotypes in combination with Cytb sequences revealed two mitochondrial origins in Chinese domestic donkeys, phenotypically expressed by the Somalian and Nubian lineages. The Somalian lineage predominated in Chinese domestic donkey breeds. Five specific Cytb gene SNPs diagnostic of each of the lineages were found in this study: 225(T-C), 237(C-T), 915(C-T), 1014(C-T), and 1134(A-G) mutations. They effectively distinguish the Nubian from the Somalian lineage in the mtDNA Cytb gene. Both lineages are from Africa and thus support the African maternal origins of Chinese domestic donkeys. No obvious geographic structure was found in Chinese domestic donkey breeds, but the population showed abundant genetic diversity.

Keywords: Chinese domestic donkeys; Cytb gene; Origin

Association of GSTM1 and GSTT1 Polymorphisms with Chronic Obstructive Pulmonary Disease in a Tunisian Population by Ramzi Lakhdar; Sabri Denden; Jalel Knani; Nadia Leban; Houria Daimi; Mohsen Hassine; Gérard Lefranc; Jemni Ben Chibani; Amel Haj Khelil (pp. 647-657).

GSTM1 and GSTT1 polymorphisms have been proposed in relationship with chronic obstructive pulmonary disease (COPD). We investigated the association between these polymorphisms and COPD (as well as its subtypes emphysema and chronic bronchitis) in 234 COPD patients and 182 healthy controls in the Tunisian population. Genotyping was performed using multiplex PCR. GSTM1-null genotype frequency was significantly higher in COPD patients than in controls (P = 0.02); however, multivariate analysis of cofounding variables showed no independent association with this genotype (P = 0.073). In contrast, the association of the GSTM1-null genotype with emphysema was significant, even after adjustment for risk factors (P = 0.011). There were no significant differences in GSTT1 genotypes between patients and controls. The GSTM1 null allele is likely not an independent risk factor for COPD but is related to emphysema, whereas the GSTT1 gene is not associated with the disease.

Keywords: Chronic obstructive pulmonary disease; Emphysema; Genetic polymorphism; Glutathione S-transferase

A Novel Ribonuclease with Antiproliferative Activity from Fresh Fruiting Bodies of the Edible Mushroom Lyophyllum shimeiji by R. Y. Zhang; G. Q. Zhang; D. D. Hu; H. X. Wang; T. B. Ng (pp. 658-668).

A 14.5-kDa ribonuclease, with an optimum pH of 6 and a temperature optimum at 70°C, was isolated from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji. It was purified by ion exchange chromatography on DEAE cellulose, Q Sepharose, and SP Sepharose, followed by FPLC gel filtration on Superdex 75, and was adsorbed on all three ion exchangers. It showed the highest ribonucleolytic potency toward poly (U), 25% as much activity toward poly (C), and undetectable activity toward poly (A) and poly (G). Its ribonucleolytic activity at 100°C was similar to that at 20°C. It suppressed proliferation of hepatoma HepG2 cells and breast cancer MCF7 cells with an IC₅₀ of 10 and 6.2 μM, respectively. It inhibited the activity of HIV-1 reverse transcriptase with an IC₅₀ of 7.2 μM.

Keywords: Antiproliferative; Lyophyllum shimeiji ; Mushroom; Ribonuclease

Cloning, Heterologous Expression, and Functional Characterization of a Chitinase Gene, Lbchi32, from Limonium bicolor by Zhi Hua Liu; Chuan Ping Yang; Xiao Tian Qi; Li Li Xiu; Yu Cheng Wang (pp. 669-679).

In the present study, an endochitinase gene, Lbchi32, was cloned from Limonium bicolor. The cDNA sequence of Lbchi32 was 1,443 bp in length and encoded 319 amino acid residues. The DNA sequence of Lbchi32 was 2,512 bp in length and contained three exons and two introns. The Lbchi32 gene was inserted into a pPIC9 vector and transferred into Pichia pastoris strains GS115 and KM71 for heterologous expression. SDS-PAGE analyses indicated that LbCHI32 was expressed in both GS115 and KM71 and that it was secreted extracellularly. The optimal reaction conditions for LbCHI32 activity are 45°C, pH 5.0, and 5 mM Ba²⁺. The LbCHI32 enzyme can efficiently degrade chitin, chitin derivatives, and the cell walls of different pathogenic fungi, including phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Valsa sordida, Septoria tritici, and Phytophthora sojae. These findings suggest that Lbchi32 has potential use in the degradation of chitin and chitin derivatives.

Keywords: Chitinase; Gene cloning; Heterologous expression; Limonium bicolor

A First Set of Polymorphic Microsatellite Loci from the Marbled Rockfish, Sebastiscus marmoratus by Tian-jun Xu; Xian-qing Quan; Yue-na Sun; Kuang-ci Zhao; Ri-xin Wang (pp. 680-683).

Lumbar Disk Degeneration Disease and Aggrecan Gene Polymorphism in Northern Iran by Farhad Mashayekhi; Gholamreza Shafiee; Masood Kazemi; Parviz Dolati (pp. 684-689).

Aggrecan is the major component of intervertebral disk matrix proteoglycan with multiple functional domains. To understand the role of aggrecan polymorphism in a part of exon 12 encoding the CS1 domain in lumbar disk degeneration disease, we have analyzed genomic DNA from 71 patients with the disease and 108 healthy individuals in northern Iran. The AGC1 alleles were determined by PCR followed by gel electrophoresis. Twelve AGC1 alleles ranging from 18 to 29 repeats were detected in patients and controls. The most frequent AGC1 allele was 27, followed by 28 in patients and controls. The shorter AGC1 alleles (≤24 repeats) were more frequent in patients than in controls (37 vs. 16%, P < 0.001). The odds ratio for lumbar disk degeneration was 3.28 (95% confidence interval 1.62–6.65) in carriers of the shorter AGC1 alleles. Our data suggest that carrying shorter AGC1 alleles with less than 24 repeats could predispose a subject to lumbar disk degeneration disease in northern Iran.

Keywords: Lumbar disk degenerative disease; Aggrecan; Polymorphism; VNTR; CS1 domain

Development of MHC Class I and II B Primers in Common Carp and its Molecular Characterization by Zhiying Jia; Xifeng Chi; Chitao Li; Lianyu Shi (pp. 690-695).

The major histocompatibility complex (MHC) has an important role in immune response and is known as the most polymorphic locus in vertebrates. We developed three pairs of polymerase chain reaction primers of the alpha-2 domain (exon 3) of MHC class I and the beta-2 (exon 3) and beta-3 domains (exon 4) of MHC class II B gene in the German mirror common carp (Cyprinus carpio L.). We analyzed the three loci in a population of 65 individuals that had suffered the serious disease of gill rot. Five to six variable nucleotide sites and two to six variable amino acid sites (71.43%) were detected in the exon sequence of the sampled populations, indicating that many of them corresponded to amino acids involved in antigen recognition. Deviation from Hardy–Weinberg equilibrium and linkage disequilibrium were differentially found in some loci, which will be important for further study of disease resistance/susceptibility and population evolution.

Keywords: MHC; Common carp; Polymorphic; Variable nucleotide site

Concordant Genetic Distinctness of the Phylogroup of the Siberian Chipmunk from the Korean Peninsula (Tamias sibiricus barberi), Reexamined with Nuclear DNA c-myc Gene Exon 2 and mtDNA Control Region Sequences by Hung Sun Koh; Minghai Zhang; Damdingiin Bayarlkhagva; Eui Jeong Ham; Jin Seong Kim; Kyung Hee Jang; Nam Jeong Park (pp. 696-705).

We reexamined Tamias sibiricus barberi from Korea by sequencing c-myc exon 2 and the mtDNA control region. In the c-myc exon, the monogenic T. s. barberi differed from the monogenic T. s. orientalis (nucleotide distance 0.48%; 3 variable sites at 168, 306, and 552), whereas T. s. orientalis was identical to T. s. sibiricus. In the control region, T. s. barberi differed from T. s. orientalis (distance 6.84%) and T. s. sibiricus (9.35%). We considered the concordant, extensive gaps between the phylogroup of T. s. barberi and other subspecies of T. sibiricus in the c-myc gene, control region, and cytochrome b gene to be evidence of a lack of intergradation through North Korea from T. s. barberi to T. s. orientalis. Our results, showing the genetic and morphological distinctness of T. s. barberi, support that this phylogroup is a distinct species.

Keywords: Tamias sibiricus barberi ; Siberian chipmunk; Korea; Phylogroup; C-myc gene; Control region

Genetic Distinctness of the Korean Hare, Lepus coreanus (Mammalia, Lagomorpha), Revealed by Nuclear Thyroglobulin Gene and mtDNA Control Region Sequences by Hung Sun Koh; Kyung Hee Jang (pp. 706-710).

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Biochemical Genetics (v.48, #7-8)