DNA Profiling and Assessment of Genetic Relationships Among Important Seedless Grape (Vitis vinifera) Varieties in India Using ISSR Markers

In the present work, 43 seedless grape (Vitis vinifera) varieties were evaluated using 14 informative ISSR primers. A total of 119 bands were scored, out of which 79% were polymorphic. The level of polymorphism varied according to the primers which was evident also from different Resolving Power values of the individual primers. Based on the DNA fingerprint data of only three primers (UBC 857, 888 and 890), all the varieties except one could be identified. UPGMA cluster analysis revealed high degree of similarity among the varieties. Distinct subgroups based on the berry colour were observed. No intravarietal differences were detected. Overall grouping was in accordance with morphological characters and pedigree, proving usefulness of ISSR markers to assess genetic relationships among grape varieties.     

DNA sequence divergence among derivatives of Escherichia coli K-12 detected by arbitrary primer PCR (random amplified polymorphic DNA) fingerprinting.

Derivatives of Escherichia coli K-12 of known ancestry were characterized by random amplified polymorphic DNA (RAPD) fingerprinting to better understand genome evolution in this family of closely related strains. This sensitive method entails PCR amplification with arbitrary primers at low stringency and yields arrays of anonymous DNA fragments that are strain specific. Among 150 fragments scored, eight were polymorphic in that they were produced from some but not all strains. Seven polymorphic bands were chromosomal, and one was from the F-factor plasmid. Five of the six mapped polymorphic chromosomal bands came from just 7% of the genome, a 340-kb segment that includes the terminus of replication. Two of these were from the cryptic Rac prophage, and the inability to amplify them from strains was attributable to deletion (excision) or to rearrangement of Rac. Two other terminus-region segments that resulted in polymorphic bands appeared to have sustained point mutations that affected the ability to amplify them. Control experiments showed that RAPD bands from the 340-kb terminus-region segment and also from two plasmids (P1 and F) were represented in approximate proportion to their size. Optimization experiments showed that the concentration of thermostable polymerase strongly affected the arrays of RAPD products obtained. Comparison of RAPD polymorphisms and positions of strains exhibiting them in the pedigree suggests that many sequence changes occurred in these historic E. coli strains during their storage. We propose that the clustering of such mutations near the terminus reflects errors during completion of chromosome replication, possibly during slow growth in the stab cultures that were often used to store E. coli strains in the early years of bacterial genetics.     

Suitability of RAPD for analyzing spined and spineless variant regenerants of pineapple (Ananas comosus L., Merr.)

A random amplified polymorphic DNA (RAPD) analysis of spineless (variant phenotype) plants obtained from micropropagated dormant pineapple (Ananas comosus L., Merr.) axillary buds was performed using arbitrary 10-mer oligonucleotide primers. This was done to investigate the genetic fidelity of the regenerants and to distinguish these variants from regenerants bearing the normal spined phenotype. Of the 58 arbitrary primers used, 29 produced bands unique to the spineless phenotype, and 30 produced bands unique to the spined phenotype. A total of 914 bands were scored, 55 of which were polymorphic to the spineless phenotype and 51 of which were polymorphic to the spined phenotype. On the basis of RAPD amplification products, genetic similarity was estimated in both types of regenerants using similarity coefficients (Nei and Li, 1979). The characteristic finger-prints generated by each probe emphasize genetic variability of regenerants. This technique is suitable for analyzing variant regenerants induced in vitro.     

RFLP和AFLP分析白菜型油菜和甘蓝型油菜遗传多样性及其在油菜改良中的应用价值

采用RFLP和AFLP标记对来自中国和欧美的7份甘蓝型油菜和22份白菜型油菜进行了遗传多样性分析。在这29份材料中,166个酶-探针组合和2对AFLP引物共检测到1477个RFLP标记和183个AFLP标记。RFLP数据显示以拟南芥EST克隆作探针比用油菜基因组克隆做探针能检测到更多的多态性位点,且采用EcoR Ⅰ或BamH Ⅰ酶切比HindⅢ酶切多态性好,白菜型油菜和甘蓝型油菜中基因的拷贝数平均都为3个左右。UPGMA聚类分析表明中国白菜型油菜的遗传多样性比甘蓝型油菜和欧美白菜型油菜丰富,欧美甘蓝型油菜与欧美白菜型油菜聚为一类,而与中国甘蓝型油菜差异更大。中国白菜型油菜丰富的遗传多样性为中国甘蓝型油菜的改良提供了宝贵的资源,揭示了利用白菜型油菜A基因组和甘蓝型油菜A基因组间亚基因组杂种优势的可能性。     

加工番茄种质资源的SSR分析

为探明加工番茄种质资源间的亲缘关系,利用SSR标记对20份加工番茄品种及育种材料进行了多样性分析。结果表明:从49对SSR引物中筛选出12对扩增稳定、条带清晰且多态性丰富的引物进行分析,共获得43个位点,多态性位点为37个,多态位点比率86%;供试材料间的遗传相似系数介于0.419~1.000之间,说明加工番茄种质资源间存在一定的遗传差异;通过UPGMA法聚类分析,将20份材料分为3大类群,其中亲缘关系较远的不同类群间及亚类间的种质资源可作为杂交育种的亲本。     

Reduction to homozygosity at the SIS/PDGF-2 locus in human mesenchymal tumors

Enhanced expression of the human SIS/PDGF-2 gene has been reported in a number of human cell lines, sarcomas, and glioblastomas. We have analyzed the SIS/PDGF-2 gene for structural alterations in fresh human tumors. DNA samples from 79 patients with solid tumors (63 mesenchymal tumors, 12 lung carcinomas, 4 breast carcinomas) were examined and compared with DNA samples from 50 leukemia patients and 14 unrelated individuals without malignant neoplasms. When DNA samples were digested with a HindIII restriction endonuclease, Southern blot analysis demonstrated two distinct bands (21kb and 18kb) after hybridization to the SIS/PDGF-2 gene probe. A pedigree analysis of a 43-member family indicated that these allelic variants segregated in a Mendelian fashion. There was, however, tumor specific allele loss in 18% of the mesenchymal tumors analyzed, which may indicate a common etiology for this tumor type.     

Analysis of polymorphic DNA markers cosegregation with arterial blood pressure in ISIAH hypertensive rats]

A search for DNA markers of hereditary arterial hypertension in ISIAH rats was performed by means of contemporary molecular genetic approaches. The backcross rat population used for the analyses was derived from a cross of the Wistar x ISIAH F1 progeny with the Wistar rats. Hybridization of the HaeIII-digested DNA samples with the (CAC)5 microsatellite probe revealed cosegregation of the basal arterial pressure value with the 4.8-kb polymorphic DNA fragment. Examination of the DNA polymorphism by means of polymerase chain reaction with arbitrary primers showed an association of the 700-bp polymorphic DNA fragment with the increase of arterial blood pressure under conditions of emotional stress.     

利用RAPD和ISSR标记分析烤烟品种间遗传关系

利用RAPD和ISSR标记对22份烤烟(Nicotiana tabacumL.)品种进行了遗传关系研究。在RAPD分析中筛选到13个引物,共扩增出167条带,其中多态性带50条,多态性比率为29.9%;在ISSR分析中筛选出7个引物,共扩增出96条带,其中多态性带44条,多态性比率为45.8%。两种标记相结合估算出的品种间遗传相似系数在0.881~0.979之间,平均为0.933。单独基于RAPD标记和ISSR标记的聚类结果有一定差异;两种标记结合起来的聚类分析结果与系谱信息吻合程度更高。定向选择可能对烤烟品种间遗传关系有较大影响;国外引进品种与国内育成品种并未完全分开,表明分子水平的遗传关系和地理来源间缺乏必然联系。     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

Exclusion of candidate genes for canine SRY-negative XX sex reversal

In mammals, the Y-linked SRY gene is normally responsible for testis induction, yet testis development can occur in the absence of Y-linked genes, including SRY. The canine model of SRY-negative XX sex reversal could lead to the discovery of novel genes in the mammalian sex determination pathway. The autosomal genes causing testis induction in this disorder in dogs, humans, pigs, and horses are presently unknown. In goats, a large deletion is responsible for sex reversal linked to the polled (hornless) phenotype. However, this region has been excluded as being causative of the canine disorder, as have WT1 and DMRT1 in more recent studies. The purpose of this study was to determine whether microsatellite marker alleles near or within five candidate genes (GATA4, FOG2, LHX1, SF1, SOX9) are associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal. Primer sequences flanking nucleotide repeats were designed within genomic sequences of canine candidate gene homologues. Fluorescence-labeled polymorphic markers were used to screen a subset of the multigenerational pedigree, and marker alleles were determined by software. Our results indicate that the mutation causing canine SRY-negative XX sex reversal in this pedigree is unlikely to be located in regions containing these candidates.     

Polymorphic CAC/T repetitive sequences in the pig genome 1

Three genomic clones were isolated from a sizeselected pig DNA library by hybridization with a DNA-fingerprint probe. Analysis at the sequence level revealed that all three clones contain interrupted stretches of triplet repeats mainly composed of CAC and CAT triplets. Evaluation of the corresponding loci for polymorphism by Southern blot hybridization showed considerable length variation. For two loci the polymorphism was also demonstrated by polymerase chain reaction (PCR) amplification. The PiGMaP reference pedigree was typed for all three loci.     

Genetic Diversity and Relationship of Yunnan Native Cattle Breeds and Introduced Beef Cattle Breeds

In this study, random amplified polymorphic DNA (RAPD) analysis was used to estimate genetic diversity and relationship in 134 samples belonging to two native cattle breeds from the Yunnan province of China (DeHong cattle and DiQing cattle) and four introduced beef cattle breeds (Brahman, Simmental, MurryGrey, and ShortHorn). Ten primers were used, and a total of 84 bands were scored, of which 63 bands (75.0%) were polymorphic. The genetic distance matrix was obtained by proportions of shared fragment. The results indicate that the Yunnnan DeHong cattle breed is closely related to the Brahman (Bos indicus), and the Yunnan DiQing cattle breed is closely related to the Simmental, ShortHorn, and MurryGrey (Bos taurus) breeds. Our results imply that Bos indicus and Bos taurus were the two main origins of Yunnan native cattle. The results also provide the basic genetic materials for conservation of cattle resources and crossbreeding of beef cattle breeds in South China.     

Evaluation of AFLPs for germplasm fingerprinting and assessment of genetic diversity in cultivars of tomato (Lycopersicon esculentum L.).

Cultivated tomato (L. esculentum L.) germplasm exhibits limited genetic variation compared with wild Lycopersicon species. Amplified fragment length polymorphism (AFLP) markers were used to evaluate genetic variation among 74 cultivars, primarily from California, and to fingerprint germplasm to determine if cultivar-specific patterns could be obtained. All 74 cultivars were genotyped using 26 AFLP primer combinations; of the 1092 bands scored, 102 AFLP bands (9.3%) were polymorphic. Pair-wise genetic similarity coefficients (Jaccard and Nei-Li) were calculated. Jaccard coefficients varied from 0.16 to 0.98 among cultivar pairs, and 72% of pair-wise comparisons exceeded 0.5. UPGMA (unweighted pair-group method with arithmetic averaging) clustering and principle component analysis revealed four main clusters, I-IV; most modern hybrid cultivars grouped in II, whereas most vintage cultivars grouped in I. Clusters III and IV contained three and two cultivars, respectively. Some groups of cultivars closely related by pedigree exhibited high bootstrap values, but lower values (<50%) were obtained for cluster II and its four subgroups. Unique fingerprints for all 74 cultivars were obtained by a minimum of seven AFLP primer pairs, despite inclusion of some closely related cultivars. This study demonstrated that AFLP markers are effective for obtaining unique fingerprints of, and assessing genetic diversity among, tomato cultivars.     

转基因抗虫棉SSR和AFLP遗传变异研究

利用SSR和AFLP两种分子标记技术,分析了52份转基因抗虫棉品种（系）的遗传多样性。结果表明：在61对SSR引物中,有4对引物在供试材料中表现出多态性,共扩增出102个标记,其中多态性标记25个,多态性百分率为24．51％,每对引物的扩增带数变化在17～30之间;在100对AFLP引物中,有9对引物在供试材料中产生多态性,共扩增出618个标记,多态性标记33个,占总数的5．34％,每对引物组合扩增的标记数分布于47～81之间。成对品种的欧式距离变化在2．00～5．57之间,平均值为4．21,单一品种欧氏距离的平均值分布在3．73～4．75之间,表明不同品种之间遗传差异不大。基于SSRs和AFLPs多态性数据的聚类分析,可以将供试材料划分为3个类群（SAGs）,但类群划分与品种地理来源不十分吻合。     

SSR标记对不同黄籽甘蓝型油菜亲本材料的遗传分析

杂交育种中,亲本选配是育种成败的关键。本研究以重庆市油菜工程技术研究中心提供的180份甘蓝型黄子油菜亲本种质为材料,应用分布于不同连锁群的60对SSR标记进行了分析,共检测出308个标记位点,每对引物在不同亲本材料之间的等位基因数在1～11个之间,平均位点为5.1个。其中多态性位点207个,多态率达67.2%。对SSR扩增结果进行UPGMA分析,在遗传距离0.566处,180个品种(系)分为3个类群,聚类结果与种质来源比较一致,本研究为甘蓝型油菜黄子杂交育种和优势组合的选配提供了理论依据。     

Bkm sequences are polymorphic in humans and are clustered in pericentric regions of various acrocentric chromosomes including the Y

Summary Probes of uncloned Bkm satellite DNA and a Drosophila clone 2(8), consisting mainly of GATA repeasts related to a major sequence component in Bkm, have been used to probe Southern blots of human male and female DNAs obtained from a Caucasian and an Australian aboriginal population and to human chromosomes in situ. Hybridization was observed to a distinct and an indistint series of bands against a smeared background. The same distinct bands are identified in the DNA samples with both probes, but are most readily detected using the uncloned Bkm probe. Most restriction bands are common to both populations and some are polymorphic. However, certain bands appear to be characteristic of the Australian aboriginal samples. There are no distinct sex-linked patterns. However all of the small acrocentric human chromosomes, including the Y chromosome show hybridization to uncloned Bkm in situ.     

家蚕胚胎细胞系的DNA指纹图谱分析

在建立可靠的家蚕细胞系基因组DNA制备和PCR扩增技术体系的基础上,筛选具有稳定多态性位点的RAPD和ISSR引物,建立家蚕细胞系基因组DNA的ISSR和RAPD分子标记技术体系,检测家蚕细胞系的DNA分子标记多态性,构建细胞系的DNA指纹图谱。筛选出了26个ISSR引物和43个RAPD引物,通过PCR扩增在家蚕胚胎细胞系和传代昆虫细胞系等9个样品中分别获得了797条和1205条多态性条带,多态性达到89．9%和76．6%,不同细胞系的DNA多态性有较大差异,三个家蚕胚胎细胞系具有各自特有的DNA标记。测定了9个样品间的Nei's相似系数和遗传距离,构建了系统发育树,结果表明本实验室建立的3个家蚕胚胎细胞系和家蚕“夏芳×秋白”聚为一簇,亲缘关系较近,而来自不同物种的五个传代昆虫细胞系聚为一簇,它们之间的遗传距离比3个家蚕胚胎细胞系之间的遗传距离更小。     

Use of RAPD markers for identifying Nelore bulls with early reproductive maturation onset

Random amplified polymorphic DNA (RAPD) markers were used for identifying animals with early (precocious) or late (non-precocious) reproductive maturation onset. Animals (n=34) were phenotyped according to spermatozoa appearance in ejaculates (group A, 20 animals) or to the expected progeny difference (EPD) values for scrotal circumference (group B, 14 animals). The RAPD markers were initially detected by amplifying two pooled samples of equimolar amounts of DNA from the eight precocious 12 and non-precocious animals of group A. Only 38 out of 320 random primers used for screening group A pooled samples detected polymorphisms. These polymorphic primers generated 443 distinguishable and reproducible bands, from which 174 were polymorphic and 269, monomorphic. These polymorphic primers were then used in RAPD reactions to amplify individual DNA samples from animals belonging to both groups, A and B. The dendrograms generated from RAPD patterns allowed phenotypic class differentiation in both cases. Therefore, RAPD markers can be used as a tool for identifying genotypes favoring early sexual maturation in Nelore breeding programs.     

Assessment of genetic instabilities induced by tissue culture in alkaligrass

Alkaligrass (Puccinellia chinampoensis Ohwi), one of the important forage grasses in saline-alkalieroded grasslands, has been proved to be invaluable for improving saline-alkali soils. However, little is known of its genetic instabilities during in vitro culture for its artificial breeding. In this paper, a simple and efficient regeneration system of mature seed-induced calli in alkaligrass was established, and the somaclonal variation in the regenerated plants was assessed by inter-simple sequence repeat (ISSR) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers. 18 randomly chosen regenerants were subjected to ISSR and REMAP analysis with the shoot from the same grain of seed as the control. ISSR analysis showed that of the 145 scored bands, 13 were polymorphic among the analyzed samples, giving rise to a genetic variation frequency of 8.97%. REMAP analysis revealed that 4 out of 127 scored bands were polymorphic, a genetic variation frequency of 3.15% occurred. Cluster analysis indicated that the genetic similarity index calculated on the basis of ISSR data or REMAP data among the 18 regenerated plants and the donor plant was 0.974 and 0.996 respectively. All the results confirmed that somaclonal variation was induced by tissue culture in alkaligrass at a higher frequency, and indicated that the regeneration system could be a viable option for genetic improvement of alkaligrass by biotechniques.