耐盐绒毛白蜡特异性SCAR分子标记的鉴定

该研究以耐盐型和盐敏感型绒毛白蜡及其F1代为材料,采用混合品系分析法进行RAPD分析。结果显示:在随机选取的150个10碱基随机引物中,仅有引物S20在耐盐基因池和盐敏感基因池间扩增出特异而可重复的592bp的多态性片段,命名为S20-592。获得的RAPD标记S20-592经克隆、测序、重新设计一对特异性引物转化成更稳定的SCAR标记。通过F1代个体验证,耐盐型个体均能扩增出此差异条带而盐敏感型个体中不能扩增出此差异条带,证明该SCAR标记的特异引物可用于耐盐绒毛白蜡物种的快速分子鉴定。     

Distinction between cold-sensitive and -tolerant jute by DNA polymorphisms

Jute is the principal coarse fiber for commercial production and use in Bangladesh. Therefore, the development of a high-yielding and environmental-stress tolerant jute variety would be beneficial for the agro economy of Bangladesh. Two molecular fingerprinting techniques, random-amplified polymorphic DNA (RAPD) and amplified-fragment length polymorphism (AFLP) were applied on six jute samples. Two of them were cold-sensitive varieties and the remaining four were cold-tolerant accessions. RAPD and AFLP fingerprints were employed to generate polymorphism between the cold-sensitive varieties and cold-tolerant accessions because of their simplicity, and also because there is no available sequence information on jute. RAPD data were obtained by using 30 arbitrary oligonucleotide primers. Five primers were found to give polymorphism between the varieties that were tested. AFLP fingerprints were generated using 25 combinations of selective-amplification primers. Eight primer combinations gave the best results with 93 polymorphic fragments, and they were able to discriminate the two cold-sensitive and four cold-tolerant jute populations. A cluster analysis, based on the RAPD and AFLP fingerprint data, showed the population-specific grouping of individuals. This information could be useful later in marker-aided selection between the cold-sensitive varieties and cold-tolerant jute accessions.     

Characterization of Zebu cattle breeds in Tanzania using random amplified polymorphic DNA markers

A total of 141 short primers, of arbitrary nucleotide sequence, were used singly in poly-merase chain reactions to amplify DNA fingerprints in pools of DNA representing three Zebu cattle breeds. Two primers, which discriminated between the breed-specific DNA pools were used further to amplify individual pool components in order to establish band frequencies of the amplified fingerprints. One of the primers (ILO 1127) amplified a RAPD fingerprint in 61%of TSZ animals but less than 6% in the other breeds, while another primer (ILO 1065) revealed a DNA sequence common to 89% of the Boran animals and less than 30% in the other two breeds. Bandsharing and mean average percentage difference calculated within and between the three breeds using RAPD fingerprint data showed a higher degree of homogeneity within than across the breeds and indicated measurable divergence between the three breeds. It is concluded that RAPD polymorphisms are useful as genetic markers for cattle breed differentiation.     

木耳交配型混合集群RAPD分析

用RAPD-混合集群分析来检测异宗结合担子菌不同交配型间的多态性。来源于同一木耳Anriculariaauricula（L．exHook．）Underw,子实体的10个单孢萌发而成的单核体进行RAPD分析证实,它们之间具有极高的同源性。将单核体按其所属的交配型分为两类,每类各5个,构成A1,A2两个基因池。用33种单引物,20种双引物对其进行扩增、分析,发现引物OPE19号能在这两个基因池间产生多态性,其中一条特异性谱带在A2基因池扩增产物中存在,在A1基因池中不存在。推测这一条谱带可能是与A2交配型基因连锁的分子标记。此研究结果不仅为由单因子控制的二极性担子菌的交配型测定提供科学依据,而且为RAPD技术在异宗结合担子菌极性研究中的应用展示了可喜的前景。     

Recombinant bovine interferon alpha causes only a temporary prolongation of the lifespan of the corpus luteum

Abstract

A method is described for developing a sheep‐ vs. goat‐specific DNA marker using sequence characterized amplified regions (SCARs) derived from a random amplified polymorphic DNA (RAPD) marker from sheep DNA samples. A sheep 645 bp DNA fragment that was absent in goat DNA was identified by analyzing pools of sheep and goat DNA with RAPD primers. This fragment was cloned and partially sequenced to design extended, strand‐specific 24‐mer oligonucleotide primers. Each primer contained the original 10 bases of the RAPD primer and the following 14 internal bases. The pair of primers resulted in the amplification of a single band of 645 bp when used to amplify sheep DNA, and in no amplification when used to amplify goat DNA. These SCAR primers successfully amplified the equivalent of DNA from one nucleated sheep cell in a sample of 5000 nucleated goat cells. This level of sensitivity is especially desirable for research involving the detection of interspecific chimerism.     

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.     

A directed search for DNA sequences tightly linked to cereal cyst nematode resistance genes in Triticum tauschii.

An improved system for identifying DNA sequences linked to a targeted region was developed by fractionating DNA sequences prior to polymerase chain reaction (PCR) analysis. In an attempt to identify DNA markers linked to a strong CCN resistance gene, Ccn-D1, in Triticum tauschii, DNA samples from individuals homozygous for resistance and susceptibility at the Ccn-D1 locus in a segregating progeny were bulked separately to produce "near isogenic" DNA pools. The polymerase chain reaction was employed to generate several DNA amplification products from each of the bulked DNA segregants using 240 random (RAPD) and 4 semirandom (consensus sequences of intron-splice junctions) primers. A DNA polymorphic fragment was apparent between the resistant and susceptible bulks using one of the semirandom primers. Hydroxylapatite chromatography of reannealed DNA (to Cot values > 100) was used to enrich low copy DNA sequences in the bulk DNA segregants (resistant and susceptible DNA pools). PCR analysis on the low copy enriched DNA pool increased the level of polymorphism detected between bulked segregants. One of the RAPD fragments present in only the resistant low copy DNA pool was cloned and mapped to the distal region of the long arm of chromosome 2D. By using the cloned RAPD fragment, csE20-2, to assay an RFLP locus in three independent F2 progenies, complete cosegregation was obtained with the Ccn-D1 locus. Joint segregation analysis from a genome-wide mapping of RFLP markers and a second CCN resistance in T. tauschii, Ccn-D2, showed this locus to be loosely linked to the proximal region of chromosome 2.     

大麻性别的RAPD和SCAR分子标记

利用随机扩增多态性DNA(randomamplifiedpolymorphicDNA,RAPD)技术获得与大麻性别连锁的分子标记.将10株雄性大麻或10株雌性大麻的单个DNA样品等量混合分别组成雄性或雌性DNA池(DNApool),以提供具有相同遗传背景的雌、雄性DNA样品.每个随机引物分别用三个不同的循环程序进行PCR扩增.在30个随机引物中,用引物401扩增得到一条约2.5kb雄性多态性片段.对该片段进行了克隆和序列分析,并根据序列分析结果将上述RAPD分子标记转化为重复性和特异性更好的SCAR(sequencecharacterizedamplifiedregions)分子标记.     

Assaying polymorphism at DNA level for genetic diversity diagnostics of the safflower (Carthamus tinctorius L.) world germplasm resources

Carthamus tinctorius (2n = 2x = 24), commonly known as safflower, is widely cultivated in agricultural production systems of Asia, Europe, Australia, and the Americas as a source of high quality vegetable and industrial oil. Twenty-two RAPD primers, 18 SSR primers, and 10 AFLP primer combinations were used to assess: (1) the genetic diversity of 85 accessions (originating from 24 countries) representing global germplasm variability of safflower and (2) the interrelationships among safflower ‘centers of similarity’ or ‘regional gene pools’ proposed earlier. The RAPD and SSR primers and AFLP primer combinations revealed 57.6, 68.0, and 71.2% polymorphism, respectively, among 111, 72, and 330 genetic loci amplified from the accessions. The sum of effective number of alleles (66.44), resolving power (59.16), and marker index (51.3) explicitly revealed the relative superiority of AFLP as a marker system in uncovering variation in safflower. Overall, AFLP markers could recognize ‘centers of similarity’ or ‘regional gene pools’. Analysis of molecular variance and Shannon’s information index provided corroborating evidences for the present and previous studies that concluded fragmentation of safflower gene pool into many gene pools. Divergent directional selection is likely to have played an important role in shaping the diversity. From the practical applications standpoint, the diversity of Iran–Afghanistan gene pool is very high, equivalent to the total diversity of the species. The Far East gene pool is the least diverse. The present comprehensive input, first of its own kind in safflower, will assist marker based improvement programmes in the crop.     

RAPD Analysis of Lathyrus sativus Somaclones

RAPD patterns were studied in seven somaclones of Lathyrus sativus having contrasting characteristics alongwith the parent cultivar P-24. Out of 81 decamer random primers used, 5 did not amplify and 24 revealed DNA polymorphism while the rest generated monomorphic banding patterns. Eight unique bands were amplified with different primers in four different somaclones. With most of the informative primers differences were observed between somaclones and also between some of the somaclones and parent cultivar P-24. More than 90% similarity in the RAPD patterns was evident among the somaclones and the parent cultivar P-24. Though it was not possible to identify a particular somaclone with a single primer, a combination of two or more primers could be employed to identify a somaclone.     

黄瓜霜霉病抗病基因的RAPD及SCAR标记

以感霜霉病黄瓜L18-10-2和抗霜霉病黄瓜129为亲本构建F2代分离群体,以F3代植株霜霉病抗性鉴定表示F2代各单株抗病性并得以区分各单株杂合或纯合感病性,采用RAPD技术和转SCAR的方法筛选黄瓜抗霜霉病基因分子标记.结果显示,在318条RAPD引物中有18条引物表现出两亲本间多态性,其中引物P18的SB-SP18561扩增片段与霜霉病抗病基因之间紧密连锁,根据交换率和Kosambi函数公式计算其遗传距离为7.85 cM.回收SBSP18561片段并克隆和测序,其准确长度为561 bp.将该RAPD标记转换为SCAR标记,长度为494 bp,命名为SSBSP18494.     

RAPD技术分析不同抗旱性苜蓿品种DNA的多态性

分别从不同抗旱性的紫花苜蓿品种中挑选抗旱性强的、抗旱性中等的和抗旱性弱的品种各三个,提取叶片基因组DNA,相同抗旱性苜蓿品种DNA等量混合构建三个池DNA。采用RAPD技术分析不同抗旱性混合DNA的多态性,并筛选出标记多态性的引物。结果表明：13组260个随机引物经五轮筛选,得到48个引物对不同抗旱性苜蓿品种池DNA扩增结果产生多态性,多态性引物占18．5％。其中5个引物能够稳定标记池DNA多态性,且特异性明显。表明不同抗旱性苜蓿品种之间具有明显的遗传多样性。     

Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated potato

The application of AFLPs, RAPDs and SSRs to examine genetic relationships in the primary northwestern European cultivated potato gene pool was investigated. Sixteen potato cultivars were genotyped using five AFLP primer combinations, 14 RAPD primers, and 17 database-derived SSR primer pairs. All three approaches successfully discriminated between the 16 cultivars using a minimum of one assay. Similarity matrices produced for each marker type on the basis of Nei and Li coefficients showed low correlations when compared with different statistical tests. Dendrograms were produced from these data for each marker system. The usefulness of each system was examined in terms of number of loci revealed (effective multiplex ratio, or EMR) and the amount of polymorphism detected (diversity index, or DI). AFLPs had the highest EMR, and SSRs the highest DI. A single parameter, marker index (MI), which is the product of DI and EMR, was used to evaluate the overall utility of each marker system. The use of these PCR-based marker systems in potato improvement and statutory applications is discussed.Abbreviations: PCR, polymerase chain reaction; AFLP, amplified fragment length polymorphism; RAPD, randomly amplified polymorphic DNA; DNA, deoxyribonucleic acid; EMR, effective multiplex ratio; DI, diversity index; MI, marker index; RFLP, restriction fragment length polymorphism.     

Identification of RAPD and ISSR markers for drought tolerance in wheat (Triticum aestivum L.)

Drought tolerance is the essential trait that needs to be incorporated in cereal crops, particularly those grown under the rainfed cultivation. Drought tolerance being contributed by several regions of the genome requires identification of these regions, using suitable molecular markers. Therefore, present investigation was aimed at analyzing the genetic diversity present among the cultivars of rainfed and the irrigated areas with respect to the drought tolerant trait. In all, 14 RAPD and 90 ISSR markers were used to identify these genomic regions. Out of 14 RAPD markers, one RAPD primer exhibited polymorphic banding pattern with 18.6 % polymorphism, clearly separating drought tolerant and drought susceptible genotypes. Out of 90 ISSR primers, only 3 ISSR primers revealed polymorphism in relation to the drought tolerance trait exhibiting 21.38 % polymorphism.     

Deciphering genetic diversity analysis of saffron (Crocus sativus L.) using RAPD and ISSR markers

The existence of genetic diversity in Crocus sativus has globally remained a mystery till date. The study investigated PCR based DNA amplification profile of saffron using ISSR and RAPD based primers. A total of 38 amplicons were generated by ISSR primers in the range from 7 to 12 with an average of 9.50 bands per primer. 20 bands were found to be polymorphic and 18 were monomorphic with an average percentage of polymorphism as 52.48%. RAPD based amplification revealed a total 161 amplicons, 107 as polymorphic and 54 as monomorphic with an average percentage of polymorphism as 66.44%. Cumulative results of RAPD and ISSR demonstrated that Nei-Li’s similarity index ranged between 0.70 and 0.97. The results of AMOVA has revealed 9% of variance among populations and 91% of variance within populations, Φ PT was found as 0.089, which indicates existence of genetic differences though limited. In conclusion, the results indicate that saffron accessions are minimally genetically differentiated, which could be capitalized in future breeding programmes to ameliorate this precious crop.     

Screening of RAPD Markers Linked to the Photoperiod-Sensitivity Gene in Rice Chromosome 6 Using Bulked Segregant Analysis

Bulked segregant analysis was used to determine randomly amplifiedpolymorphic DNA (RAPD) markers in a specific interval in themiddle of chromosome 6 of rice for tagging the photoperiod sensitivitygene.Two pools of F₂ individuals (japonica cv. Nipponbare and indicacv. Kasalath) were constructed according to the genotypes ofthree restriction fragment length polymorphism (RFLP) markerslocated at both ends and the middle of the targeted interval.Then another pair of pools were constructed based on the "graphicalgenotype," which was made with our high density linkage map.RAPD analysis was performed using these DNA pools as templates,and polymorphic fragments were detected and mapped. Using 80primers, either singlyor pairwise, we tested 2,404 primer pairsand established 14 markers tightly linked to the photoperiodsensitivitygene. The obtained RAPD markers were converted intosequence-tagged sites bycloning and sequencing of the polymorphicfragments and they can be used directlyfor construction of physicalmaps. This bulked segregant method can be applied for any speciesand any region of interest in which detailed linkage maps orphysical maps are needed.     

水稻抗褐飞虱基因SCAR标记的获得

采用282个随机引物对药用野生稻1665和栽培稻桂99远缘杂交的抗褐飞虱近等基因系B3F4分离群体的不抗池DNA和抗池DNA进行了特异性RAPD标记筛选,从中筛选到一个具有明显的特异性扩增带谱的RAPD标记S1159,序列分析表明,S1159序列长度为1408bp,与基因库中已报道的水稻第四号染色体的BAC克隆(编号OSJNBa0070O11)序列(67114-69100)有51.86%的同源性。为了提高所找到的RAPD标记S1159在应用上的稳定性,将RAPD标记转化为SCAR标记检测近等基因系群体,结果表明与RAPD标记结果一致,说明该研究得到的RAPD标记具有较好的稳定性和重复性,为进一步的研究打下了良好的基础。     

Development of random amplified polymorphic DNA markers for tropical tasar silkworm Antheraea mylitta

Antheraea mylitta (Drury) is a tropical tasar-silk producing insect. Its populations occupying different ecological and geographical regions show a certain degree of phenotypic variability, for which they are known as "eco-races." The eco-races are exploited for tasar silk production, and they are classified on the basis of their geographical distribution and morphology, which is often misleading when their systematic position is considered. To understand the genetic variability among the different eco-races, we used the random amplified polymorphic DNA (RAPD) method. Eighty random decamer primers were taken for RAPD amplifications. In total, 415 reproducible bands were used to generate a distance matrix, and for the subsequent clustering with unweighted pair-group method with arithmetic average. The number of polymorphic bands detected by each primer ranged from 5 to 24, with a mean value of 14.1 per primer. Percentage polymorphism was 81.9, and genetic distance values ranged from a minimum of 0.0108 between Modal and Nalia eco-races to a maximum of 0.0244 between Modal and Andhra local. The RAPD profiles obtained using A14, BC07, and C17 primers substantially differentiate all 10 commercially important eco-races, and the phylogenetic tree obtained from the data closely follows their geographical separations.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

大豆曲茎性状的遗传分析和RAPD标记研究

曲茎遗传材料NG94-156与遗传背景不同的3个正常茎品种杂交,获得1个F2群体和2个重组自交家系(F7:8)。后代分离分析的结果表明,NG94-156的曲茎性状受两对隐性重叠基因控制。利用4个亲本和1个重组自交家系筛选260个RAPD随机引物,其中有1个引物S-506扩增出的多态性条带有较好的重复性。经过连锁分析,RAPD标记S-5061600与控制曲茎的基因的遗传距离为6.94 cM。