应用RAPD标记进行茶树优异种质遗传多态性、亲缘关系分析与分子鉴别

采用RAPD分子标记技术,对茶树优异种质资源的遗传多态性、亲缘关系和分子鉴别进行了研究。结果表明,20个引物在15份优异资源中得到1050个RAPD位点,平均52．5个位点／引物,70个位点／资源。在所获得的137条可重现谱带中,8条是单态的,129条是多态的,多态性程度达94．2％;引物的多态性相对频度为0．24～0．83,总平均为0．47;遗传距离在0．16～0．62之间,平均为0．37,这可能与我国是茶树的原产地和起源中心有密切关系。RAPD数据的类平均法聚类结果显示,15份资源可划分为3个类群,从相似性系数讨论了资源间的亲缘关系。应用12个引物产生的20个特异标记的存在和11个特异的缺失,可以鉴别所有15份优异茶树种质资源。RAPD可以作为茶树优异种质资源遗传多态性、系统演化和分子鉴别研究的有效手段之一。     

茶树种质资源遗传多样性的RAPD分析

用随机扩增多态DNA（RAPD）标记对来自全国产茶省的31份种质资源、福建地区的10份种质资源以及湖南安化云台山种有性后代居群的30个单株的遗传多样性进行了检测。不同生态条件下的31份不同种质资源中,21个RAPD引物扩增出188条谱带,多态性比率为90.43%;福建10份种质资源,21个RAPD引物共扩增出154条谱带,多态性比率为81.81     

Identification of commercial chicory cultivars for hydroponic forcing and their phenetic relationships revealed by random amplified polymorphic DNAs and amplified fragment length polymorphisms

One‐hundred and twenty‐four amplified fragment length polymorphism (AFLP) and 49 random amplified polymorphic DNA (RAPD) markers have been used to distinguish between 20 and 23 commercial chicory cultivars, respectively. These were all Cichorium intybus var. foliosum F₁ hybrids, currently used in hydroponic forcing. Five‐hundred and twenty RAPD primers (OPERON) were tested, of which 156 resulted in reproducible patterns and 26 yielded polymorphisms. Two‐hundred and fifty‐six AFLP primer‐combinations were tested and six combinations were selected for identification purposes. Similarity indices were measured and clustering has been done using pairwise comparison. Both types of marker provide similar conclusions. Two major clusters are formed, representing late and early cultivars. All cultivars were identified using 10 informative RAPD primers or three AFLP primer combinations. A low degree of polymorphism was detected between some early cultivars, suggesting a narrow genetic base in their breeding strategy.     

Evaluation of the genetic diversity among elite tea (Camellia sinensis var. sinensis) accessions using RAPD markers

The diversity of 27 superior tea (Camellia sinensis var. sinensis) accessions from Korea, Japan and Taiwan was examined with RAPD-PCR (Random Amplified Polymorphic DNA Polymerase Chain Reaction) markers. Out of the 50 primers screened, 17 primers generated 58 polymorphic and reproducible bands. A minimum of 3 primers was sufficient to distinguish all the 27 accessions studied. The Shannon's index used to partition diversity into inter- and intra-group, revealed that 71 percent of variability resided within groups and 29 percent between groups. Diversity was greatest within the Korean group followed by Taiwan and Japan. The relatively high diversity observed in Korea might reflect the larger genetic base of its plantations while the low diversity in Japan could be explained by the long and intensive tea selection programme in this country. A dendrogram based on the UPGMA-link method using Jaccard's distances and multivariate Factorial correspondence analysis clustered the tea accessions into two main groups, regrouping the Taiwan cultivars on the one side and the Korean and Japanese accessions on the other side. This suggests that the Taiwan tea studied here may have a different origin from that of Korea and Japan. This revised version was published online in July 2006 with corrections to the Cover Date.     

石斛种质资源遗传多样性的RAPD分析

利用RAPD技术对10种石斛种质资源的遗传多样性及亲缘关系进行了分析。从100条10bp的RAPD引物中筛选获得17条多态性引物,对石斛属的10个种的基因组DNA进行扩增。共获得200条多态性带,平均每个引物产生11.8个多态性条带。材料间遗传相似系数变化范围为0.356~0.676。根据RAPD标记的结果,采用UPGMA法进行聚类分析,将石斛属的10个种区分开来,划分为4类。结果表明：RAPD标记技术较好地从分子水平上揭示石斛种质资源的遗传背景、亲缘关系。     

RAPD and SCAR markers for powdery mildew resistance gene er in pea

In pea, a single recessive gene (er) on linkage group 6 confers resistance to powdery mildew caused by Erysiphe pisi. The present study aims to identify molecular markers linked to the er gene. Screening of the powdery mildew‐resistant cultivar ‘DMR11’ and its susceptible nearisogenic line for polymorphism revealed linkage of two RAPD primers (OPO‐02 and OPU‐17) to the er gene and a sequence characterized polymorphic region (SCAR) primer, ScOPD‐10₆₅₀ with er in a population of 83 F₂ plants in the order: OPU‐17 ‐ er ‐ ScOPD‐10₆₅₀ ‐ OPO‐02. The markers ScOPD‐10₆₅₀ and OPU‐17 being coupled with the allele causing resistance would substantially increase the efficiency of marker‐assisted selection in peabreeding for powdery mildew.     

Homoeological relationships between the f chromosome of Brassica rapa and the e chromosome of Brassica oleracea

Eight plants of the putative double monosomic addition line (DMAL, 2n= 20) were developed by crossing a monosomic chromosome addition line of radish [f(A)‐type monosomic addition line (MAL) (2n= 19)] carrying the f chromosome of Brassica rapa (2n= 20, AA) with another [e(C)‐type MAL (2n= 19)] having the echromosome of Brassica oleracea (2n= 18, CC). The homoeological relationships between the two alien chromosomes were investigated by morphological, cytogenetic and random amplified polymorphic DNA (RAPD) analysis. Seventeen morphological traits that were not present in the radish cv. ‘Shogoin’ were observed in both MALs and these traits were substantially exhibited in DMAL plants. At the first metaphase of pollen mother cells (PMCs), the two parental MALs showed a chromosome configuration of 9II +1I, demonstrating impossibility of recombination between the R and the added chromosomes. The DMALs formed 10II in approximately 73% of PMCs, with one bivalent showing loose pairing between two chromosomes differing in size. In an attempt to identify the two MALs by RAPD‐specific markers using 26 selected random primers, 13 and 20 bands were specific for the f(A)‐type and the e(C)‐type MALs, respectively; 12 bands were common to both MALs (26.7%). In conclusion, the f chromosome of B. rapa is homoeologous to the e chromosome of B. oleracea. The genetic domain (genes) for 17 morphological traits are linked to each homoeologous chromosome bearing 27% of the corresponding RAPD markers.     

Morphological traits and high resolution RAPD markers for the identification of the main strawberry varieties cultivated in Argentina

Eight genotypes of the main Fragaria×ananassa cultivars grown in Argentina were analysed using the random amplified polymorphic DNA (RAPD) technique combined with electrophoresis in polyacrylamide gels. The high resolution of this procedure allowed the detection, with only 13 random primers, of 37 genotype‐specific bands that can be used as markers for verifying the identity of cultivars. By using this approach, three different accessions of the cultivar ‘Pájaro’ exhibited differences in amplification profiles, confirming the need for DNA analysis to prevent misidentification of cultivars. In addition, RAPD bands and morphological traits were used to assess genetic relatedness among cultivars. Comparison of both dendrograms revealed that there is no correlation between the clustering obtained with molecular and morphological characters.     

Identification of PCR-based markers linked to the powdery-mildew-resistance gene Pl1 from Malus robusta in cultivated apple

The availability of molecular markers linked to mildew resistance genes would enhance the efficiency of apple-breeding programmes. This investigation focuses on the identification of random amplified polymorphic DNA (RAPD) markers linked to the Pl₁ gene for mildew resistance, which has introgressed from Malus robusta into cultivated apples. The RAPD marker technique was combined with a modified ‘bulked seg-regant analysis’ mapping strategy. About 850 random decamer primers used as single primers or in combinations were tested by PCR analysis on the basis of resistant and susceptible DNA pools. Selected primers producing RAPD fragments were applied in an additional selection step to M. robusta and genotypes representing intermediate breeding stages of the breeding population 93/9, for which a 1:1 segregation could be observed for the resistance trait. Seven RAPD markers, all representing introgressed DNA sequences from M. robusta, were identified and arranged with the Pl₁ locus in a common linkage group. The two most tightly-linked RAPD markers, OPAT20₄₅₀ and OPD2₁₀₀₀ were mapped with a genetic distance of 4.5 and 5 cM, respectively, from the Pl₁ gene. Both markers are suitable for marker-assisted selection in apple breeding. The polymorphic DNA fragment OPAT20₄₅₀ was cloned and sequenced, and longer primers for the generation of a sequence-characterized amplified region (SCAR) marker have been constructed; this marker was easier to score than the original RAPD marker.     

Genetic diversity among tea cultivars from China, Japan and Kenya revealed by ISSR markers and its implication for parental selection in tea breeding programmes

Tea plant [Camellia sinensis (L.) O. Kuntze] is an important beverage crop in the world. In recent years many clonal tea cultivars have been released, and they play major roles in improving the production and quality of tea. It is important to understand the genetic diversity and relatedness of these cultivars to avoid inbreeding and narrow genetic basis in future tea breeding. In the present study, genetic diversity and relationship of 48 tea cultivars from China, Japan and Kenya were evaluated by inter‐simple sequence repeat (ISSR) markers. A total of 382 ISSR bands were scored, of which 381 (99.7%) were polymorphic. The ISSR primers showed high ability to distinguish between tea cultivars according to their high Resolving Power (R_P) with an average of 7.4. The mean of Nei’s gene diversity (H) and Shannon’s information index (I) were 0.22 and 0.35, respectively. More abundant diversity was revealed among cultivars in China than those in Japan and Kenya. Within Chinese populations, the level of diversity in east China was higher than that in other regions. The coefficient of genetic differentiation (G_ST) was 0.202, which indicates a high degree of genetic variation within populations. This result was further confirmed by analysis of molecular variance, which revealed the variance component within the populations (92.07%) was obviously larger than that among populations (7.93%). The level of gene flow (N_m) was estimated to be 2.0. This could be explained by frequent natural cross‐pollination and seed dispersal among tea populations. The pairwise similarity coefficient between the cultivars varied from 0.162 to 0.538. A dendrogram of 48 tea cultivars was constructed where all the tested cultivars were divided into two groups. Our data show that the genetic relationship among tea cultivars can be determined by the ISSR markers. This will provide valuable information to assist parental selection in current and future tea breeding programmes.     

Analysis of molecular variance and population structure in southern Indian finger millet genotypes using three different molecular markers

The genetic relationship among 42 genotypes of finger millet collected from different geographical regions of southern India was investigated using random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), and simple sequence repeats (SSR) markers. Ten RAPD primers produced 111 polymorphic bands. Five ISSR primers produced a total of 61 bands. Of these, 23 bands were polymorphic. The RAPD and ISSR fingerprints revealed 71.3 and 37.4% polymorphic banding patterns, respectively. Thirty-six SSR primers yielded 83 scorable alleles in which 62 were found to be polymorphic. Out of 36 SSR primers used, 14 primers (46.6%) produced polymorphic bands. The SSR primer UGEP7 produced a maximum number of six alleles. Mean polymorphic information content (PIC) of RAPD, ISSR and SSR were 0.44, 0.28, and 0.14, respectively. Molecular variances among the population were 2, 11, and 1% for RAPD, ISSR, and SSR markers, respectively. SSR produced 99% molecular variance within individuals. RAPD and ISSR markers produced a low level of molecular variance within individuals. The STRUCTURE (model-based program) analysis revealed that the 42 finger millet genotypes could be divided into a maximum of four subpopulations. Based on the Bayesian statistics, each RAPD and SSR marker produced three subpopulations (K=3), while ISSR marker showed four subpopulations (K=4). This study revealed that RAPD and SSR markers could narrow down the analysis of population structure and it may form the basis for finger millet breeding and improvement programs in the future.     

Assessment of genetic relationships among Cucumis spp. by SSR and RAPD marker analysis

The first successful production of a sterile interspecific hybrid obtained from a cross between Cucumis hystrix Chakr. (2n = 2x = 24) and Cucumis sativus var. sativus L. (2n =2x = 14), and its subsequent fertility restoration through chromosome doubling provide an effective means for investigating genetic relationships among Cucumis spp. In this study, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to investigate relationships among C. s. var. sativus L., C. s. var. hardwickii (R.) Alef., C hystrix, C. hytivus Chen & Kirkbride (the amphidiploid species from chromosome doubling of the C. sativus x C. hystrix interspecific hybrid, 2n = 38), C. melo (2n =2x = 24) and C. metuliferus Meyer and Naudin (2n =2x= 24). A total of 109 SSR bands and 398 RAPD primed sites were used to calculate Jaccard's distance coefficients for cluster analysis using a unweighted pair‐group method using an arithmetic averaging (UPGMA) algorithm. The genetic relationships identified using SSR and RAPD markers were highly concordant, such that the correlation between SSR and RAPD genetic distance (GD) estimates was r = 0.94. SSR and RAPD analysis of 22 accessions allowed for their grouping into two distinct groups designated as CS and CM. While group CS consisted of 11 C. sativus genotypes, and the C. hytivus and C. hystrix accessions, group CM included six C. melo genotypes and C. metuliferus. The GD values between C. hystrix and C. sativus ascribed by SSR and RAPD matrices were 0.59 and 0.57, respectively. These GDs were smaller than those detected between C. hystrix and C melo (0.87 and 0.70 derived from SSR and RAPD markers, respectively).     

Development and application of SCAR markers for sex identification in the dioecious species <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

There is an urgent need for early sex identification to support field planting in Ginkgo biloba L., due to the different economic and medicinal values between male and female trees. An easy, rapid and reliable molecular method for sex type determination of G. biloba was reported in the paper. Random amplification of polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) were used to search for specific molecular markers linked to the sex locus. A total of 48 primers were used for screening of specific RAPD markers in six male and three female samples. Only one primer, S10, showed different amplification band patterns associated with sex types. Then the sex-specific bands, S10-BandA and S10-BandB, were cloned and sequenced. Based on the sequences two pairs of SCAR primers, GBA and GBB, were designed. The GBA primers amplify a single 571 bp band in male samples but not in female samples, and DNA amplification using GBB primers could generate a 688 bp band only in the female individuals. Finally, the SCAR primers were used to test 16 sex-unknown samples. SCAR primers developed in this paper can be used as effective, convenient and reliable molecular markers for sex identification in G. biloba.     

Purity control of F1-hybrid tomato cultivars by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were applied in purity control of hybrid seed production of tomato (Lycopersicon esculentum Mill.). DNA from three commercial F₁-hybrid cultivars and their parental lines was subjected to RAPD screening with 50 primers. Two of four primers which detected polymorphism between the parents tested, generated paternal-specific RAPDs, enabling a clear distinction to be made between hybrids and their maternal parents. In addition, combination of the polymorphic DNA products generated by these primers exhibited hybrid-specific patterns, enabling each cultivar to be identified. This result indicates the practical usefulness of RAPD markers in hybrid-tomato-seed purity-control tests and cultivar identification. The approach is advantageous in its rapidity and simplicity, particularly as an alternative for those cultivars for which lengthy and costly phenotypic tests are currently used.     

Assessment of genetic diversity in cabbage cultivars using RAPD and SSR markers

Genetic relationship and diversity among seven cabbage cultivars were analyzed using RAPD and SSR markers. These cultivars are of great commercial value in India and are confirmed for their reaction to black rot caused by Xanthomonas campestris pv. campestris. However, so far the extent of genetic diversity and relatedness has not been studied in these cultivars. A total of 17 selected RAPD primers generated 90 bands, 76 of which were polymorphic (84.44%). In addition, 27 selected SSR primers generated 67 amplified bands with 59 of which were polymorphic (87.6%). Though both the marker techniques were able to discriminate the cultivars effectively, analysis of combined data of markers (RAPD and SSR) resulted in better distinction of cultivars. By combining both the markers, a total of 157 bands were detected of which 135 bands (85.98%) were polymorphic, i.e. an average of 5.95 bands per primer. High level of polymorphism (> 85%) recorded with two different marker systems indicated a high level of genetic variation existing among the cultivars. Genetic relationship estimated using similarity co-efficient (Jaccard’s) values between different pairs of cultivars varied from 0.21 to 0.77 in RAPD, 0.42 to 0.82 in SSR, and 0.43 to 0.89 with combined markers. A high correspondence had been recorded between the values of genetic variations generated by UPGMA, clustering, and scatter plot diagrams. The cultivars ‘January King Sel. Improved’ and ‘Golden Acre’ are highly divergent cultivars as demonstrated by both the marker systems.     

Genetic relationship of Mediterranean mandarins (Citrus deliciosa Tenore) using RAPD markers

Summary Random amplified polymorphic DNA (RAPD) analysis was carried out to evaluate polymorphism and genetic similarity between 39 Mediterranean mandarin genotypes. One hundred eleven amplification products were identified using 21 random primers. An average of 2.2 RAPD markers was obtained for each primer, corresponding to 42% of the amplification products. Genotype-specific RAPD markers were also found, mainly in known hybrids. UPGMA cluster analysis revealed the low level of genetic variation between accessions of Mediterranean mandarins, whereas their hybrids with other Citrus species showed greater genetic dissimilarity. Twenty accessions yielded very similar patterns, suggesting either that they could be a single clone, or that the technique was not able to detect genomic variation. However, for the other specimens genetic polymorphism can easily be detected by RAPD, although the genetic variation between accessions was quite low. The large number of hybrids and the low polymorphism between accessions support the hypothesis that Mediterranean mandarins are all true hybrid of Common mandarins (Citrus reticulata Blanco).     

利用DNA分子標誌鑒別台灣茶樹品種及評估種原之遺传歧異性

爲評估台灣茶樹種原之遺傳歧異性,本硏究由100條ISSR引子中篩選出12條可産生多型性條帶明顯的引子,這些引子共可産生67個的多型性條帶罁恳环N原之分子標誌數據進行UPGMA法分群分析結果,可將台灣133個茶樹種原區分成六大群,包括油茶群、赤芽山茶群、野生茶樹群、大葉變種與小葉變種混合群、大葉、小葉及大葉、小葉雜交種混合群及小葉變種群。而主成分向量分析的結果與利用群聚分析得到的親緣關係樹形圖結果相符合。台灣茶樹種原高比例的遺傳歧異度是由台灣的野生茶樹所貢獻,部分重要栽培種間的相似性仍極高。爲了探討制茶過程對分子級品種鑒定之影響及DNA分子標誌應用于成茶品種鑒定之可行性,本硏究分析不同發酵程度的茶類,在制茶過程中對DNA質量之影響,試驗結果顯示高溫殺菁過程嚴重造成成茶DNA的降解。利用各種類別成茶與新鮮茶葉(對照)所抽取之DNA樣品進行PCR擴增反應,結果發現分子量小於1,000bp的ISSRDNA條帶表現較穩定。     

Analysis of genetic diversity in Agave tequilana var. Azul using RAPD markers

By federal law in Mexico, A. tequilana Weber var. Azul is the only variety of agave permitted for the production of any tequila. Our objective was to assay levels of genetic variation in field populations of A. tequilana var. Azul using randomly amplified polymorphic DNA (RAPD) markers. Ten plants were collected from each of four different fields, with two fields being located in each of two principal regions of Mexico for the cultivation of A. tequilana var. Azul. The two regions are separated geographically by approximately 100km. Genetic relationships between A. tequilana var. Azul and two other varieties of A. tequilana Weber, ‘Chato’ and ‘Siguin’, were also investigated using RAPDs. Among the three varieties, 19 decamer primers produced 130 markers, of which 20 (15.4%) were polymorphic betweenA. tequilana var. Chato and A. tequilana var. Siguin. The results of RAPD analysis suggest that A. tequilana var. Siguin is more closely related to A. tequilana var. Azul than is A. tequilana var. Chato. Among the 40 field selections of A. tequilana var. Azul, only 1 of124 RAPD products (0.8%) was polymorphic and 39 of 40 plants were completely isogenic. This is one of the lowest levels of polymorphism detected to date for the analysis of a crop species, and is proposed to be the result of the promotion of a single conserved genotype over many years due to an exclusive reliance on vegetative propagation for the production of new planting materials. The significance of these results is discussed in relation to breeding programs focused on the improvement of A. tequilana var. Azul. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of random amplified polymorphic DNA (RAPD) markers in Hevea brasiliensis

The applicability of random amplified polymorphic DNA (RAPD) markers in the cultivated rubber tree, Hevea, was evaluated using 43 decamer oligonucleotide primers in a set of 24 clones selected in different South-East Asian countries. A total of 220 0.35–3.5 kb DNA fragments were amplified, of which 111 were polymorphic. Of these, 80 fragments (RAPD markers) which were repeatable and clearly scorable across all genotypes were used to estimate genetic distances among the clones tested. The estimated genetic distances ranged from 0.05 (RRII 308 and PB 5/51) to 0.75 (RRIC 100 and SCATC 88–13). A mean genetic distance of 0.5 indicates a rather high genetic variability among the tested clones. As expected, because of the breeding history of Hevea, UPGMA cluster analysis and Principal Coordinate Analysis (PCoA) indicated the absence of a distinct geographical grouping. The possible application of RAPD markers for clone identification and also for analysis of genetic relationships among Hevea clones is discussed.     

黄瓜序列特征性扩增区域标记(SCAR)的开发

黄瓜的分子标记连锁图谱研究已经到了比较和整合的阶段,然而可用于黄瓜作图的锚定标记却并不多.本研究利用华北类型和欧洲温室型黄瓜自交系S94和S06作为PCR模板,通过将二者差异的RAPD和SRAP条带克隆、测序并根据测序结果设计特异引物,成功获得了118个SCAR标记.经4个典型黄瓜种质材料PCR验证,引物多态性比例高达10%以上.本研究获得的SCAR可望用于黄瓜遗传图谱的构建和整合.