Development of a novel and efficient strategy for practical identification of Pyrus spp (Rosaceae) cultivars using RAPD fingerprints

Accurate and reliable cultivar identification of crop species is essential to guarantee plant material identity for purposes of registration, cultivar protection and production. To facilitate identification of plant cultivars, we developed a novel strategy for efficient recording of DNA molecular fingerprints in genotyped plant individuals. These fingerprints can be used as efficient referential information for quick plant identification. We made a random amplified polymorphic DNA (RAPD) marker analysis of 68 pear cultivars. All pear genotypes could be distinguished by a combination of eight 11-mer primers. The efficiency of the method was further verified by correct identification of four cultivars randomly chosen from the initial 68. The advantages of this identification include use of fewer primers and ease of cultivar separation by the corresponding primers marked on the cultivar identification diagram. The cultivar identification diagram can efficiently serve for pear cultivar identification by readily providing the information needed to separate cultivars. To the best of our knowledge, this is the most efficient strategy for identification of plant varieties using DNA markers; it could be employed for the development of the pear industry and for the utilization of DNA markers to identify other plant species.     

A new strategy employed for identification of sweet orange cultivars with RAPD markers

We optimized RAPD techniques by increasing the length of RAPD primers and performing a strict screening of PCR annealing temperature to distinguish 60 sweet orange cultivars from the Research Institute of Pomology at the Chinese Academy of Agricultural Sciences. A new approach called cultivar identification diagram (CID) was used to improve the efficiency of RAPD markers for cultivar identification. Thirteen effective primers were first screened from 54 RAPD arbitrary 11-mer primers based on their amplification products and amplified polymorphic bands; they were then used for PCR amplification of all 60 cultivars. All cultivars were manually and completely separated by the polymorphic bands appearing in DNA fingerprints from 13 primers; a CID of the 60 sweet orange cultivars was then constructed. This CID separated all the cultivars from each other, based on the polymorphic bands; the corresponding primers were marked in the correct positions on the sweet orange CID. The CID strategy facilitates the identification of fruit cultivars with DNA markers. This CID of sweet orange cultivars will be very useful for the protection of cultivar rights and for early identification of seedlings in the nursery industry.     

A novel strategy for identification of 47 pomegranate (Punica granatum) cultivars using RAPD markers

DNA marker can be used for precise plant cultivar identification. However, DNA markers have often not been used effectively for the identification of plant cultivars due to a lack of an effective analysis strategy. We used a novel strategy for effective identification of plant individuals based on a new way of recording DNA fingerprints of the genotyped plants; a cultivar identification diagram can be manually generated and used as key reference information for quick identification of plant and/or seed samples. Forty-seven pomegranate varieties popularly cultivated in various provinces of China were subjected to RAPD marker analysis. Using the cultivar identification diagram strategy, they were clearly separated by the fingerprints of 11 RAPD primers. The utility and accuracy of the cultivar identification diagram analysis results were confirmed by the identification of three randomly chosen groups of cultivars among the 47 varieties.     

牡丹品种鉴定用ISSR引物的筛选与开发

用于牡丹品种鉴定的DNAISSR-PCR反应体系已经建立。利用DNAISSR分子标记分析少量牡丹品种时,容易获得各品种的特有ISSR标记。然而,中国牡丹品种约有1500个,在小批量品种范围内找到的品种特有ISSR标记有可能出现在其它品种中。因此,利用DNAISSR分子标记对数量庞大的中国牡丹品种进行区分和鉴定时,寻找品种特有标记成为突出的技术难题。标记是由引物通过PCR扩增产生的。因此,关键在于找到理想的ISSR引物。对已知的ISSR引物的筛选未获得良好的PCR扩增结果。报道牡丹鉴定用ISSR引物的设计与开发新途径。     

Identification and analysis of genetic variation among rose cultivars using random amplified polymorphic DNA

Identified germplasm is an important component for efficient and effective management of plant genetic resources. Traditionally, cultivars or species identification has relied on morphological characters like growth habit or floral morphology like flower colour and other characteristics of the plant. Studies were undertaken for identification and analysis of genetic variation within 34 rose cultivars through random amplified polymorphic DNA (RAPD) markers. Analysis was made by using twenty five decamer primers. Out of twenty five, ten primers were selected and used for identification and analysis of genetic relationships among 34 rose cultivars. A total of 162 distinct DNA fragments ranging from 0.1 to 3.4 kb was amplified by using 10 selected random decamer primers. The genetic similarity was evaluated on the basis of presence or absence of bands. The cluster analysis indicated that the 34 rose cultivars form 9 clusters. The first cluster consists of eight hybrid cultivars, three clusters having five cultivars each, one cluster having four cultivars, two clusters having three cultivars each and two clusters having one cultivar each. The genetic distance was very close within the cultivars. Thus, these RAPD markers have the potential for identification of clusters and characterization of genetic variation within the cultivars. This is also helpful in rose breeding programs and provides a major input into conservation biology.     

Genetic variability of introduced and local Spanish peach cultivars determined by SSR markers

A set of 94 peach cultivars including Spanish native peach and foreign commercial cultivars were analyzed using 15 SSR markers, selected for their high level of polymorphism. The number of alleles obtained varied from two to 11 with an average of 6.73 giving 185 different genotypes. All the cultivars showed a unique genetic profile, each one using different genotypic combination of all loci. BPPCT001 was the most informative locus showing also the highest discrimination power. Only six loci allowed the unambiguous separation of all the Spanish native cultivars studied, and the genotypic combination of only eight loci permitted the total differentiation of the 94 peach cultivars analyzed. The six selected loci (BPPCT001, BPPCT006, BPPCT008, PS9f8, UDP98-022, and UDP98-412) seem to be very useful for future Spanish peach identification works, and they will help to establish a molecular data base for native peach cultivars. UPGMA analysis was performed from the genetic distance matrix, and allowed the arrangement of all genotypes according to their genetic diversity. The genetic diversity among cultivars, observed in this work, led to their separation according to their regional origin, their morphological characteristics, and especially according to their fruit traits. Analysis of molecular variance was performed for seven populations from different regions of Spain and USA to examine the distribution of genetic variation of the studied accessions, showing that the major variation occurred within populations in each geographic site. The results reveal the existence of two diversity regions in Spain for peach germplasm.     

Microsatellite DNA and RAPD fingerprinting,identification and genetic relationships of hybrid poplar (<Emphasis Type="Italic">Populus x canadensis</Emphasis>) cultivars

Microsatellite DNA markers of ten SSR loci and 248 RAPD loci (resolved by 26 RAPD primers) were used for DNA fingerprinting and differentiation of 17 widely grown Populus x canadensis syn. Populus x euramericana (interspecific Populus deltoides x Populus nigra hybrids) cultivars ("Baden 431", "Blanc du Poitou", "Canada Blanc", "Dorskamp 925", "Eugenei", "Gelrica", "Grandis", "Heidemij", "I-55/56", "I-132/56", "I-214", "Jacometti", "Ostia", "Regenerata", "Robusta", "Steckby" and "Zurich 03/3"), and determination of their genetic interrelationships. Informativeness of microsatellite and RAPD markers was also evaluated in comparison with allozyme markers for clone/cultivar identification in P. x canadensis. High microsatellite DNA and RAPD genetic diversity was observed in the sampled cultivars. All of the 17 P. x canadensis cultivars could be differentiated by their multilocus genotypes at four SSR loci, and were heterozygous for their parental species-specific alleles at the PTR6 SSR locus. Except for "Canada Blanc" and "Ostia", which had identical RAPD patterns, all cultivars could also be differentiated by RAPD fingerprints produced by each of the two RAPD primers, OPA07 and OPB15. For microsatellites, the mean number of alleles, polymorphic information content, observed heterozygosity, observed number of genotypes and the number of cultivars with unique genotypes per locus was 5.2, 0.64, 0.67, 5.7 and 2.2, respectively. For RAPD markers, the number of haplotypes per locus, and the number of cultivars with unique RAPD profiles per locus were 1.06 and 0.72, respectively. Overall, microsatellite DNA markers were the most informative for DNA fingerprinting of P. x canadensis cultivars. On the per locus basis, microsatellites were about six-times more informative than RAPD markers and about nine-times more informative than allozyme markers. However, on the per primer basis, RAPD markers were more informative. The UPGMA cluster plots separated the 17 cultivars into two major groups based on their microsatellite genotypic similarities, and into three major groups based on their RAPD fragment similarities. Both the microsatellite and RAPD data suggest that the cultivars "Baden 431", "Heidemij", "Robusta" and "Steckby" are genetically closely related. The inter-cultivar genetic relationships from microsatellite DNA and RAPD markers were consistent with those observed from allozyme markers, and were in general agreement with their speculated origin. Microsatellite DNA and RAPD markers could be used for clone and cultivar identification, varietal control and registration, and stock handling in P. x canadensis.     

CAAT box- derived polymorphism (CBDP): a novel promoter -targeted molecular marker for plants

The availability of a simple, reproducible and cost-effective molecular marker is a prerequisite for plant genetic analysis. We have developed a novel promoter-targeted marker, CAAT box- derived polymorphism (CBDP) using the nucleotide sequence of CAAT box of plant promoters. CBDP, like random amplified polymorphic DNA (RAPD), uses single primer in polymerase chain reaction (PCR) for generating markers. However unlike RAPD, the CBDP primers are 18 nucleotides long and consist of a central CCAAT nucleotides core flanked by the filler sequence towards the 5′ end and di- or trinucleotides towards the 3′ end. In this study, a small set of 25 CBDP primer was designed and initially tested in a representative set of eight cultivars of jute for generation of polymorphic markers. Further, to achieve high reproducibility, a touchdown PCR was employed with an annealing temperature of 50ºC. All the CBDP primers generated polymorphic markers in jute cultivars, and an UPGMA dendrogram based on Jaccard’s similarity grouped them into two clusters represented by Corchorus capsularis and C. olitorius, respectively. Interestingly, such grouping of jute cultivars was consistent with genetic relationships established earlier for these cultivars using other DNA markers. Moreover, these CBDP primers also generated polymorphic markers in representative sets of cotton (Gossypium species) and linseed (Linum usitatissimum ) cultivars. Given the high success rate of CBDP primers in generating markers in the tested species and advantages like ease in marker development and assay with reproducible profiles, they could potentially be exploited in other species as well for assessing genetic diversity, cultivar identification, construction of linkage map and marker- assisted selection.     

Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean

Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F₂ families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.     

Interpretation of randomly amplified polymorphic DNA marker data for fingerprinting sweet potato (Ipomoea batatas L.) genotypes

In this paper we present a method for the generation of randomly amplified polymorphic DNA (RAPD) markers for sweet potato. These were applied to produce genetic fingerprints of six clonal cultivars and to estimate genetic distances between these cultivars. The level of polymorphism within the species was extremely high. From the 36-decamer random primers used, 170 fragments were amplified, of which 132 (77.6%) were polymorphic. Ten primers resulted in no detected amplification. Of the remaining 26 primers for which amplification was achieved, only one did not reveal polymorphism. Six primers used alone enabled the discrimination of all six genotypes. Pattern analysis, which employed both a classification and ordination method, enabled the grouping of cultivars and the identification of primers which gave greatest discrimination among the cultivars.     

Identification and genetic variation among Hibiscus species (Malvaceae) using RAPD markers

Germplasm identification and characterization is an important link between the conservation and utilization of plant genetic resources. Traditionally, species or cultivars identification has relied on morphological characters like growth habit or floral morphology like flower colour and other characteristics of the plant. Studies were undertaken for identification and determination of genetic variation within the two species of Hibiscus and 16 varieties of Hibiscus rosa-sinensis L. through random amplified polymorphic (RAPD) markers. Primer screening was made by using the DNA of variety "Prolific". Genetic analysis was made by using ten selected decamer primers. A total of 79 distinct DNA fragments ranging from 0.3 to 2.5 kb were amplified by using ten selected random decamer primers. The genetic similarity was evaluated on the basis of presence or absence of bands. The cluster analysis indicated that the 16 varieties and two species formed one cluster. The first major cluster consisted of three varieties and a second major cluster consisted of two species and 13 varieties. The genetic distance was very close within the varieties and also among the species. Thus, these RAPD markers have the potential for identification of species/varieties and characterization of genetic variation within the varieties. This is also helpful in Hibiscus breeding programs and provides a major input into conservation biology.     

莲品种DNA指纹图谱的构建

为了克服单纯依据形态特性鉴定品种的局限性, 我们开展了莲品种DNA指纹图谱构建研究, 旨在对其品种的快速准确鉴定及专利权保护等起一定作用。本研究以圆明园保存的72个莲品种为实验材料, 用来自不同地点的1,409份野生莲(Nelumbo nucifera)和58份美洲黄莲(N. lutea)群体样本作遗传背景参照。从104对核微卫星引物(nSSR)中筛选出15对, 从17对叶绿体微卫星(cpSSR)引物中筛选出2对, 共17对引物作为72个莲品种DNA指纹鉴定的条码。15对nSSR引物共检测到94个等位基因(平均6.27个), 其中11个属于美洲黄莲, 65个属于野生莲, 18个不能区分; 多态信息含量(PIC)介于0.3899-0.8023之间 (平均0.5748)。2对cpSSR引物共检测到13个单倍型, 其中9个属于野生莲, 4个属于美洲黄莲。全部17对引物标记结果显示, 共有19个品种含有美洲黄莲遗传组分, 其中8个母系来源于美洲黄莲; 有36个品种(涉及12对引物)具有至少1个特有基因型; 最少8对引物组合可完全区分开68个品种。有2组共4个品种组内全部17对引物均不能区分。本研究通过核心引物组合法使68个莲品种获得特异性DNA指纹。推荐13对nSSR和2对cpSSR共15对引物作为莲品种鉴定的核心条码, 并建议将形态特征与DNA指纹相结合作为莲品种的鉴定标准。     

Molecular phylogeny of date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia by DNA fingerprinting

Genetic diversity among 13 different cultivars of date palm (Phoenix dactylifera L.) of Saudi Arabia was studied using random amplified polymorphic DNA (RAPD) markers. The screening of 140 RAPD primers allowed selection of 37 primers which revealed polymorphism, and the results were reproducible. All 13 genotypes were distinguishable by their unique banding patterns produced by 37 selected primers. Cluster analysis by the unweighted paired group method of arithmetic mean (UPGMA) showed two main clusters. Cluster A consisted of five cultivars (Shehel, Om-Kobar, Ajwa, Om-Hammam and Bareem) with 0.59–0.89 Nei and Li's coefficient in the similarity matrix. Cluster B consisted of seven cultivars (Rabeeha, Shishi, Nabtet Saif, Sugai, Sukkary Asfar, Sukkary Hamra and Nabtet Sultan) with a 0.66–0.85 Nei and Li's similarity range. Om-Hammam and Bareem were the two most closely related cultivars among the 13 cultivars with the highest value in the similarity matrix for Nei and Li's coefficient (0.89). Ajwa was closely related with Om-Hammam and Bareem with the second highest value in the similarity matrix (0.86). Sukkary Hamra and Nabtet Sultan were also closely related, with the third highest value in the similarity matrix (0.85). The cultivar Barny did not belong to any of the cluster groups. It was 34% genetically similar to the rest of the 12 cultivars. The average similarity among the 13 cultivars was more than 50%. As expected, most of the cultivars have a narrow genetic base. The results of the analysis can be used for the selection of possible parents to generate a mapping population. The variation detected among the closely related genotypes indicates the efficiency of RAPD markers over the morphological and isozyme markers for the identification and construction of genetic linkage maps.Communicated by H.F. Linskens     

ISSR鉴定亲缘关系非常近的芒果栽培品种

用ISSR技术鉴定7个吕宋芒品种(系)和柳州吕宋芒。从30个引物中筛选出6个多态性好的ISSR引物建立DNA指纹图谱用于区分吕宋芒品种(系)。分析DNA指纹图谱,发现这6个引物中每个引物都能区分吕宋系列品种(系),表明ISSR-PCR技术对芒果品种(系)的鉴定非常有效,能区分亲缘关系很近的品种(系)。基于69条多态性条带的聚类分析结果,发现吕宋芒和其它供试的7个品种(系)同源性低,而这7个品种(系):高州吕宋芒、湛江吕宋芒、田阳香芒、金钱芒、柳州吕宋芒、粤西一号、攀西红吕宋同源性较高,可归为一类。     

Genetic diversity assessment in Portugal accessions of <Emphasis Type="Italic">Olea europaea</Emphasis> by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.     

Genetic variation and cultivar identification in Cymbidium ensifolium

As a popular flowering species with many cultivars, Cymbidium ensifolium (L.) is commercially important in horticulture. However, so far little has been known about genetic diversity and conservation genetics of this species. Understanding of the genetic variation and relationships in cultivars of C.?ensifolium is a prerequisite for development of future germplasm conservation and cultivar improvement. Here we report assessment of genetic variations in C.?ensifolium cultivars using the DNA fingerprinting technique of inter-simple sequence repeats (ISSR). A total of 239 ISSR loci were identified and used for evaluation of genetic variation with a selection of 19 ISSR primers. Among these ISSR loci, 99.16% were polymorphic with wide genetic variation as shown by Nei??s gene diversity (H?=?0.2431) among 85 tested cultivars. ISSR fingerprinting profiles showed that each cultivar had its characteristic DNA pattern, indicating unequivocal cultivar identification at molecular level. Eighteen cultivar-specific ISSR markers were identified in seven cultivars. The cultivar Sijiwenhan was confirmed as hybrid by four ISSR primers. Several cultivars with same name but different geographical origins were distinguished based on their ISSR profiles. A dendrogram generated with ISSR markers could group 73 of 85 cultivars into four major clusters. Further analysis of ISSR variation revealed that about 69% of total genetic variation in this species is due to genetic divergence inside geographical groups. Our results suggest that both germplasm collection and in?situ conservation are important for future planning of C.?ensifolium species conservation.     

Genetic fingerprinting of Australian cotton cultivars with RAPD markers.

RAPD (random amplified polymorphic DNA) markers generated by 30 random decamer primers were used to fingerprint 12 released cultivars and a breeding line of Gossypium hirsutum and 1 cultivar of G. barbadense presently under cultivation in Australia. Among a total of 453 developed markers, 69 (15.2%) were only present (unique) in the G. barbadense cultivar Pima S-7. Of the remaining markers, 128 (33.3%) were fixed in all 13 G. hirsutum cultivars. In pairwise comparisons of the degree of band sharing, nine closely-related cultivars showed 92.1-98.9% genetic similarity. Cluster analysis of genetic distance estimates between each of the cultivars revealed phylogenetic relationships in broad agreement with the known lineage of the cultivars. Ten of the G. hirsutum cultivars can be characterized individually based upon cultivar-specific RAPD markers, thus making it possible to differentiate closely related cultivars by molecular markers.     

Molecular diversity and relationships among <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> cultivars based on inter-simple sequence repeat (ISSR) markers

Spring orchid (Cymbidium goeringii) is a popular flowering plant species. There have been few molecular studies of the genetic diversity and conservation genetics on this species. An assessment of the level of genetic diversity in cultivated spring orchid would facilitate development of the future germplasm conservation for cultivar improvement. In the present study, DNA markers of intersimple sequence repeats (ISSR) were identified and the ISSR fingerprinting technique was used to evaluate genetic diversity in C. goeringii cultivars. Twenty-five ISSR primers were selected to produce a total of 224 ISSR loci for evaluation of the genetic diversity. A wide genetic variation was found in the 50 tested cultivars with Nei’s gene diversity (H = 0.2241) and 93.75% of polymorphic loci. Fifty cultivars were unequivocally distinguished based on ISSR fingerprinting. Cultivar-specific ISSR markers were identified in seven of 50 tested cultivars. Unweighted pair-group mean analysis (UPGMA) and principal coordinates analysis (PCA) grouped them into two clusters: one composed the cultivars mainly from Japan, and the other contained three major subclusters mainly from China. Two Chinese subclusters were generally consistent with horticultural classification, and the third Chinese subcluster contained cultivars from various horticultural groups. Our results suggest that the ISSR technique provides a powerful tool for cultivar identification and establishment of genetic relationships of cultivars in C. goeringii.     

Genetic diversity of durum wheat as determined by AFLP in fluorescence

The DNA of fifteen Italian cultivars of durum wheat (Triticum turgidum L. ssp. durum) were analyzed by in fluorescence amplified fragment length polymorphism (fAFLP) in order to obtain the characteristic fingerprintings of genotypes and assess their genetic relatedness. Among 64 combinations of fluorescence labelled primers, three different combinations were chosen as producing a total of 6630 AFLP fragments, 2277 (34.3 %) of them being polymorphic. By using this fAFLP methodology a DNA fingerprinting of each durum wheat cultivar was generated for genotype identification. Analysis of the genetic relationships show the low variability among durum wheat cultivars.     

Diversity and compatibility of peanut (Arachis hypogaea L.) bradyrhizobia and their host plants

A high degree of genetic diversity among 125 peanut bradyrhizobial strains and among 32 peanut cultivars collected from different regions of China was revealed by using the amplified fragment length polymorphism (AFLP) technique. Eighteen different peanut bradyrhizobial genotypes and six peanut cultivars were selected for symbiotic cross-inoculation experiments. The genomic diversity was reflected in the symbiotic diversity. The peanut cultivars varied in their ability to nodulate with the strains used. Some cultivars had a more restricted host range than the others. Also the strains displayed a range of nodulation patterns. In yield formation there were clear differences between the plant cultivar/bradyrhizobium combinations. There was good compatibility between some peanut bradyrhizobial strains and selected cultivars, with inoculation resulting in well-nodulated, high-yielding symbiotic combinations, but no plant cultivar was compatible with all strains used. The strains displayed a varying degree of effectiveness, with some strains being fairly effective with all cultivars and others with selected ones. The AFLP genotypes of the strains did not explain the symbiotic behavior, whereas the yield formation of the plant cultivars was more related to the genotype. It is concluded that to obtain optimal nitrogen fixation efficiency of peanut in the field, compatible plant cultivar-bradyrhizobium combinations should be selected either by finding inoculant strains compatible with the plant cultivars used, or plant cultivars compatible with the indigenous bradyrhizobia.