Hybrid detection and characterization of Curcuma spp. using sequence characterized DNA markers

The tropical flower Curcuma alismatifolia of the family Zingiberaceae is widely valued as a cut flower and potted plant due to its range of vibrant colors and large long-lasting inflorescences. Much of this diversity has been cultivated through extensive hybridization of wild varieties since hybrids often exhibit dramatic phenotypic differences from the parents. This phenotypic diversity though has led to difficulties identifying and classifying the relatedness of Curcuma varieties particularly between hybrids. One Curcuma variety called ‘Patumma’ is of particular importance since it has strong stalks, large symmetric inflorescences, is moderately resistant to fungal blight, has a high yield, and has been used to produce numerous high quality hybrids. Since Patumma is one of the key Curcuma varieties from which many hybrid crosses and induced mutation varieties were developed, we chose it as a target to be characterized using a molecular marker. Starting with a set of 11 unique decamer primers, polymorphic bands ranging from 100 to 2500 base pairs were used to examine 20 Curcuma varieties from which banding patterns of interest were selected for conversion to the more reproducible and robust sequence characterized amplified region (SCAR) markers. In particular, one SCAR marker amplified a region 600 bp in length which was conserved in all Patumma varieties and hybrids and as an independent test did not amplify an additional series of 24 distinct Curcuma varieties. Since new varieties of Curcuma are often dissimilar from their progenitors, this genomic analysis allows a cost-effective morphologically independent characterization of Curcuma hybrids.     

Application of RAPD and SI-typing techniques to confirm genetic integrity of radish (Raphanus sativus L.) varieties

The random amplified polymorphic DNA (RAPD) and self-incompatibility (SI) typing techniques were applied to settle a lawsuit between a Seed Company and a farmer concerning seed impurity in radish (Raphanus sativus L.). After sowing “Halra Woldong” (HW) a winter radish variety, two morphologically different radish plants grew. Genomic DNA from each of the two types were prepared and analyzed using ninety-six RAPD markers and four variety-specific markers were found. The polymorphic RAPD band patterns were compared with those of the twenty Korean radish varieties. The genetic integrity of the two varieties in question was identified and further confirmed by SI-typing of S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes.     

Combination of SCAR primers and Touchdown-PCR for sex identification in Pistacia vera L.

A combined method of sequence characterized amplified regions (SCAR) primers and Touchdown-PCR was used for the development of a female DNA marker in Pistacia vera L. The random decamer primer OPO-08 amplified a 905-bp fragment in all female trees, but also in several males. SCAR primers designed on the basis of the RAPD female molecular marker amplified a 905-bp female and a 909-bp male fragment. Sequencing these fragments revealed high homology, with several point mutations, four deletions in female and one in the male sequences. A second internal set of SCAR primers designed on the basis of a polymorphic locus, in combination with Touchdown-PCR technique, amplified a specific female 297-bp product. The diagnostic reliability of the new female specific marker was verified on 54 different genotypes. The method reported here offers a simple and reproducible way for early gender determination in P. vera.     

Development of SCAR markers to differentiate between mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.)

Summary

Mume (Prunus mume Sieb. et Zucc.) and apricot (P. armeniaca L.) are similar in fruit and tree morphology, and exhibit high cross- and graft-compatibility with each other. It is therefore difficult to differentiate mume and apricot cultivars on the basis of morphological and phenotypical characteristics. Molecular markers were developed to differentiate nine mume from ten apricot cultivars. Four dominant, random amplified polymorphic DNA (RAPD) markers that can discriminate between mume and apricot cultivars (designated OPA15₆₂₈, OPO10₅₅₀, OPO20₂₅₉, and OPU03₄₁₅) were identified from 21 decamer primers. Two RAPD markers (OPO10₅₅₀ and OPU03₄₁₅) were developed into dominant sequence-characterised amplified region (SCAR) markers (SCO10 and SCU03). These SCAR markers could differentiate between all mume and apricot cultivars.     

利用RAPD和SCAR标记鉴定草莓品种

 利用RAPD和SCAR标记对32份草莓材料进行了鉴定, 结果表明: 8个RAPD引物共扩增出85个标记, 其中71个为多态性标记, 多态性比率为83。5%。25份草莓材料的RAPD图谱差异较大, 易于区分。利用具有多态性的RAPD标记, 对28份草莓试材的亲缘关系进行分析, 初步鉴定出同名异物和同物异名品种。两个RAPD标记被转化为片段长度分别为378 bp和214 bp的显性SCAR标记, 其多态性与相应RAPD标记一致。利用这两个SCAR标记对草莓品种进行了初步鉴定。     

Molecular analysis of genetic stability in micropropagated apple rootstock MM106

Random amplified polymorphic DNA (RAPD) markers were used to assess the genetic stability of 10 micropropagated plants regenerated through axillary buds of clonal apple (Malus pumila Mill.) rootstock MM106. Eleven random decamer primers were successfully used to analyse genomic DNA from mother plants and in vitro plant material. A total of 129 scorable fragments were amplified with an average of 11.73 bands per primer. Among them, 99 were monomorphic and 30 were polymorphic with 23.2% polymorphism. Among these 30, 12 were found monomorphic across seven plants and parent. Three plants could be regarded as off-types. Our results show that RAPD markers could be used to detect the genetic similarities and dissimilarities in micropropagated material.     

梅单瓣复瓣花的相关分子标记初探

 应用RAPD分子标记技术和SCAR 分子标记技术研究了梅单瓣花与复瓣花相关的分子标记。结果表明从34 个随机引物中筛选到一个多态性引物OPP01 , 扩增出只有单瓣花类型具有的分子量为1899 bp的DNA 特异片段, 即OPP01-DS-1899 ; 根据该序列设计大小分别为25 bp 和24bp 的一对引物SSD-1 和SSD-2 ,将RAPD 标记转为SCAR 标记, 该标记大小为1079 bp 。     

Identification of persimmon (Diospyros kaki) cultivars and phenetic relationships between Diospyros species by more effective RAPD analysis

Randomly amplified polymorphic DNA (RAPD) markers were used to identify cultivars of persimmon (Diospyros kaki Thunb.) and to estimate the phenetic relationships between Diospyros species. Long random primers (15 and 20 bases) were used to increase the effectiveness of recognizing polymorphism. Twenty-five of 34 (74%) long random primers amplified polymorphic bands of 25 persimmon cultivars, indicating that the effectiveness improved compared with RAPD analysis using 10-base primers. The 25 primers amplified a total of 444 polymorphic bands, which distinguished all 25 cultivars. RAPD markers (133) clarified the phenetic relationship between the six species and subspecies of Diospyros: D. lotus, D. lotus subsp. glabbra and D. taitoensis formed one group (lotus group), and persimmon was related in order of closeness to the lotus group, D. oleifera, and D. rhombifolia.     

Conversion of chromosome-specific RAPDs into SCAR-based anchor markers for onion linkage maps and its application to genetic analyses in other Allium species

Integration of previously developed Allium cepa linkage maps requires the availability of anchor markers for each of the eight chromosomes of shallot (A. cepa L. common group Aggregatum). To this end, eight RAPD markers originating from our previous research were converted into SCAR markers via cloning and sequencing of RAPD amplicons and designing of 24-mer oligonucleotide primers. Of the eight pairs of SCAR primers, seven resulted in the amplification of single bands of the original RAPDs, and the remaining primer set amplified an additional band. The results of Southern hybridization using RAPD amplicons from genomic DNA of Japanese bunching onion (Allium fistulosum L.)—shallot monosomic addition lines indicated that five SCAR markers were single shallot chromosome-specific markers and were not detected in genomic DNA of A. fistulosum. The eight SCAR primer pairs were applied to other Allium species and exhibited three types of amplification profiles, namely RAPD amplicons observed only in shallot, in shallot and Allium vavilovii, and in several Allium species. A mapping study using 65 F₂ plants generated by the selfing of one interspecific cross A. cepa × Allium roylei individual integrated the SCAR marker SAOE17₅₀₀ into chromosome 5 as expected. The results of the present study show that the eight SCAR primer sets specific to shallot can facilitate the mapping in A. cepa and can also serve as anchor points between maps of different Allium species.     

Development of DNA markers for hybrid identification in Leucadendron (proteaceae)

A rapid and reliable method to accurately identify hybrids at an early age is essential to the success of Leucadendron breeding programs because identification based on morphology can be difficult or impossible when the seedlings are young. DNA based PCR-RFLP and random amplified microsatellite polymorphism (RAMP) markers were developed for this purpose. Unexpected non-parental fragments appeared during the PCR-RFLP analysis of the nuclear ITS region of L. uliginosum 05 × L. procerum 04 hybrids. Mixing DNA from both parents in a single PCR also produced the non-parental fragment, suggesting that PCR recombination had introduced a novel restriction site into the products from the hybrids. Sequencing of individual amplified ITS products from the hybrids confirmed this conclusion. To avoid this complication, RAMP markers were developed for accurate hybrid identification in Leucadendron. RAMP analysis generated a considerable number of polymorphic products, and showed more discrimination in identifying Leucadendron hybrids than did PCR-RFLP.     

31份野生树莓资源的RAPD分析

用RAPD技术分析了31份野生树莓资源的亲缘关系,从100个A系列随机引物中选出了15个多态性高、分辨能力强的引物。将这15个引物应用于31份野生树莓材料,共扩增出182条谱带,其中170条为多态性标记(占93.41%),平均每个引物扩增多态性谱带12.13条,表明不同树莓资源基因型间存在着极为丰富的遗传多样性。基于RAPD的扩增结果建立UPGMA聚类分析树状图,31份野生树莓资源在遗传距离0.5100处被划分为4个类群。从分子水平研究了树莓属资源的亲缘关系,为树莓种质资源的保存和利用及其杂交亲本的选配提供了依据。     

Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids

Paphiopedilum armeniacum, Paphiopedilum micranthum and Paphiopedilum delenatii are endangered orchid species. These three Paphiopedilum species and their hybrids are difficult to distinguish morphologically. In this study, rDNA-ITS (internal transcribed spacer) sequences were used to design species-specific SCAR (sequence characterized amplified regions) markers to distinguish P. armeniacum, P. micranthum, P. delenatii and their respective hybrids. The developed markers efficiently amplified 600 bp DNA product for P. armeniacum and its hybrids (SCAR-600armF/Pap-ITS2R), 300 bp product for P. delenatii and its hybrids (SCAR-300delF/Pap-ITS2R) and 700 bp product for P. micranthum and its hybrids (SCAR-700micF/Pap-ITS2R). The effectiveness of designed species-specific markers was also confirmed by using multiplex polymerase chain reaction amplification with a combination of developed three SCAR markers.     

Assessment of diversity in Podophyllum hexandrum by genetic and phytochemical markers

For successful conservation and domestication of a species, evaluation of its genetic diversity by different markers is important. Morphological characteristics, phytochemical variation and random amplified polymorphic DNA (RAPD) profiles were generated in different accessions of Podophyllum hexandrum in order to determine the genetic diversity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity among the accessions used in the study. There was also high diversity in the concentration of marker compounds in the collected samples as revealed by HPLC analysis. It is shown that the approaches used in the work successfully discriminate between the accessions of this species and thus they constitute interesting tools to analyze molecular, biochemical and phenotypic diversity within this species. Similarity measurement using UPGMA followed by cluster analysis resulted in formation of many groups based on geographical distribution that generally reflected expected trends between the genotypes. There were also some important exceptions like PW-S, an accession from Wastoorwan, Khrew showing close resemblance to PG-S and PG-B collected from Gulmarg but grown at two different gene banks at Srinagar and Bonera. Further an accession PSH-B from Keller was significantly diverse from the rest of the native genotypes phytochemically, morphologically and at molecular level. RAPD data analysis was found to be significant predictor of phytochemical markers in cultivated P. hexandrum germplasm. Twelve accessions grown in gene bank repository were subjected to RAPD analysis and were assessed for content of podophyllotoxin and podophyllotoxin β-d-glycoside by HPLC. Individual regressions of podophyllotoxin and podophyllotoxin β-d-glycoside by RAPD analysis against HPLC has been found to determine linear values. Strong correlation and a strong association of values of the phytochemical variables and the DNA polymorphism data has been recorded.     

中国野生葡萄果实抗白腐病基因的分子标记

 以感病×抗病的葡萄种间杂交组合白玉霓×塘尾的F₁ 代17 个单株及塘尾自交一代16 个单株为试材, 采用BSA 法和RAPD 技术, 通过对155 个随机引物的筛选, 获得了一个与中国野生葡萄抗白腐病基因连锁的RAPD 标记OPP09-760 , 并在中国野生葡萄8 个种的32 个株系及欧洲葡萄16 个品种中得到验证。进一步将该DNA 片段克隆并测序, 根据两端序列, 设计了一对长26 bp 的特异引物分别扩增供试材料, 获得了与该RAPD 标记相同大小的DNA 片段, 成功地将RAPD 标记转化为SCAR 标记, 可以用作抗白腐病基因的分子辅助选择的依据。     

燕山板栗种质资源遗传多样性的RAPD分析

为研究燕山板栗的遗传多样性,采用随机扩增多态DNA(randomamplifiedpolymorphicDNA,RAPD)技术对36份燕山板栗种质进行了分析。分析了燕山板栗的遗传丰富度,并对包括36个燕山板栗品种和8份外来板栗品种在内的44份板栗种质进行聚类分析。结果表明,RAPD能有效地区分品种间的差异,用16个随机引物经PCR扩增共得到132个片段,其中有83个多态性片段,占62.9%;不同遗传位点之间遗传多样度最大可达0.444,最小值为0.096,平均多样度为0.187;UPGMA法聚类,将44份板栗种质聚成4个大的类群,36份燕山板栗可分为3个大的类群,外来种质聚为一类。燕山板栗明显不同于外来品种。在RAPD图谱中,找到了19个品种(类型)的特异性标记和标记组合,可作为品种(类型)分子鉴别的依据。     

Genetic diversity analysis in the traditional and improved ginger (Zingiber officinale Rosc.) clones cultivated in North-East India

Identification of clonal or genotypic variations is a prerequisite for ginger (Zingiber officinale Rosc.) improvement programmes. Genetic diversity analysis was carried out in a set of forty-nine ginger clones cultivated in North-East India using random amplified polymorphic DNA (RAPD) markers. The set included clones of released varieties and clones collected from various parts of North East India. Jaccard's genetic similarity, cluster analysis and principal component analysis identified five clusters. Cluster V included four clones traditionally cultivated in the Indian state of Meghalaya known for production of high-quality ginger indicating that the clones were a good candidate for ginger improvement. Specific bands for these clones were also identified. Principal component analysis of the molecular data supported grouping of the clones into six hypothetical populations based on their source or location of collection.     

Development of ISSR- and RAPD-derived SCAR markers for identification of Gladiolus germplasm

Gladiolus is an economically important ornamental crop, cultivated for its beautiful flowers throughout the world. The correct genotype identification of plant material is very significant for the floriculture industry. The aim of this study was to develop sequence-characterised amplified region (SCAR) markers from RAPD and ISSR fragments for identification and authentication of Gladiolus germplasm. The SCAR markers developed could be easily employed as valuable tools to identify newly developed Gladiolus cultivars. The SCAR markers, viz. ScG12, ScG34, and ScG36, are specific to the DNA from all 62 Gladiolus cultivars, as they did not amplify the DNA of other taxa of the family Iridaceae, including Iris, Amaryllis, and Narcissus. All three SCAR markers distinguished Gladiolus from other taxa of the family Iridaceae, whereas marker ScAm was specific to the ‘Amethyst’ cultivar. Our results confirmed that this particular SCAR marker distinguished the ‘Amethyst’ cultivar from the other 62 Gladiolus cultivars investigated in the present study. This development of SCAR markers based on RAPD and ISSR markers seems to be the maiden attempt for Gladiolus cultivars.     

Identification of gentian cultivars using SCAR markers based on intron-length polymorphisms of flavonoid biosynthetic genes

Gentian is one of the most important ornamental flowers in Japan. Gentiana triflora, G. scabra and their interspecific hybrids are the main breeding materials. Gentian cultivars are easily proliferated vegetatively, therefore it is important to develop a reliable discrimination method to prevent the illegal propagation and distribution of various high-value cultivars. Here, we report five sequence characterized amplified region (SCAR) markers based on the length polymorphisms in introns of four gentian flavonoid biosynthetic genes. These SCAR markers effectively discriminated nine gentian cultivars and nine breeding lines. This method could be applied in identifying gentian cultivars/lines and therefore will aid in protecting breeders’ rights.     

Studies on genetic variation in cherry germplasm using RAPD analysis

Random amplified polymorphic DNA (RAPD) variation among eight cherry species and two interspecific progenies were analyzed. Out of 130, 48 arbitrary oligonucleotide primers were screened for PCR amplification to generate polymorphisms. The phylogenetic analysis was carried out using two distance-matrix methods and a dendrogram was generated to show the relationships among species and cultivars. The results showed that there were 840 amplified loci in total; 23 sweet cherry and four sour cherry cultivars were clustered together with 569 and 247 polymorphic loci respectively which accounted for 67.74% and 29.40% of the total variation. Prunus tomentosa T., Prunus fruticosa var. aucta P. and Prunus humilis B. formed a monophyletic group. A relationship between Prunus pseudocerasus L. and Colt, which formed another closely related group, was observed while Prunus avium L., Prunus cerasus L. and other cherry species were more divergent. The range of genetic distance was from 0.0623 to 0.2719 among the Prunus species, which were genetically distinct. The topology of the tree was generally in agreement with taxonomic classification. The results indicated that with the exception of the sweet cherry variety “Hongdeng”, there were one or more cultivar-specific RAPD markers in cherry species and cultivars. Using these specific markers, cherry species and varieties could be identified and there is therefore the potential to select for good characteristics of hybrids at an early stage.     

Characterization of pea accessions by SRAP's markers

Sequence-related amplified polymorphism (SRAP) and morphological markers to estimate the genetic relations among forty pea varieties (Pisum sativum L.) were studied. Data on 15 morphological traits were collected and analyzed. A total of 162 polymorphic SRAP's bands were scored using seven combinations of primers. Cluster analysis and both principal component and principal coordinate analysis were carried out. The varieties were grouped in four clusters through procrustes generalized analysis. Relationships among varieties revealed by molecular markers were significantly correlated with those based on the agronomic traits, suggesting that the two systems give similar estimates of genetic relations among the varieties. Parents selection depend on the specific objectives in further breeding programs.