Identification of sex in Simmondsia chinensis (Jojoba) using RAPD markers

Simmondsia chinensis (Link) Schneider, a multipurpose dioecious shrub of arid zones, has emerged as a cash crop. It is being cultivated for its seeds which store liquid wax whose properties are similar to spermaceti (Sperm whale oil), a substitute for petro products and precious high-priced lubricants. Jojoba is a slow-growing desert shrub having a male biased (5:1; male:female ratio) population. Since there is no method available to determine the sex at the seedling stage, current investigations have been carried out to generate a sex-specific random amplified polymorphic DNA (RAPD) marker in jojoba which is based on the PCR amplification of random locations in the genome of plant. Of the 72 primers tested, only one random decamer primer, OPG-5, produced a unique ∼1,400 base pairs fragment in male DNA. To validate this observation, this primer was re-tested with the individuals of male and female samples of four cultivars. The unique ∼1,400 bp fragment was present in male individuals of all the four cultivars and completely absent in respective female individuals tested. To the best of our knowledge, this is the first report to ascertain the sex of jojoba plants at an early stage of development of the taxon.     

菠菜性别相关的ISSR标记

菠菜为雌雄异株植物,用CTAB法提取其雌、雄株成株幼嫩叶片DNA,分别构建雌、雄株DNA池,以之为模板,用已优化的ISSR体系扩增,在74条ISSR引物中,I62扩增出一条约1 200 bp雌性连锁标记,回收纯化该特异扩增片段,将其连接于pUCm-T载体,转化进大肠杆菌JM109菌株,并检测及测序。回收克隆和测序后发现该片段全长1 176 bp,富含AT,AT占57.0%。根据测序结果设计1对25 bp的特异引物将这个雌性连锁的ISSR标记转化为稳定性和特异性更好的SCAR标记。该特异引物对随机选取的雌雄菠菜单株进行PCR扩增,在雌株中均有1 176 bp的特异条带,而雄株中均无。此特异条带的获得为菠菜性别相关基因的克隆奠定基础。     

Development of male-specific SCAR marker in date palm (Phoenix dactylifera L.)

Genetics of control mechanisms that underlies sex differentiation in date palm is not known. Sex of the plants becomes known only at the time of first flowering, which takes around 5 years. In comparison, molecular diagnosis (if available/feasible) promises quick and reliable identification of sex types very early when plantlets are growing in seedbeds. To develop such an assay, genomic DNA from 45 individual plants (25 female and 20 male) belonging to different varieties of date palm was subjected to PCR amplification using 100 random amplified polymorphic DNA (RAPD) and 104 intersimple sequence repeat (ISSR) primers. Initially, two bulk genomic DNA samples (each made by pooling DNA from ten male and female plants, separately) were used. A primer showing sex-specific band in bulked samples was further used for amplification of the genomic DNA of the individual samples of that bulk. Only one RAPD primer, OPA-02, amplified a fragment of ~1.0 kb in all the individual samples of male genotypes, whereas this fragment was absent in all the female genotypes. This male-specific fragment was cloned and sequenced (GenBank accession no. JN123357), and a sequence-characterized amplified region (SCAR) primer pair was designed that amplified a 406-bp fragment in both female and male genotypes and a unique fragment of 354 bp in only male genotypes. The SCAR marker was further validated using 25 female and ten male date palm plants belonging to different varieties collected from different locations.     

送春与多花兰种间杂交后代的ISSR分析

该文使用简单重复序列间( ISSR)分子标记,对送春与多花兰种间杂交后代进行了研究。结果表明：从80个ISSR引物中筛选出14个扩增效果稳定的ISSR引物,对两亲本和59个F1代个体进行了ISSR扩增,得到107个扩增位点,扩增的片段大小位于90~2100 bp之间,平均每个引物扩增7.64条条带,得到11种类型的带。 ISSR标记在送春×多花兰的F1代中表现出一定的多态性,分离频率为44.86％,分离位点有83.33％符合孟德尔1︰1或3︰1的分离规律,产生偏孟德尔分离的位点占12.50％,余下的4.17％属于特殊分离带型。可能导致后代变异的位点为偏孟德尔分离的6条带、缺失的8条带或新生成的2条带。聚类图中父本和母本与F1代个体间的遗传距离较远,59个杂交后代先聚集成一组,再同母本相聚为一组,最后才同父本聚在一起,59个杂种均偏母本型。送春与多花兰的杂交后代在植株形态、染色体、遗传物质方面都具备双亲特点,61个个体间的ISSR分子量标记结果和植株形态学特征都说明,59个F1代杂种包含送春和多花兰的遗传特性是真杂种;F1代杂种既有双亲的互补特征带,又有双亲的重组片断即产生新的特异带,这说明送春与多花兰的杂交后代具有遗传变异的特点。该研究结果可以有效地对杂交后代进行定向选择,为兰花的杂交育种提供了分子依据。     

Identification of RAPD markers linked to sex determination in guggal [<Emphasis Type="Italic">Commiphora wightii</Emphasis> (Arnott.)] Bhandari

Decamer RAPD primers were tested on dioeceious and hermaphrodite plants of Commiphora wightii to identify sex-specific molecular markers. Sixty different random decamer primers were screened out of which only three primers were found to be associated with sex expression. A ~1,280-bp fragment from the primer OPN06 was found to be present in all the female individuals. Another primer OPN 16 produced a unique ~400-bp amplification product in only hermaphrodite individuals. The third marker, OPA20 amplified a ~1,140-bp fragment from female and hermaphrodite DNAs, but failed to do so from the male plant DNAs.     

Development of strain-specific SCAR markers for authentication of Ganoderma lucidum

In this study we report the application of sequence-characterized amplified region (SCAR) markers in Ganoderma lucidum for strain identification, the first such study in this medicinal mushroom. One fragment unique to strain No. 9 was identified by inter-simple sequence repeats (ISSR), and then sequenced. Based on the specific fragment, one SCAR primer pair designated as GL₆₁₂F and GL₆₁₂R was designed to amplify a 612-bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer pair. The results showed that this SCAR marker can clearly distinguish strain No. 9 from other related Ganoderma lucidum strains. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for Ganoderma lucidum.     

Terminal repeat retrotransposon in miniature (TRIM) as DNA markers in Brassica relatives

We have developed a display system using a unique sequence of terminal repeat retrotransposon in miniature (TRIM) elements, which were recently identified from gene-rich regions of Brassica rapa. The technique, named TRIM display, is based on modification of the AFLP technique using an adapter primer for the restriction fragments of BfaI and a primer derived from conserved terminal repeat sequences of TRIM elements, Br1 and Br2. TRIM display using genomic DNA produced 50–70 bands ranging from 100 to 700 bp in all the species of the family Brassicaceae. TRIM display using B. rapa cDNA produced about 20 bands. Sequences of 11 randomly selected bands, 7 from genomic DNA and 4 from cDNA, begin with about 104 bp of the terminal repeat sequences of TRIM elements Br1 or Br2 and end with unique sequences indicating that all bands are derived from unique insertion sites of TRIM elements. Furthermore, 7 of the 11 unique sequences showed significant similarity with expressed gene. Most of the TRIM display bands were polymorphic between genera and about 55% (132 of 239 bands) are polymorphic among 19 commercial F1 hybrid cultivars. Analysis of phylogenetic relationships shows clear-cut lineage among the 19 cultivars. Furthermore, a combination of 11 polymorphic bands derived from only one primer combination can clearly distinguish one cultivar from the others. TRIM display bands were reproducible and inheritable through successive generations that is revealed by genetic mapping of 6 out of 27 polymorphic TRIM markers on the genetic map of Brassica napus. Collective data provide evidence that TRIM display can provide useful DNA markers in Brassica relatives because these markers are distributed in gene-rich regions, and are sometimes involved in the restructuring of genes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank accession nos.: AC190326 (TRIM display fragment gCP01); AC190327 (TRIM display fragment gCP02); AC190328 (TRIM display fragment gCP02) AC190329 (TRIM display fragment gCP04) AC190330 (TRIM display fragment gDP01); AC190331 (TRIM display fragment gDP02); AC190332 (TRIM display fragment gDP03); AC190333 (TRIM display fragment gDP04); AC190334 (TRIM display fragment gDP05); AC190335 (TRIM display fragment gDP06); AC190336 (TRIM display fragment gDP07).     

Characterisation of microsatellites from Actinidia chinensis

We have identified a set of informative microsatellite markers for genome analysis in kiwifruit and related Actinidia species. A small-insert genomic library was constructed from Actinidia chinensis DNA, and screened for microsatellites. About 1.2% of the total colonies hybridised to a (GA)₈ probe, 0.4% to (GT)₈, and 0.1% to a mixture of three different trinucleotide repeat probes, (CAA)₅, (GAA)₅ and (CTA)₅. From the DNA sequences of 35 hybridising clones, 18 primer pairs were designed, and used to amplify genomic DNA from 38 individual plants, representing 30 different accessions of ten Actinidia species. The banding patterns for most of the dinucleotide repeats showed a high degree of polymorphism in the diploid and tetraploid A. chinensis, and in the hexaploid A. deliciosa (kiwifruit). Heterozygosity levels of up to 100% were found among eight diploid accessions of A. chinensis examined, and the number of different-sized bands among all the species varied from 3 to 36 for each microsatellite. One simple CT microsatellite gave 21 bands with sizes suggesting that the number of repeats ranged from 9 to 37. The highest number of bands (36) and the largest size variation (>100 bp) were observed with a complex microsatellite harbouring four different repeat motifs. The majority of primer pairs amplified bands from most of the ten Actinidia species tested. The most polymorphic primer pairs were used successfully to fingerprint a range of closely related varieties of kiwifruit (A. deliciosa).Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - VNTR variable number of tandem repeats     

Use of random amplified polymorphic DNA to identify several novel markers for sex identification in the crested serpent eagle and crested goshawk

The crested serpent eagle (Spilornis cheela hoya) has no distinct sexual dimorphic traits. In the current study, we report the results of an EE0.6 (EcoRI 0.6-kb fragment) sequence applied to S. cheela hoya and a novel random amplified polymorphic DNA (RAPD) marker that can be used to sex individuals within the species S. cheela hoya and Accipiter trivigatus formosae (crested goshawk). We used sex-specific primers for the avian CHD1 (chromo-helicase-DNA-binding 1) gene and the EE0.6 sequence in PCR assays to determine sex. In addition, 120 random primers were used for RAPD fingerprinting to search for novel sex-specific fragments of S. cheela hoya. The OPBB08 random primer generated a 1241-bp sex-specific fragment in all female S. cheela hoya. From the nucleotide sequence, PCR primers were designed to amplify 553-, 895-, and 194-bp sex-specific fragments present in all female S. cheela hoya. One of these primer pairs (ScBB08-7F/R) also amplified a male/female common fragment that can be used as an internal control (543 bp). Moreover, one of the primer pairs (ScBB08-5aF/5bR) could be used to identify genders of A. trivigatus formosae. In conclusion, we identified novel sex-specific DNA markers of S. cheela hoya and A. trivigatus formosae that can be used for rapid and accurate sex identification.     

DNA probes for the Y-chromosome ofSilene latifolia,a dioecious angiosperm

Summary In order to obtain markers for the Y chromosome ofSilene latifolia, we pooled equal weights of leaf tissue from 18 female siblings into one sample and repeated the process with 18 male siblings. Pooling was intended to provide a common genetic background for each sample, leaving the absence or presence of the Y chromosome as the primary difference between the two samples. DNA was extracted from each sample and subjected to polymerase chain reaction (PCR) amplification with arbitrary 10 bp primers. Four of 60 primers used gave an amplification with the male DNA not found among those from the female DNA. Each of these was subsequently shown to provide a reliable marker for the Y chromosome.     

Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers

An efficient micropropagation protocol produced large number of plants of the three elite banana (Musa spp.) cultivars Robusta (AAA), Giant Governor (AAA) and Martaman (AAB) from shoot tip meristem. The genetic relationships and fidelity among the cultivars and micropropagated plants as assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers, revealed three somaclonal variants from Robusta and three from Giant Governor. A total of 5330 RAPD and 2741 ISSR fragments were generated with 21 RAPD and 12 ISSR primers in micropropagated plants. The percentage of polymorphic loci by RAPD and ISSR were found to be 1.75, 5.08 in Robusta and 0.83, 5.0 in Giant Governor respectively. Among the two marker systems used, ISSR fingerprinting detected more polymorphism than RAPD in Robusta and Giant Governor with most of the primers showing similar fingerprinting profile, whereas Martaman revealed complete genetic stability.     

Genetic relatedness of Portuguese almond cultivars assessed by RAPD and ISSR markers

Randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to analyse the genetic diversity of Portuguese Prunus dulcis cultivars and their relationship to important foreign cultivars. Of the primers tested, 6 (out of 60) RAPD and 5 (out of 18) ISSR primers were selected for their reproducibility and high polymorphism. Out of 124 polymerase chain reaction fragments that were scored, 120 (96.8%) were polymorphic. All the plants could be discriminated and constitute a very heterogeneous group. Five unidentified almond plants found in the region of Foz Côa (north Portugal) and wild almond (P. webbii) from Italy and Spain were also included. Four main groups of plants could be distinguished: P. dulcis cultivars; one Foz Côa plant; P. webbii; and P. persica (outgroup). The segregating Foz Côa plant may represent a feral individual or a hybrid between P. dulcis and P. webbii.Abbreviations dNTP Deoxynucleotide triphosphate - CTAB Cetyltrimethylammonium bromide - ISSR Inter-simple sequence repeats - PCR Polymerase chain reaction - RAPD Randomly amplified polymorphic DNA - RASTM Regional Agricultural Services of Trás-os-Montes - TE Tris-EDTA buffer - UPGMA Unweighted pair group method with arithmetical averagesCommunicated by P. Puigdoménech     

Comparative Assessment of ISSR and RAPD Marker Assays for Genetic Diversity Analysis in Jojoba [Simmondsia chinensis (Link) Schneider]

A collection of male and female plants of ten Jojoba [Simmondsia chinensis (Link) Schneider] genotypes was analyzed with 50 RAPID and 55 ISSR markers to compare the efficiency and utility of these techniques for detecting genetic polymorphism. RAPID and ISSR analysis yielded 442 and 566 scorable amplified products, respectively, of which 60.7 and 69.3% were polymorphic. ISSRs revealed efficiency over RAPDs due to high EMR (effective multiplex ratio), DI (diversity index, mean PIC per primer) and MI (marker index) values. Jaccard similarity matrices among male plants, among female plants and between male and female plants of the ten jojoba genotypes varied between 0.705-0.784. Dendrograms generated by cluster analysis (UPGMA, NTSYS-pc) supported by bootstrap values using RAPID and ISSR datasets led to grouping of most of male and females genotypes in separate clusters. While pattern of clustering remained more or less same, the two dendrograms did differ with respect to the grouping of a few male and female genotypes. The value of the Mantel test shows poor correlation (r = 0.41) between ISSR and RAPID marker datasets.     

Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis

The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in the dioecious species, Pistacia vera. Progenies from two female parents pollinated by a common male parent were studied. Two bulks of DNA were made in each cross, one from males and one from females, by pooling an equal weight of fresh leaves from each individual contributing to the bulk prior to DNA extraction. DNA was extracted from each bulked sample and from each of the contributing individuals. DNA was also extracted from 14 cultivars of P. vera and from 94 open-pollinated, fewweeks-old P. vera seedlings of unknown sex. Seven hundred different decamer oligonucleotide primers were used to perform DNA amplification, with 1 of these (OPO08) producing a 945 bp amplification band that was present only in the bulked female samples and absent in the bulked male samples of the two crosses. The relationship between band presence and female sex expression was conserved in every individual obtained from the two crosses and in the 14 cultivars unrelated to the crosses. We propose that this band is tightly linked to the gene(s) that control sex determination in pistachio. The OPO08₉₄₅ RAPD marker could be used in a breeding program to screen the gender of pistachio plants long before they reach reproductive maturity, resulting in considerable savings of time and economic resources. In order to verify that assumption we screened 94 additional seedlings with the OPO08 primer and obtained results consistent with a 11 male:female ratio.     

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。     

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.     

基于ISSR分子标记的叶子花亲缘关系分析和指纹图谱构建

为分析品种遗传多样性和遗传距离并构建品种聚类图和指纹图谱,该研究从DNA模板浓度、引物浓度、退火温度和循环次数等方面优化了叶子花ISSR-PCR反应体系和反应程序,利用11个ISSR引物对131个叶子花品种进行PCR扩增,扩增产物经琼脂糖凝胶电泳检测。结果表明:优化的ISSR-PCR反应体系中DNA模板浓度为0.5 ng·μL^-1,引物浓度为0.5μmol·L^-1,引物UBC813、UBC814、UBC815、UBC823、UBC824、UBC835、UBC840、UBC841、UBC843、UBC844和UBC876的最佳退火温度分别为52.3、55.9、54.3、54.3、53.6、56.2、56.2、51.9、54.4、54、50℃,循环次数为32。用11个ISSR引物对131个叶子花品种扩增出161条带,其中多态性条带156条,多态性比率为96.89%。单个引物的等位基因数、有效等位基因数、Nei’s基因多样性指数和Shannon’s信息指数分别为1.86~2.00、1.33~1.68、0.21~0.39和0.34~0.57,平均值分别为1.969、1.478、0.294和0.447。引物UBC841的鉴别率最高(80.92%),可有效鉴别106个品种,与引物UBC876结合可将131个叶子花品种完全鉴别开,建立了各品种的指纹图谱。叶子花品种的遗传距离范围为0.00~0.60,平均值为0.365,遗传多样性较低,在遗传距离0.58处,131个品种分为6大类群,聚类分析显示同一个种的品种大多聚在一类,但同一个种仍有品种未聚在一类或亚类、也有多个种的品种聚在一类。该研究较为准确地揭示了叶子花种质资源的遗传多样性,建立的指纹图谱为叶子花品种登记、知识产权保护以及品种鉴定提供了可靠技术和有效手段。     

AFLP analysis of nephthytis (Syngonium podophyllum Schott) selected from somaclonal variants

This study analyzed genetic differences of 19 cultivars selected from somaclonal variants of Syngonium podophyllum Schott along with their parents as well as seven additional Syngonium species and six other aroids using amplified fragment length polymorphism (AFLP) markers generated by 12 primer sets. Among the 19 somaclonal cultivars, ‘Pink Allusion’ was selected from ‘White Butterfly’. Tissue culture of ‘Pink Allusion’ through organogenesis resulted in the development of 13 additional cultivars. Self-pollination of ‘Pink Allusion’ obtained a cultivar, ‘Regina Red Allusion’, and tissue culture propagation of ‘Regina Red Allusion’ led to the release of five other cultivars. The 12 primer sets generated a total of 1,583 scorable fragments from all accessions, of which 1,284 were polymorphic (81.9%). The percentages of polymorphic fragments within ‘White Butterfly’ and ‘Regina Red Allusion’ groups, however, were only 1.2% and 0.4%, respectively. Jaccard's similarity coefficients among somaclonal cultivars derived from ‘White Butterfly’ and ‘Regina Red Allusion’, on average, were 0.98 and 0.99, respectively. Seven out of the 15 cultivars from the ‘White Butterfly’ group and three out of six from the ‘Regina Red Allusion’ group were clearly distinguished by AFLP analysis as unique fragments were associated with respective cultivars. The unsuccessful attempt to distinguish the remaining eight cultivars from the ‘White Butterfly’ group and three from the ‘Regina Red Allusion’ group was not attributed to experimental errors or the number of primer sets used; rather it is hypothesized to be caused by DNA methylation and/or some rare mutations. This study also calls for increased genetic diversity of cultivated Syngonium as they are largely derived from somaclonal variants.     

Identification of closely related citrus cultivars with inter-simple sequence repeat markers

 Inter-simple sequence repeat (ISSR) markers generated by 22 primers were tested for their ability to distinguish among samples from 94 trees of 68 citrus cultivars. Within each of the six cultivar groups studied, most of these cultivars are so closely related that they are difficult to distinguish by other molecular-marker techniques. ISSR markers involve PCR amplification of DNA using a single primer composed of a microsatellite sequence anchored at the 3′ or 5′ end by 2–4 arbitrary, often degenerate, nucleotides. The amplification products were separated on non-denaturing polyacrylamide gels and detected by silver staining. ISSR banding profiles were very repeatable on duplicate samples. Different citrus species had very different fingerprint patterns. Within Citrus sinensis (L.) Osbeck and C. paradisi Macf., in which all cultivars have originated by the selection of mutants, ISSR markers distinguished 14 of 33 sweet orange and 1 of 7 grapefruit cultivars. Five of six lemon cultivars were discriminated by ISSR markers. Many differences were found among mandarin cultivars; however, all five satsuma cultivars analyzed had identical ISSR fingerprints. Four of five citrange cultivars were distinguishable, but ‘Troyer’ and ‘Carrizo’ had identical ISSR fingerprints. ‘Kuharske Carrizo’ citrange, which has better citrus nematode resistance than other ‘Carrizo’ citrange accessions, had unique ISSR fingerprints. Three ISSR markers that differentiated certain sweet orange cultivars were hybridized to Southern blots of sweet orange DNA digested with different restriction endonucleases. The sweet orange cultivars tested could be distinguished by these ISSR-derived RFLP markers. Moreover, one ISSR marker unique to ‘Ruby’ blood orange was observed in its progeny trees. Received: 9 September 1996 / Accepted: 4 April 1997