叶绿体SSR标记：柑橘体细胞杂种胞质遗传分析的一种新方法

从水稻(Oryza sativa L．)、烟草(Nicotiana tabacum L．)和黑松(Pinus thunbergii Parl．)等植物的22对叶绿体SSR引物中筛选出5对能用于柑橘叶绿体SSR分析的引物,应用这5对引物对9个组合的柑橘体细胞杂种的叶绿体遗传进行了分析。结果表明：这些组合再生的杂种中叶绿体都呈现随机分离,该现象与以前报道的RFLP分析结果一致,而且其可靠性已被CAPS分析所证实。表明柑橘叶绿体SSR同RFLP及CAPS一样可靠,并且更简单高效、易于操作,特别适合对柑橘等植物体细胞杂种进行早期胞质遗传组成分析。     

叶绿体S S R标记:柑橘体细胞杂种胞质遗传分析的一种新方法

从水稻(Oryza sativa L.)、烟草(Nicotiana tabacum L.)和黑松(Pinus thunbergiiParl.)等植物的22对叶绿体SSR引物中筛选出 5对能用于柑橘叶绿体SSR分析的引物,应用这5对引物对9个组合的柑橘体细胞杂种的叶绿体遗传进行了分析.结果表明:这些组合再生的杂种中叶绿体都呈现随机分离,该现象与以前报道的RFLP分析结果一致,而且其可靠性已被CAPS分析所证实.表明柑橘叶绿体SSR同RFLP及CAPS一样可靠,并且更简单高效、易于操作,特别适合对柑橘等植物体细胞杂种进行早期胞质遗传组成分析.     

Citrullus simple sequence repeats markers from sequence databases

Publically available cDNA sequence data of Citrullus lanatus were searched for simple sequence repeats (SSRs). Nineteen microsatellites were identified and primer pairs were designed to amplify those loci. Primers were evaluated for their ability to detect polymorphisms within a set of several watermelon varieties and local landraces, C. colocynthis, and interspecific hybrids. Eighteen polymorphic SSR loci were identified. These polymorphic loci can be used for varietal identification and other uses.     

冬枣×宁梨巨枣的子代SRAP鉴定及遗传多样性分析

应用SRAP分子标记方法对冬枣×宁梨巨枣的子代进行了分子鉴定及遗传多样性分析。采用构建基因池的方法对SRAP分子标记引物进行筛选,从88对引物中筛选出15对多态性好、主带清晰的引物,并对子代进行了真实性鉴定及多态性分析。结果表明:(1)15对引物共产生95个多态性条带,平均每对引物产生6.3个多态性条带,显示了较高的多态性比率。(2)80个子代中44个具有父本特征带,鉴定为真杂种。子代遗传多样性及UPGMA聚类分析表明,子代个体与亲本间的遗传相似系数在0.55~0.98之间,个体差异明显。该研究结果为枣树杂交育种提供了重要的分子证据。     

Transferability and genome specificity of a new set of microsatellite primers among Brassica species of the U triangle

We present a new set of 12 highly polymorphic simple sequence repeat primer sequences for use with Brassica species. These new primers, and four from A.K.S. SzewcMcFadden and colleagues, were tested in four Brassica species (B. rapa, B. napus, B. oleracea and B. nigra). Most primers successfully amplified products within all species and were polymorphic. Due to the risk of gene flow from GM oilseed rape to its wild relatives, hybrid formation in the Brassicaceae is of great interest. We identify six primer pairs as specific to the A, B or C genomes that could be used to identify such hybrids.     

Effectiveness of bovine microsatellites in resolving paternity cases in American bison, Bison bison L.

A set of 33 cattle microsatellite primer pairs was tested with the DNA of American bison from a captive population in Belgium and evaluated for usefulness in parentage testing. Two primer sets did not amplify and three were monomorphic. Among the polymorphic markers, the number of alleles ranged from two to nine. Heterozygosity, polymorphism information content (PIC) and probability of exclusion (PE) values were low by comparison with those obtained with the same markers in cattle. Two methods of estimating PE were used, one which assumed equal allele frequencies between parental sexes and another which took into account differences in allele frequencies between parental sexes. An internationally accepted set of nine microsatellites gives cumulative PE values of 0·98 and 0·97, respectively, for the two methods. The potential of this marker set to identify bison × cattle hybrids is discussed. Because bison and cattle have a common ancestor, these microsatellites are a useful way to establish genetic distances and can lead to the construction of phylogenetic trees.     

甜橙与酸橙体细胞杂种核质组成鉴定

采用流式细胞术(flow cytometry,FCM)、简单重复序列(simple sequence repeat,SSR)和酶切扩增多型性序列(cleaved amplifiedpolymorphic sequence,CAPS)等技术分析酸橙(Citrus aurantium L.)叶肉原生质体和甜橙(C.sinenis Osbeck cv.Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种.FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体.用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本.在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种.通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样.结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本.讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用.     

Cross-species microsatellite amplification in Vasconcellea and related genera and their use in germplasm classification.

To generate inexpensive and efficient DNA markers for addressing a number of population genetics problems and identification of wild hybrids in Vasconcellea, we have evaluated the use of simple sequence repeat (SSR) primers previously developed for other species. A set of 103 Vasconcellea accessions and some individuals of the related genera Carica and Jacaratia were analyzed with 10 primer pairs directing amplification of chloroplast microsatellites in Nicotiana tabacum and 9 nuclear SSR primer pairs recently identified in Vasconcellea x heilbornii. Heterologous amplification of chloroplast SSRs was successful for 8 of the 10 loci, of which 6 showed polymorphism. Seven of the 9 nuclear SSR primer pairs were useful in Vasconcellea and often also in Jacaratia and Carica, all revealing polymorphism. Exclusive haplotypes for each described taxon were identified based on chloroplast microsatellite data. Clustering based on separate nuclear and chloroplast data resulted in a clear grouping per taxon, but only low resolution was obtained above species level. The codominancy of nuclear SSRs and the general high polymorphism rate of SSR markers will make them more useful in future population genetics studies and diversity assessment in conservation programs.     

Molecular characterization of cytoplasmic and nuclear genomes in phenotypically abnormal Valencia orange (Citrus sinensis) + Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids

Organelle DNA inheritance of four 10-year-old somatic hybrid trees between Valencia orange [Citrus sinensis (L.) Osbeck] and Meiwa kumquat (Fortunella crassifolia Swingle) was analyzed by cleaved amplified polymorphic sequence (CAPS) and restriction fragment length polymorphisms (RFLPs). Five chloroplast (cp) and three mitochondrial (mt) universal primer pairs were amplified, but no polymorphisms were detected. When the polymerase chain reaction products were digested by 15 restriction enzymes, four polymorphic cpDNA-CAPS and two mtDNA-CAPS markers were found. Both the cpDNA and mtDNA in the somatic hybrids were derived from Valencia orange (the embryogenic suspension parent). Genomic DNA of the somatic hybrids and corresponding parents was digested by five restriction endonucleases and hybridized with one chloroplast probe (RbcL- RbcL) and nine mitochondrial probes (coxI, coxII, c oxIII, c ob, atpA, tyr, proI, atp6 and atp9). The results indicated that three hybrid plants shared one strong cpDNA band with both parents and that the remaining one plant had two additional novel bands besides the shared band, while their mtDNA was identical to that of Valencia orange plus non-parental bands. When data on the mtDNA banding patterns were combined with observations on phenotypic performance in the field, it was found that the more complex mtDNA banding pattern coincided with increased vigor of the plant. The stability of the organelle genomes was studied by extracting the genomic DNA of one hybrid plant at monthly intervals for 1 year and then analyzing it using RFLPs. Before the dieback of the shoots, two fragments of the mtDNA were lost while the cpDNAs remained stable. Ploidy analysis by flow cytometry showed that all of the hybrids were stable tetraploids. Four simple sequence repeat primer pairs were applied to detect microsatellite alleles of the four hybrid plants, both parents and the 12 DNA samples from one plant. The results showed that all hybrids had biparental bands uniformly, which indicated that they had the same nuclear background. These results suggest that the mtDNA pattern is correlated with the phenotypic abnormality of Valencia and kumquat somatic hybrid plants and that nuclear-cytoplasm incompatibility may be the cause of dieback.     

乌鳢和斑鳢微卫星制备及其对自交与杂交F1代的初步鉴定

通过磁珠富集法分离了乌鳢(Channa argus)和斑鳢(C.maculate)的微卫星DNA序列,根据这些微卫星DNA序列的两翼序列,采用Primer 3.0或Primer 5.O软件设计出乌鳢与斑鳢的微卫星引物各31对.用62对微卫星引物对乌鳢和斑鳢自交与杂交F1代的4个组合进行扩增,10对引物无扩增产物,其余5...     

PCR primers for human chromosomes: reagents for the rapid analysis of somatic cell hybrids

Rapid analysis of somatic cell hybrids can be facilitated by using the polymerase chain reaction (PCR) to assay for genes assigned to specific human chromosomes. We describe PCR primer pairs for genes on the short and long arms of the 22 autosomes and the X chromosome. Some of the primers were designed from the 3' untranslated region of cDNA sequences, whereas others were derived from genomic sequence. Each primer set was tested for its specificity and mapped to a chromosome by screening a somatic cell hybrid panel. Two of the primer pairs (APOC2 and G6PD) detect CA dinucleotide repeat polymorphisms.     

Studies on Paeonia cultivars and hybrids identification based on SRAP analysis

Plants of Paeonia are valuable for their ornamental and medicinal values. Genetic relations and hybrids identification among different sections of Paeonia were studied using sequence related amplified polymorphism (SRAP) markers. A total of 29 cultivars including 2 intersectional hybrids, 13 sect. Moutan and 14 from sect. Paeonia were used. A total of 197 bands were produced using 24 primer combinations, among which 187 bands showed polymorphism. From the bands amplified, we can identify the peony cultivars using unique SRAP markers and specific primer combinations. Fourteen peony cultivars were distinguished among each other by using totally 35 SRAP markers, which were generated by 16 primer pairs. Two specific primer pairs of Me8/Em8 and Me8/Em1 can be used to identify cultivars from different sections. The mean genetic similarity coefficient (GS), the gene diversity (GD), and the Shannon's information index of peony cultivars were 0.45, 0.19 and 0.32, respectively. Both UPGMA (unweighted pair-group method of arithmetic average) dendrogram and PCA (principle component analysis) analysis showed clear genetic relationships among the 29 peony cultivars, and within section and its intersectional hybrids. The above results are valuable for estimating and analyzing genetic background of Paeonia, parent selection in crossing breeding programs, molecular marker assisted selection (MAS) breeding for further germplasm innovation programs.     

AFLP标记技术在鉴定甘蓝种子真实性及品种纯度中的应用

利用AFLP方法对在农业生产上大面积推广使用的5个甘蓝杂交组合及其10个亲本共15个材料进行了分析研究,得到了清晰的DNA扩增指纹图谱.在AFLP分析中,共采用了32个EcoRI+3/MseI+3引物组合,每个引物组合扩增出的条带数在43～62条之间,平均为54.5条.并从这32个引物组合中筛选出一个引物组合,能够用来对供试5个甘蓝杂交种进行真实性及品种纯度鉴定.从而证明了AFLP技术在甘蓝种子真实性及品种纯度鉴定中的可行性.该技术应用于甘蓝种子真实性及品种纯度鉴定在国内尚属首次,其稳定性重复性好,检测分辨率高,很适合甘蓝种子真实性及品种纯度鉴定.是今后种子真实性及品种纯度鉴定的发展方向,应加大其研究力度.     

甜橙与酸橙体细胞杂种核质组成鉴定(英文)

采用流式细胞术(flow cytometry, FCM)、简单重复序列(simple sequence repeat, SSR)和酶切扩增多型性序列(cleaved amplified polymorphic sequence, CAPS)等技术分析酸橙(Citrus aurantium L. )叶肉原生质体和甜橙(C. sinenis Osbeck cv. Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种。FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体。用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本。在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种。通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样。结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本。讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用。     

Portable microsatellite primers for Ficus (Moraceae)

? Premise of the study: Highly portable microsatellite primers were developed for Ficus to facilitate investigation of genetic structure of complete regional floras using a single set of markers. ? Methods and Results: Pyrosequencing of five species of Ficus produced a library of 5723 potential primers. Potential primers found in at least two species and presenting identical annealing temperatures were tested on a set of five additional Ficus species. A set of 20 primer pairs producing well-defined and easily readable peaks was retained and tests showed their potential utility for analyzing population genetic structure of 24 Ficus species from Taiwan. Numbers of alleles per locus ranged from one to six in the least variable species and from one to 17 in the most variable species. ? Conclusions: The results indicate that our set of primers can be used to analyze polymorphism and compare levels of polymorphism among Ficus species.     

Use of 5´-anchored primers for the enhanced recovery of specific microsatellite markers in Brassica napus L.

Microsatellite markers have assumed great significance in biological research. The isolation and characterisation of microsatellites involves DNA library construction and screening, DNA sequencing, primer design and PCR optimisation. When a microsatellite is situated close to the beginning or end of a cloned fragment, specific primers cannot be designed for one of the flanking sequences, thus hindering the utilisation of such microsatellites as markers. The present approach was to use one 5′-anchored primer complementary to the microsatellite sequence in combination with one specific Cy5- labelled primer with a view to retrieving useful microsatellites, which would otherwise be lost. Six pairs of a 5′ anchored primer and a specific primer were used across a set of 31 Brassica napus winter cultivars and one accession each of five additional Brassica species. Using laser fluorometry a single labelled product was observed after amplification with each of four primer pairs, and one primer pair gave two labelled products. Three products corresponded in size with the products expected if 5′ anchoring was effective, indicating the amplification of locus-specific full-length products including all of the microsatellite repeats. All six primer pairs showed polymorphisms across the Brassica species examined, but only one primer pair showed polymorphisms within B. napus, making it useful for genetic analysis in rapeseed cultivars. The other primer pairs could be useful in studying gene introgression into B. napus or for investigating interspecific crosses involving different Brassica species. Received: 5 August 1999 / Accepted: 1 November 1999     

用于绿豆种质资源遗传多样性分析的SSR及STS引物的筛选

目前能够用于绿豆(Vigna radiate)种质资源遗传多样性分析的PCR引物极其有限。通过12份农艺性状差异较大的绿豆种质对绿豆以及小豆(Vigna angularis)、豇豆(Vigna unguiculata)、菜豆(Phaseolus vulgaris)等近缘食用豆中的PCR引物进行筛选,结果表明41对绿豆SSR引物中能够有效扩增的有35对,6对有多态性;28对绿豆STS引物中有23对能够有效扩增,2对有多态性;8对小豆SSR引物能够有效扩增的有6对,但均无多态性;27对豇豆SSR引物能够有效扩增的有17对,1对有多态性;24对菜豆SSR引物能够有效扩增的有9对,1对有多态性。这些多态性引物的获得将有助于中国绿豆种质资源的遗传多样性分析。     

Towards Plant Species Identification in Complex Samples: A Bioinformatics Pipeline for the Identification of Novel Nuclear Barcode Candidates

Monitoring of the food chain to fight fraud and protect consumer health relies on the availability of methods to correctly identify the species present in samples, for which DNA barcoding is a promising candidate. The nuclear genome is a rich potential source of barcode targets, but has been relatively unexploited until now. Here, we show the development and use of a bioinformatics pipeline that processes available genome sequences to automatically screen large numbers of input candidates, identifies novel nuclear barcode targets and designs associated primer pairs, according to a specific set of requirements. We applied this pipeline to identify novel barcodes for plant species, a kingdom for which the currently available solutions are known to be insufficient. We tested one of the identified primer pairs and show its capability to correctly identify the plant species in simple and complex samples, validating the output of our approach.     

Development and use of chloroplast microsatellites in Phaseolus spp. and other legumes

Chloroplast microsatellites (cpSSRs) provide a powerful tool to study the genetic variation and evolution of plants. We have investigated the usefulness of 39 primer pairs tagging cpSSR loci on a set of eight different genera of Leguminosae (Papilionoideae subfamily) and five species belonging to the genus Phaseolus . Thirty-six 'universal' primer pairs were retrieved from the literature, one was re-designed and a further two were designed de novo . The cpSSR loci analysed were highly polymorphic across the individuals examined. Twenty-seven primer pairs were polymorphic in the overall sample, 18 within Phaseolus , and 16 in both P. vulgaris and P. coccineus . Analysis of the plastome sequences of four Leguminosae species (obtained from GenBank) showed that in the loci targeted by universal primer pairs: (i) the originally tagged cpSSRs can be lost; (ii) other cpSSRs can be present; and (iii) polymorphism arises not only from differences in the numbers of cpSSR repeats, but often from other insertion/deletion events. Multilocus linkage disequilibrium analysis suggests that homoplasy is not a major problem in our dataset, and principal component analysis indicates intelligible relationships among the species considered. Our study demonstrates that this set of chloroplast markers provides a useful tool to study the diversity and the evolution of several legumes, and particularly P. vulgaris and P. coccineus .     

Genetic diversity and its relationship to hybrid performance and heterosis in rice as revealed by PCR-based markers

Ten elite inbred lines (four japonica, six indica), chosen from those widely used in the hybrid rice breeding program at Human Hybrid Rice Research Center in China, were crossed to produce all possible hybrids excluding reciprocals. The 45 F₁ hybrids along with the ten parents were evaluated for eight traits of agronomic importance, including yield potential, in a replicated field trial. The ten parents were analyzed with 100 arbitrary decamer oligonucleotide primers and 22 microsatellite (simple sequence repeats, SSRs) primer sets via polymerase chain reaction (PCR). Out of the 100 random primers used, 74 were informative and amplified 202 non-redundant bands (variants) with a mean of 2.73 bands per polymorphic primer. All 22 microsatellite primer sets representing 23 loci in the rice genome showed polymorphisms among the ten parents and revealed 90 alleles with an average of 3.91 per SSR locus. Cluster analysis based on Nei's genetic distance calculated from the 291 (202 RAPDs, 89 SSRs) non-redundant variants separated the ten parental lines into two major groups that corresponds to indica and japonica subspecies, which is consistent with the pedigree information. Strong heterosis was observed in hybrids for most of the traits examined. For the 43 diallel crosses (excluding 2 crosses not heading), yield potential, its components (including panicles per plant, spikelets per panicle and 1000-grain weight) and their heterosis in F₁ hybrids showed a significant positive correlation with genetic distance. When separate analyses were performed for the three subsets, yield potential and its heterosis showed significant positive correlations with genetic distance for the 15 indica x indica crosses and the 6 japonica x japonica crosses; however, yield potential and its heterosis were not correlated with genetic distance for the 22 indica x japonica crosses. Results indicated that genetic distance measures based on RAPDs and SSRs may be useful for predicting yield potential and heterosis of intra-subspecific hybrids, but not inter-subspecies hybrids.