金柑属及其近缘属植物亲缘关系的SSR分析

用SSR标记分析了金柑属(Fortunella)及柑橘属(Citrus)植物的亲缘关系。选取的22对柑橘属SSR引物中,有14对能在所有材料中扩增出带纹,表明柑橘属SSR引物在金柑属植物上具有一定的通用性。在18个材料中扩增得到275个位点,平均每对引物扩增出19.7个位点。用NTSYS-PC软件进行分析,结果表明,金柑属植物与宽皮柑橘的亲缘关系很近;SSR聚类结果支持长寿金柑种的地位;支持宁波金弹、融安金弹、蓝山金柑均为金弹的栽培种的结论。为金柑属种质的保存、育种和分类提供了分子水平上的依据。     

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.     

草莓属种质资源亲缘关系的SSR 标记分析

 用20对SSR引物对草莓属83份资源包括14个种和自然五倍体以及未鉴定的材料进行PCR扩增。在20个SSR位点共获得363个等位基因。每个位点扩增等位基因8 ~ 34个,平均18.2个,各位点多态性信息含量（PIC）在0.6691 ~ 0.9431,平均为0.8598。聚类分析表明,同属一个种的草莓资源被紧密地聚在一起。以种为单位,3个八倍体种智利草莓、弗州草莓和栽培种凤梨草莓亲缘关系很近,而八倍体种与其他低倍性野生种亲缘关系较远;在低倍性草莓种中,伞房草莓、五叶草莓、日本草莓、蝦夷草莓、高原草莓、黄毛草莓和绿色草莓聚在一组,具有较近的亲缘关系,而森林草莓、东北草莓、东方草莓、麝香草莓和自然五倍体草莓聚在一组,具有较近的亲缘关系。自然五倍体草莓可能起源于森林草莓或东北草莓与东方草莓或麝香草莓的自然杂交。     

SSR与AFLP标记在甜瓜连锁图谱上的分布

利用3-2-2×TopMark杂交组合,得到152株重组自交系群体,构建甜瓜分子遗传图谱。该图谱包括71个SSR标记和94个AFLP标记,由17个连锁群构成,覆盖基因组总长度1 222.9 cM,标记之间平均距离为7.41 cM。筛选了428对SSR分子标记（其中360对为EST-SSR引物,68对为g-SSR引物）,得到94对具有多态性的SSR标记,其片段大小在150～300 bp之间,多态性比例占21.29 %;筛选了256对EcorI/MseI AFLP引物组合,得到22对AFLP多态性引物,共含有146个AFLP多态性位点,其片段大小在100～600 bp之间。SSR标记较平均的分布在染色体上,AFLP标记在LG1、LG12及LG14上出现聚集现象。     

大白菜品种鉴定的SSR核心引物筛选及其应用

为筛选出一套大白菜（Brassica rapa L. ssp. pekinensis）简单重复序列（Simple sequence repeat,SSR）的核心引物,以54份大白菜骨干自交系为材料,从已发表的2 129对白菜SSR引物中,初步筛选出分布于白菜整个基因组、具有唯一扩增位点、扩增稳定性及重复性好的多态性SSR引物51对。进一步根据引物的多态性信息量（PIC）、等位基因数量、位点的物理位置和主成分分析（PCA）等分析结果,确定了一套包含28对SSR引物的核心引物组合。比较核心引物组合、51对多态性SSR引物和123个标记（72个SNP标记与51对多态性SSR引物）对54份大白菜骨干自交系的聚类分析结果,三者具有较高的一致性;该套引物可将262份大白菜高代自交系区分开,具有较好的鉴别能力。利用这套核心引物构建了242份大白菜品种的指纹图谱。该研究获得的28对SSR引物可以用于大白菜的遗传多样性分析和品种核酸指纹库构建等研究,可为大白菜新品种的特异性、一致性、稳定性检测体系的建立和新品种保护提供技术支持。     

利用苹果SSR引物分析山楂属植物遗传关系

SSR引物在不同物种间具有通用性,从141对苹果属(Malus spp.)SSR引物中筛选出10对适合于山楂属(Crataegus spp.)植物的SSR引物,并对8个种37份山楂种质资源的遗传关系进行了分析。10对SSR引物共检测到91个多态性谱带,每个位点的等位基因数为3～13个,平均为9.1个。位点杂合度为0.432～0.790,平均为0.688。10对SSR引物可以将20份山楂资源区分开,17份不能区分的资源分为3组,第1组为3个伏山楂品种,第2组和第3组分别包括大果山楂的2个和12个品种。基于SSR标记构建的聚类树状图将供试37份山楂资源分成2个类群,第1类群包括6个山楂野生种,第2类群包括供试的所有伏山楂、山楂和大果山楂资源。该聚类结果与传统形态学分类一致。     

甜樱桃SSR标记的选择性扩增微卫星( SAM) 法筛选

 以甜樱桃‘红灯’为试材, 应用选择性扩增微卫星( SAM) 法分离、克隆了100个SSR序列, 其中81个非重复, 可用。加上搜索数据库所获得的1个SSR序列, 一共82个序列用于特异引物的设计。仅从69个序列的77个基因座设计出特异引物。合成38对特异引物, 对其中的36个基因座进行检测。其中19对引物扩增出相应大小的片段, 另外8对引物扩增出非预期片段。最后, 以27个甜樱桃种质的基因组DNA为模板, 从27对可扩增出带的引物中, 筛选出多态性引物24对, 获得了24个甜樱桃基因座特异性SSR标记。     

不同产地中国李资源遗传多样性SSR分析

利用均匀分布在8个染色体连锁群上的16对SSR引物对来自3类产区的24份中国李品种的遗传多样性进行分析,结果表明：各引物的多态信息含量（PIC）在0.547 ~ 0.783间变化,其中引物CPSCT005最低,引物CPSCT022最高。不同引物间有效等位基因数（Ne）、Nei’s基因多样性（H）和Shannon’s指数（I）分析均存在显著差异,且均为引物CPSCT039最高,引物CPSCT031最低。3类产区所有引物分析表明,Nei’s基因多样性（H）、Shannon’s指数（I）和有效等位基因数（Ne）的关系是：南方品种和北方品种相差不大,均大于国外品种,且南方品种与国外品种先聚到一类。16对SSR引物总共扩出条带86条,其中多态性条带81条,多态性比率为94.19%,平均每对引物扩增位点5.38个。品种聚类分析表明,在遗传距离0.35处,24份中国李材料可分为2大类,大部分北方品种聚为一类,南方品种和国外品种聚为一类,从分子水平支持了以前提出的国外品种起源于中国的说法。     

Detection of variation among clonal selections of grapevine (Vitis vinifera L.) ‘Kishmish Chernyi’ by AFLP analysis

Summary

Clonal selection is an important method for varietal improvement in grapevine. Ampelometric and morphological markers fail to differentiate clones from their parent genotype. Molecular markers offer the opportunity to identify the clonal material. In this study, five clones of the grapevine variety ‘Kishmish Chernyi’ were analysed using microsatellite (SSR) and AFLP markers. These clones differed significantly in their bunch characteristics including berry size, shape, and colour. Microsatellite (SSR) analysis using 24 primers could not distinguish between these clones. The allele profiles of the clones and the parent variety were identical. AFLP analysis using 13 primer pair combinations yielded 592 markers ranging in size from 50 – 500 bp. Of these, 79 markers (13%) were polymorphic. The majority of the polymorphic markers (75/79) were detected in the clone ‘Sharad Seedless’. Three AFLP primer combinations detected unique markers in three clones which could be useful for future identification.     

Analysis of genetic diversity in Portuguese Ceratonia siliqua L. cultivars using RAPD and AFLP markers

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.     

Molecular marker analyses of pistachio rootstocks by Simple Sequence Repeats and Sequence-Related Amplified Polymorphisms

Summary

Simple Sequence Repeat (SSR) and Sequence-Related Amplified Polymorphism (SRAP) molecular marker systems were used to analyse four commercially important pistachio rootstocks: two species of Pistacia atlantica (cv. ‘Standard Atlantica’), P. integerrima (cv. ‘Pioneer Gold’) and two interspecific hybrids of the same, ‘Pioneer Gold II’ (‘PGII’) and ‘University of California at Berkeley 1’ (‘UCB-1’). A total of 35 putative alleles were detected by 12 SSR primer pairs with an average of 2.9 alleles per locus. The number of putative alleles ranged from 2 to 5 in the pistachio rootstocks tested. The number of bands produced by the SRAP protocol was highly variable, ranging from 11 to 38, with an average of 25.2 per primer combination. Eight primer combinations resulted in 104 (51%) polymorphic markers in these samples. SSR and SRAP markers successfully identified all pistachio rootstocks tested from their unique fingerprints. Both SSR and SRAP molecular markers confirmed that the observed variation in ‘UCB-1’ rootstock is genetic.Thus, there will always be variation among ‘UCB-1’ hybrid seedling progeny due to the segregation of alleles when propagated by seed.We also found evidence of contaminating pollen other than from P. integerrima in some hybrid ‘UCB-1’ rootstock progeny produced by closed pollination. Only alleles from the cultivar ‘Standard Atlantica’ were observed in abnormal ‘UCB-1’ rootstock in the nursery. We found that the poor performance of the scion cv. ‘Kerman’ on ‘UCB-1’ rootstock was not due to ‘UCB-1' rootstocks displaying abnormal behaviour in the nursery. We have successfully developed two efficient marker systems for genome analyses in pistachio, which can be used for identification and management in pistachio rootstock production.     

应用RAPD分析新疆主要梨品种的遗传关系

利用RAPD分析了新疆18个梨品种间的亲缘关系。从160个随机十聚体引物中筛选出12条RAPD引物。18份材料共扩增133条带,平均每条引物扩增约11条带,其中14条带为所有材料的共有带,有119个多态性位点,占总带数的89.47%,Nei相似系数为0.5～0.923。用PHYLIP构建MP树,认为(1)对选用的形态学经典分类已较明确的新疆梨、白梨、砂梨、西洋梨、秋子梨,RAPD分析数据完全与其相符;(2)几个芽变品种间高度相似;(3)杂种新梨1号与砀山梨、杂种新梨6号与香梨、杂种早酥梨与苹果梨分别并类;(4)苹果梨独立于供试的5个种之外;(5)库尔勒香梨归属于新疆梨。     

大花蕙兰遗传多样性及亲缘关系的AFLP分析

对来源于日本、韩国和美国的42个大花蕙兰品种和两个国产兰属原生种进行了遗传多样性和亲缘关系的AFLP分析, 9对多态性引物在50～500 bp内共扩增出1 597条带, 其中多态性带1 565条, 多态性比率9810%。单引物对扩增的带数156～193条, 平均每对引物扩增带数177条。42个品种具特征带或缺失带。大花蕙兰品种间的遗传多样性丰富, 品种间的相似系数0.3399 ～0.8223, 平均相似系数0.5783。UPGMA聚类结果将供试品种分为4大类, 与根据花枝类型或花径大小、花色等形态指标分类的结果相吻合, 同一产地来源甚至同一育种公司选育出的品种能基本上聚类在一起, 反映出了品种间的亲缘关系。     

Characterization of white sapote (Casimiroa edulis Llave & Lex.) germplasm using floral morphology,RAPD and AFLP markers

Floral morphology, random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) were used to characterize and verify genetic diversity within a white sapote cultivar collection and to develop molecular markers for germplasm identification. On the basis of floral morphology, the cultivars were classified into three types: type I included 23 cultivars with large ovaries and small anthers; type II included 13 cultivars with small ovaries and large anthers; and type III included one cultivar, named ‘Maltby’, with a large ovary and large anthers. DNA was isolated from 39 cultivars of white sapote and subjected to RAPD and AFLP analysis using 24 and 7 primers, respectively. One hundred and sixty-eight RAPD and 286 AFLP bands were used to assess genetic characterization among white sapote. Sixty percent of the RAPD and 77% of the AFLP amplification products were polymorphic among accessions. RAPD or AFLP markers differentiated all white sapote cultivars effectively. Moreover, each flower type was characterized as specially associated with two RAPD bands. UPGMA dendrograms based on RAPD and AFLP data, showed the majority of the cultivars from flower type I and flower type II clustering together. Finally 101 RAPD markers and 220 AFLP markers were used to construct a neighbor-joining dendrogram. This showed that the 37 cultivars could be classified into six distinct clusters, between which the similarity coefficient was as low as 0.00–0.55, even though the cultivars were morphologically very similar. The remaining two cultivars namely ‘Smathers’ and ‘Maltby’ were found genetically very distant from the other cultivars in RAPD, AFLP or combined RAPD and AFLP based dendrograms. The results suggested that the level of genetic variation among white sapote cultivars is diverse and the morphological and molecular data may lead to representation of the cultivar relationships as well as flower type discrimination.     

Application of five DNA marker techniques to distinguish between five apple (Malus × domestica Borkh.) cultivars and their sports

Summary

Genetic variation between five apple cultivars (‘Golden Delicious’, ‘Gala’, ‘Jonagold’, ‘?ampion’, and ‘Idared’) and ten of their sports (‘Golden Delicious Reinders’, ‘Goldrosio’, ‘Gala Must’, ‘Gala Schniga Schnitzer’, ‘Jonagored’, ‘Jonagold Excel’, ‘Szampion Arno’, ‘Szampion Reno Malinowy’, ‘Idaredest’, and ‘Red Idared’) was investigated using five types of DNA markers: Inter-Simple Sequence Repeats (ISSR), Simple Sequence Repeats (SSR), Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), and Inter-Primer Binding Site (iPBS) amplification. In total, 941 polymorphic amplified fragments were obtained using 12 ISSR, 12 SSR, ten AFLP, 19 iPBS, and 15 S-SAP primers or primer pairs. Four of the above-described techniques (except for SSRs with the primer pairs used in this study) were able to distinguish between the sports and their parental cultivar. The most effective technique to distinguish between the genotypes analysed was S-SAP, which detects variations in DNA regions flanking retrotransposon insertion sites.The combined use of ISSR,AFLP, iPBS, and S-SAP markers identified and distinguished all of the sports tested.     

栽培番茄品种指纹图谱的AFLP分析

以62份栽培番茄为材料,利用65对AFLP引物进行选择性扩增,选出5对多态性丰富、带型清晰的引物进行扩增,共获得85个多态性位点,占总扩增位点的35.41%;选出清晰的16个位点构建了62份栽培番茄的AFLP指纹鉴别图谱。结果表明,由引物组合P55M48、P58M68、P21M49、P77M51、P58M60所构建的DNA指纹鉴别图谱可将供试材料完全区分开。     

Assessment of genetic relationships among 29 introduced and 49 local sweet cherry accessions in Turkey using AFLP and SSR markers

Summary

The characterisation of sweet cherry (Prunus avium L.) genetic resources in Turkey may help to increase their use in breeding programmes worldwide, as Turkey is the centre of origin of sweet cherry. Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were therefore used to analyse genetic diversity among a total of 78 local and introduced sweet cherry cultivars. Four AFLP primer combinations, and six SSR primer pairs for sweet cherry were used for genetic diversity analysis. A genetic similarity matrix was calculated using the combined data from AFLP and SSR analyses with simple matching coefficient. Genetic similarities among the sweet cherry genotypes studied were higher than 42%. No two accessions had an identical AFLP and SSR marker profile, indicating that all 78 genotypes were unique. An UPGMA dendrogram, based on the similarity matrix, revealed 18 separate Groups at or above the 70% similarity level. While some Groups consisted of both introduced and local genotypes, other Groups had only local genotypes. This result suggests that there was broad genetic diversity among the local Turkish sweet cherry genotypes, which was not present in the introduced sweet cherry accessions. The genetic variation present in local Turkish sweet cherry genotypes may be useful for future breeding programmes. We found that the use of both SSR and AFLP marker systems was effective for distinguishing between genetically-close sweet cherry genotypes. These marker systems can be used to complement pomological and morphological markers during the characterisation and identification of sweet cherry genotypes.     

萱草部分野生种和栽培品种亲缘关系的AFLP分析

 借助AFLP标记对35份萱草野生种和栽培品种进行亲缘关系研究, 结果表明, 7对引物组合对萱草共扩增出条带380条, 其中多态性条带357条, 平均多态性达到93.39% , 单对引物扩增条带19～85条, 平均每对引物组合扩增多态性条带51条。7对引物扩增出的多态性条带均超过90.0%。种质资源相似系数为0.3822～0.9656, 平均相似系数为0.7039。UPGMA聚类结果将供试材料分为3类, 即早花、中花和晚花类, 同一产地的品种基本能聚在一起。AFLP标记技术能较好地从分子水平揭示萱草种质资源的亲缘关系。     

Molecular characterization and genetic relationship among almond cultivars assessed by RAPD and SSR markers

RAPDs and SSRs were used to study the genetic diversity of Iranian almond cultivars and their relationship to important foreign cultivars and three related species. Eight unidentified almond Shahrodi cultivars and three wild almonds (Prunus communis, Prunus orientalis and Prunus scoparia) were also included. Of the primers tested, 42 (out of 80) RAPD and 18 (out of 26) SSR primers were selected for their reproducibility and high polymorphism. A total of 664 polymorphic RAPD bands were detected out of 729 bands. The number of presumed alleles revealed by the SSR analysis ranged from 3 to 10 alleles per locus with a mean value of 6.64 alleles per locus. Both techniques discriminated the genotypes very effectively, but only RAPDs were able to discriminate the cultivars Monagha and Sefied. Results demonstrated an extensive genetic variability within the tested cultivars as well as the value of SSR markers developed in peach for characterization of almond and related species of Prunus. Dice similarity coefficient was calculated for all pair wise comparisons and was used to construct a UPGMA dendrogram. For both markers a high similarity in dendrogram topologies was obtained although some differences were observed. All dendrograms, including that obtained by the combined use of both the marker data, depicted the phenetic relationships among the cultivars and species, depending upon their geographic region and/or pedigree information. Almond cultivars clustered with accession of P. communis showing their close relationship. P. orientalis and P. scoparia were clustered out of the rest of P. dulcis.     

枳属36份特异种质的AFLP指纹图谱构建与分析

 从40个Pstl／MseI酶切的AFLP引物对中筛选出多态性高的7个，对36份枳属特异种质进行了指纹分析。7个引物对共获得539条带，其中146条带具有多态性。各种质材料之间的遗传相似性在0．23～ 0．98之间，只需结合其中的3个引物对即可区分所有的材料。根据7个AFLP引物对所得谱带的聚类分析表明，聚类结果与地理来源没有明显的联系，这可能正反映了近时间内地区之间枳资源的频繁交流。