月季种质鉴定和多样性分析

 利用RAPD 技术对28 个月季品种和2 个蔷薇品种进行了遗传多样性分析, 从145 个引物中筛选出12 个引物, 可以扩增出清晰、稳定和多态性高的产物。其中3 个引物从30 个材料中扩增出3 个品种特异的RAPD 分子标记, 并将其中的一个成功地转换成了SCAR 标记。利用筛选出的12 个引物扩增出的65 条多态性片段作了聚类分析, 对这些月季品种的亲缘关系和进化作了初步探讨。     

香榧性别鉴定的RAPD和SCAR标记

为了对香榧幼苗进行早期的性别鉴定,利用RAPD标记技术对香榧品种中的雌性和雄性群体进行了分析。在300条RAPD随机引物中,用引物S250扩增得到了1条大约600bp雄性特异性片段,将其命名为S250-600。进一步对该RAPD标记片段进行回收、克隆和测序,获得了593bp的该标记的核苷酸特定序列。根据对该片段的序列分析结果,设计了1对引物,成功地将RAPD标记转化为SCAR标记,命名为SW-593。将该序列递交至GenBank数据库,接收号为:EU670673。利用该SCAR标记对香榧的雌性和雄性个体进行性别鉴定,结果表明,该SCAR标记在雄性个体中均能稳定出现,说明此SCAR标记是一个与香榧雄性基因连锁的标记,可以用来对香榧品种进行大规模的性别鉴定。     

核桃早实性相关性状的SCAR标记

用RAPD技术对新疆核桃早实特性的分子标记进行了研究。用180个10-mer随机引物分别扩增早实和晚实近等基因池DNA筛选出5个多态性引物,结果只有引物OPG15(5′-ACTGGGACTC-3′)扩增得到1条约710bp的早实核桃特异片段。对该片段进行克隆和序列分析,并根据序列分析结果将该RAPD标记转化为重复性和特异性更好的SCAR标记。研究设计出了1对早实核桃特异SCAR引物P1(5'-ACTGGGACTCCAATTGTATC-3')和P2(5'-ACTGGGACTCTCAACTAT-3'),用这对特异引物对14份材料进行PCR扩增,结果所有晚实核桃材料无任何扩增,但早实材料均扩增出了预期大小759bp的特异带。     

桃果实有毛/无毛性状的SCAR标记

 以桃品种‘京玉’和‘美味’的正反交69株F1 群体为试材, 利用RAPD技术扩增出了与桃果实有毛/无毛性状(G/ g) 连锁的2 258 bp的多态性片段, 经克隆、测序后, 根据获得的序列重新设计了两对引物进行SCAR转化。引物对BFP94 /BFP95在有毛和无毛个体中均扩增出了2 258 bp的片段, RAPD显示的多态性消失。利用引物对BFP96 /BFP98成功将RAPD标记转化成了SCAR标记, 并命名为SCP2022258。该标记仅在果实有毛的个体中出现, 与有毛/无毛性状的连锁距离为718 cM, 且扩增稳定, 为桃果实有毛/无毛性状育种的分子标记辅助选择奠定了基础。     

西瓜品种和育种材料的DNA指纹分析

利用RAPD技术对国内外26份西瓜材料进行遗传亲缘关系研究、构建每份材料的特异指纹。在720个RAPD随机引物中筛选出15个引物用于聚类分析,将供试26份材料分为6个类群1个美国生态型类群、2个中间生态型类群、1个东亚生态型类群、2个野生类群。将京欣一号杂种纯度鉴定的RAPD特异标记K14-1500和N04-600成功转化为SCAR标记,并应用于京欣一号纯度鉴定实践。     

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 。     

桃果实白肉/黄肉性状的RAPD标记向SCAR标记的转化研究

以桃品种京玉和美味的正反交69株F1群体为试材,利用RAPD技术扩增出了与桃果实白肉/黄肉性状连锁的899bp的多态性片段,经克隆、测序后,根据获得的序列重新设计了一对引物进行SCAR转化。利用引物对BFP15/60成功将RAPD标记转化成了SCAR标记,并命名为SCU03-900。该标记仅在果肉为白肉的个体和重组型个体中出现,与白肉/黄肉性状的连锁距离为21cM,且扩增稳定,为桃果实白肉/黄肉性状育种的分子标记辅助选择奠定了基础。     

罗汉果性别相关的SCAR标记筛选

以罗汉果雌雄单株各30份为试材,采用随机引物筛选法,筛选与罗汉果性别相关的RAPD标记,并根据特异片段的测序结果,将RAPD标记转化为特异性和稳定性更好的SCAR标记,以期为罗汉果的性别早期鉴定和分子辅助育种研究提供参考依据。结果表明:在330条随机引物中获得了分别与罗汉果雌性和雄性相关的RAPD引物S2124和S343。根据特异序列测序结果,对雌性特异带S2124-1K和雄性特异带S343-1.4K各自设计并合成了2对SCAR引物,经验证,SCAR引物SC2124-1K-14在62℃和SC2124-1K-8在56℃均可扩增出1 019bp的雌性特异带,SC343-1.4K-12在64℃和SC343-1.4K-4在56℃均可扩增出1 373bp的雄性特异带。这些特异的SCAR标记具有较好的应用价值,可为罗汉果的性别早期鉴定提供分子依据。     

番茄抗根结线虫病基因(Mi)分子标记的评估

利用前人研究获得的2对与番茄根结线虫病基因（Mi）连锁的分子标记,对15份番茄材料进行苗期抗根结线虫病鉴定,以检测其在育种实践中的有效性。结合室内分子标记鉴定和苗期人工接种鉴定,筛选出对番茄根结线虫病免疫的材料1份,高抗根结线虫病的材料3份,感根结线虫病的材料11份。结果表明,引物SCAR1和SCAR2均能检测出抗根结线虫病的特异条带,但SCAR1标记与苗期人工接种的病情指数表现出更大的相关性。说明SCAR1标记更适合应用于番茄抗根结线虫病的辅助选择中。     

黄瓜抗炭疽病相关基因AFLP标记的SCAR转换

将黄瓜抗炭疽病相关基因的一个共显性AFLP标记成功地转换成了简单实用的SCAR标记。对AFLP标记片段进行序列测定,根据序列特点设计了特异的SCAR引物,引物长18～21 bp。PCR结果表明,引物对（1）可以扩增出131 bp和125 bp两条带,引物对（2）可以扩增出178 bp和172 bp两条带,分别为抗炭疽病的特征带和感炭疽病的特征带。将该两个标记命名为SCEM131/125和SCEM178/172,两对引物在F2单株和抗感病材料验证中符合率高达97.27%,可以用于黄瓜炭疽病的分子鉴定。     

Development of sequence characterized DNA markers linked to a temperature dependence for flower induction in lychee (Litchi chinensis Sonn.) cultivars

In this paper, we present a method to find DNA markers for traits of interest in lychee cultivars (Litchi chinensis Sonn.) using high-annealing temperature random amplified polymorphic DNA (HAT-RAPD) as an initial screening method. Using 5 arbitrary random primers, a wide range of polymorphic bands ranging from 200 to 5200 bp were produced. Bands of interest were then selected for sequencing and conversion to the more reproducible and robust sequence characterized amplified region (SCAR) markers. Specifically, SCAR markers were found that distinguished lychee varieties requiring a sustained interval at low temperatures for flower induction versus those varieties that do not require such an environment, and another SCAR marker was found that amplified only the economically important Kom cultivar. These sequences shared similarity to known transposons suggesting a mechanism by which the temperature insensitivity may have initially developed.     

香石竹品种的RAPD 标记

 用随机扩增多态DNA (RAPD) 技术, 选用4 个随机引物, 对87 个大花型香石竹品种总DNA进行随机扩增。4 个引物均得到了稳定的RAPD 图谱。扩增出的片段分子量在500～4300 bp 之间, 每个随机引物扩增出的条带数在8～12 条之间, 共扩增出2113 个条带, 平均每个引物扩增的DNA 条带数为915 条。根据DNA 谱带计算品种间遗传距离, 对87 个品种进行了聚类分析, 分为10 个组群, 组群间差异较大, 组群内差异较小。     

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.     

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.     

不同山楂品种亲缘关系的RAPD分析

为探讨山楂品种间的亲缘关系,采用RAPD技术对20个不同品种的山楂材料进行了基因组DNA多态性分析。从120个引物中筛选出15个10bp的随机引物对所选山楂品种的DNA样品进行PCR扩增,共得到216条谱带,177条表现多态性,多态性比率达81.9%,其中包含27条特异性谱带,揭示了山楂植物丰富的遗传多样性。且利用NTSYS软件和UPGMA法对扩增结果进行了品种间相似系数的计算及聚类分析,结果表明相似系数在0.71～0.87,实生楂与其他山楂品种亲缘关系较远。     

13个阿月浑子品种的RAPD分析

为探明引进的阿月浑子品种间的亲缘关系,采用RAPD 标记技术对引入的12 个国外品种和‘新疆优株’进行了分析。用筛选的21 条10 bp 随机引物进行PCR 扩增可扩增出137 个位点,其中多态位点122 个,多态位点比率89.05%;品种间遗传距离在0. 2015 ~ 0.5163 之间,表明各品种间存在一定的遗传差异。UPGMA 聚类分析表明,13 个阿月浑子品种在遗传距离0.40 处可划分为3 个类群,第1 类包括 ‘Kerman’、‘Larnaka’、‘M-38’、‘M-P3’、‘Ashoury’、‘N’、‘Joley’、‘Aegina’、‘Avidon’、‘B’和‘Peter’11 个品种;第2 类为‘Xinjiang select tree’品种;第3 类为‘Mateur’品种。     

河南17 个桂花品种的RAPD 分析

 采用改良CTAB 法提取河南17 个桂花品种的基因组DNA , 利用RAPD 技术对其进行鉴定和分类研究。从100 个10 bp 的随机引物种筛选出15 个扩增效果较好的引物进行扩增, 共产生121 条带, 其中87 条为多态性带。根据扩增结果进行聚类分析, 得出反映各品种间亲缘关系的树状图。各个品种群内的不同品种间的亲缘关系接近, 而不同品种群间亲缘关系较远。RAPD 对基因组的分析结果与传统分类学的结果基本相符。     

杧果主要品种遗传多态性的AFLP标记研究

 采用扩增片段长度多态性AFLP标记对国内外31个杧果主要品种进行分类与亲缘关系研究。结果表明: 筛选出的14对引物组合在31份杧果种质中共扩增出了1 761条带, 其中多态性带的比例为97%。平均每对引物产生125.8条带和121.6条多态性带, 总的多态性带率为97%; 基于AFLP标记, 以0.48的相似系数为阈值, 所有供试　果材料被分为7大组群, 第一组群内以0.52为阈值, 又可分为6组。与形态标记相比, 基于AFLP标记的杧果分类体系更能反映杧果品种间的亲缘关系。     

中国北方引种的部分越橘品种的RAPD分析

利用RAPD技术对我国北方引种的15份越橘(Vaccinium spp.)栽培品种进行了遗传多样性分析。从70条随机引物中筛选出15条引物,共扩增出94个位点,其中多态性位点83个(88.3%)。材料间遗传相似系数变化为0.5426～0.8936,表明各品种间具有一定的遗传差异。UPGMA聚类分析结果可将供试材料分为2大类、5个亚类。聚类结果与各品种的杂交系谱相对应,从分子水平揭示了品种间的亲缘关系。