SRAP标记及其在植物遗传育种中的应用

SRAP标记是基于选择性扩增开放性阅读框的新型分子标记,具有简便、高效、重复性好、高共显性等优点,在植物育种中已经得到广泛应用.本文介绍了SRAP标记基本原理和特点,对SRAP标记在遗传多样性研究、遗传连锁图谱构建、比较基因组以及分子标记辅助选择育种等方面的应用进行了综述.     

美洲黑杨遗传差异的SRAP和EST-SSR分析

采用SRAP和EST-SSR分子标记对美洲黑杨I-69及与其有亲缘关系的4个美洲黑杨品种进行遗传差异分析,比较两种分子标记在遗传差异性分析中的适用性,为美洲黑杨的鉴别提供准确的分子技术依据。结果表明:(1)以SRAP标记筛选出21对引物组合,共扩增出287条谱带,多态性条带209条,多态性比率72.8%,遗传相似系数为0.548 1~0.769 2。(2)以EST-SSR标记筛选出17对引物,共扩增出86条谱带,多态性条带69条,多态性比率80.2%,遗传相似系数为0.444 4~0.717 2。(3)对SRAP和EST-SSR以及两者混合数据形成的3个遗传相似矩阵进行相关性分析结果显示,SRAP和EST-SSR分别同综合数据之间呈显著相关(r=0.844 2,r=0.830 8)。(4)聚类分析发现,两种分子标记的聚类结果有一定差异,SRAP聚类结果同综合数据分析的结果一致,说明SRAP标记更适用于杨树亲缘关系较近材料的遗传差异分析。     

虎杖种质资源的分子标记研究

本文应用RAPD、ISSR和SRAP标记对26份虎杖种质资源遗传多样性进行检测.在22个引物中有17个引物(77.3%)扩增产物具多态性,多态性水平相对较高.22个引物共得到98条扩增DNA片段,其中90.8%具有多态性.每个多态性引物平均可扩增出5.24个多态性片段.聚类分析表明,利用BAPD、ISSR和SRAP技术相结合可将全部供试材料区分开,26份材料在Gs值0.54水平上全部聚为一类,以所有材料间的平均遗传相似遗传系数0.71为阈值,将其分为11类.虎杖种质资源在分子水平上确实存在较大遗传差异,RAPD、ISSR和SRAP标记可作为构建虎杖DNA指纹图谱的有效工具.     

基于SRAP分子标记的冰草遗传连锁图谱构建

构建高密度遗传连锁图谱是冰草抗性、品质、产量等重要性状QTL精细定位及标记辅助育种研究的基础。该试验以四倍体杂交冰草F2群体的202个分离单株及其亲本为材料,利用SRAP分子标记技术和Join Map 4.0作图软件对冰草的遗传连锁图谱进行了构建。结果表明:(1)共筛选出22对多态性好、标记位点清晰稳定的SRAP适宜引物,对冰草杂种F2分离单株的基因组DNA进行PCR扩增,共获得510个SRAP多态性标记位点,其比率占88.2%。(2)偏分离分析表明,偏分离标记比率仅为14.12%,符合遗传作图的要求。(3)成功构建了冰草的SRAP分子标记遗传连锁图谱,该图谱有14个连锁群、510个标记,连锁群间长度范围86.4～179.0cM,覆盖基因组总长度1 912.9cM,标记间平均间距3.75cM,为高密度遗传图谱。     

SRAP结合SCoT标记分析番木瓜种质的遗传多样性

利用SRAP和SCoT两种分子标记相结合对中国22个番木瓜主要栽培品种(系)进行遗传多样性研究。SCoT标记检测获得的遗传多样性参数值和遗传相似系数范围均高于SRAP标记检测的结果,表明SCoT标记检测多态性的能力高于SRAP标记。基于两种标记数据合并后的UPGMA聚类结果显示,番木瓜种质间的遗传相似系数为0.65~0.90,种质间遗传多样性水平较低;在遗传相似系数为0.82时,将所有参试材料划分为3个类群。应用Mantel检测对SRAP和SCoT及两种标记合并进行相关性分析,表明三者之间具有显著的相关性,且相关性很高。聚类结果从分子水平反映出中国番木瓜主要栽培品种(系)的遗传基础狭窄。     

新型标记SRAP在棉花F2分离群体及遗传多样性评价中的适用性分析

将新型分子标记SRAP(Sequence-related Amplified Polymorphism)应用于棉花的遗传研究,并建立了完整的PCR反应体系,此体系稳定可靠、扩增效果好、可重复性强。采用30个SRAP引物组合对海岛棉品种“Pima90”和陆地棉品种“邯郸208”进行比较扩增,29个引物组合可以获得多态性扩增,显示了较高的多态性。对上述两个品种的F2群体进行检测,共产生149个多态性条带,平均每个组合产生5．14个,单引物组合最多可产生13个多态性条带。用SRAP标记对11份陆地棉材料进行遗传多样性检测,30个引物组合中15个组合有多态性,得到22个多态性条带,显示了较高的多态性比率。研究结果表明,SRAP标记可在棉花分子生物学领域中广泛应用。     

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。     

RSAP、SSR和SRAP分析马铃薯遗传多样性的应用比较

本研究分别利用RSAP、SSR和SRAP对15份马铃薯种质进行遗传多样性分析,比较3种分子标记的分析效力。结果表明,平均每对引物扩增出的多态性位点RSAP(5.75个)SSR(6.33个)SRAP(7.75个);RSAP+SSR+SRAP联合聚类的结果与SRAP和RSAP聚类结果基本一致,相关性分别达到极显著和显著水平,与SSR的聚类结果基本相似,呈显著相关性。在分析马铃薯遗传多样性中,SRAP标记的效果最优,SSR标记效果略优于RSAP标记。RSAP分子标记能较好地分析马铃薯的遗传多样性,而以RSAP+SSR+SRAP联合分析,则能更好地评估种质的遗传多样性和亲缘关系。     

红掌品种亲缘关系SRAP分析

利用相关序列扩增多态性（SRAP）分子标记,从100对引物组合中筛选出 26对多态性高、条带清晰的SRAP引物,对33个红掌品种进行遗传多样性和亲缘关系分析。结果如下：（1）26对引物共扩增出366条条带,其中有314条多态性条带,多态性比率为85.79%。引物组合产生的条带数在9～23之间,平均每对引物组合扩增出14.1条和12.1条多态性条带。（2）根据SRAP扩增结果,利用UPGMA法进行聚类分析,33份材料的遗传相似系数在0.55～0.94之间,在遗传相似系数0.786处可将33个红掌品种分为5个类群。结果表明,供试品种遗传多样性丰富,本研究为品种鉴定和杂交育种提供了参考信息。     

相关序列扩增多态性(SRAP)标记及其应用研究进展

SRAP是一项基于PCR技术的分子标记技术,利用其独特的引物设计对基因组的开放阅读框(ORFs)进行特异扩增,利用个体以及物种的内含子、启动子和间隔序列的不同,产生基于内含子和外显子的SRAP多态性。阐述了SRAP的原理和流程,详细论述了SRAP标记目前在植物遗传多样性、作物品种鉴定、遗传图谱构建等方面的研究进展及应用前景。     

SRAP技术研究烟粉虱遗传多样性

采用AFLP、SRAP2种标记方法分别对2个烟粉虱Bemisia tabaci Gennadius种群(一品红、甘蓝)的多态性进行分析。结果表明,(1)2种方法平均每对引物组合产生的条带数分别为29.4和21.8。(2)AFLP法每对引物组合产生10～23条多态性带,平均17.20条,多态性带的比例平均为57.93%。SRAP法每对引物组合产生5～18条多态性带,平均13.3条,多态性带的比例平均为60.59%。(3)前者的基因多样性范围为0.1503～0.2838,平均为0.2297;后者的基因多样性范围为0.0977～0.2911,平均为0.2332。证明利用SRAP技术和AFLP技术研究烟粉虱的遗传多样性是有效的。     

Development of SRAP,SNP and Multiplexed SCAR molecular markers for the major seed coat color gene in <Emphasis Type="Italic">Brassica rapa L.</Emphasis>

Seed coat color inheritance in B. rapa was studied in F(1), F(2), F(3), and BC(1) progenies from a cross of a Canadian brown-seeded variety 'SPAN' and a Bangladeshi yellow sarson variety 'BARI-6'. A pollen effect was found when the yellow sarson line was used as the maternal parent. Seed coat color segregated into brown, yellow-brown and bright yellow classes. Segregation was under digenic control where the brown or yellow-brown color was dominant over bright yellow seed coat color. A sequence related amplified polymorphism (SRAP) marker linked closely to a major seed coat color gene (Br1/br1) was developed. This dominant SRAP molecular marker was successfully converted into single nucleotide polymorphism (SNP) markers and sequence characterized amplification region (SCAR) markers after the extended flanking sequence of the SRAP was obtained with chromosome walking. In total, 24 SNPs were identified with more than 2-kb sequence. A 12-bp deletion allowed the development of a SCAR marker linked closely to the Br1 gene. Using the five-fluorescence dye set supplied by ABI, four labeled M13 primers were integrated with different SCAR primers to increase the throughput of SCAR marker detection. Using multiplexed SCAR markers targeting insertions and deletions in a genome shows great potential for marker assisted selection in plant breeding.     

High throughput genome-specific and gene-specific molecular markers for erucic acid genes in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.) for marker-assisted selection in plant breeding

A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5' primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.     

三色堇与角堇种间遗传连锁图谱构建

该研究以二倍体三色堇和角堇为亲本杂交产生的66株F2代分离群体为作图群体,采用SRAP标记技术进行基因分型,利用JoinMap4.0软件构建了首张三色堇与角堇的种间遗传连锁图谱.结果表明:(1)从256对SRAP引物组合中筛选获得50对多态性好、标记位点清晰且稳定的引物组合.(2)通过对三色堇F2代群体的PCR扩增,共...     

应用SRAP标记分析黄瓜的遗传差异

利用49对SRAP引物对4种不同类型28份黄瓜种质资源进行了遗传差异分析.结果表明,有35对引物扩增出多态性,在28份资源间共产生724条扩增带,平均每对引物组合产生20.69条;共检测出337个多态性位点,多态性比率为46.5%,每对引物平均为96.3个.利用NTSYS软件分析遗传相似系数,UPGMA方法聚类分析表明,28份资源可聚为两大类.试验结果表明SRAP标记位点多,重复性好,可以揭示不同类型黄瓜种质之间的遗传基础.     

湖北河岸带植物中华蚊母树遗传多样性的SRAP分析

利用SRAP分子标记技术,对湖北省河岸带植物中华蚊母树的4个自然居群和1个迁地居群的遗传多样性和遗传结构进行了分析。结果表明,中华蚊母树物种具有较高水平的遗传多样性,7对SRAP引物进行PCR扩增的多态性位点百分率(PPF)为80.43%,每个位点的等位基因数(A)为2,有效等位基因数(Ae)为1.34,总遗传多样性Nei’s基因多样性指数(Hp)为0.215 9,Shannon信息多样性指数(I)为0.350 9。在居群水平上,5个居群总的遗传变异Ht为0.218 8,居群内的遗传变异Hs为0.193 4,居群间的遗传分化系数Gst为0.116 1,表明在总的遗传变异中有88.39%变异存在于居群内,仅11.61%存在于居群间,居群间的基因流Nm为3.807 2,表明居群间有较大程度的基因交流。UPGMA聚类分析和主成分分析显示中华蚊母树主要分为两个居群组,在长江三峡沿岸香溪和乐天溪由于遗传距离比较近聚为一小类再与高家堰聚为一大类,而沿渡河和三峡植物园居群聚为另一大类,表明迁地居群三峡植物园的中华蚊母树与来自巴东沿渡河居群的样本亲缘关系最近,且三峡植物园迁地保护居群基本保育了其遗传多样性总水平。同时在分析讨论了中华蚊母树遗传多样性与其繁育系统、生境及其起源进化的关系的基础上,评价了中华蚊母树的保护策略,并在评价保护成果的基础上,提出了今后进一步保育的策略。结果还表明SRAP标记是分析中华蚊母树遗传多样性和遗传结构非常可靠的一种标记,而且这是使用SRAP标记研究中华蚊母树的首次报道。     

SRAP技术在遗传的研究进展

SRAP是一种新型的DNA分子标记,具有简便、稳定、中等产率和容易得到选择条带序列的特点。SRAP利用独特的引物设计对开放读码框(ORFs)进行扩增,上游引物长17bp,对外显子进行特异扩增,下游引物长18bp,对内含子区域、启动子区域进行特异扩增,因个体不同及其物种的内含子、启动子与间隔长度不等而产生多态性。本文阐述SRAP的原理和操作流程,综述了SRAP标记目前在植物遗传图谱构建、遗传多样性、基因定位、基因克隆、杂种优势利用等方面的研究进展及应用前景。     

Inheritance of seed coat color genes in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.) and tagging the genes using SRAP,SCAR and SNP molecular markers

Seed coat color inheritance in Brassica napus was studied in F₁, F₂, F₃ and backcross progenies from crosses of five black seeded varieties/lines to three pure breeding yellow seeded lines. Maternal inheritance was observed for seed coat color in B. napus, but a pollen effect was also found when yellow seeded lines were used as the female parent. Seed coat color segregated from black to dark brown, light brown, dark yellow, light yellow, and yellow. Seed coat color was found to be controlled by three genes, the first two genes were responsible for black/brown seed coat color and the third gene was responsible for dark/light yellow seed coat color in B. napus. All three seed coat color alleles were dominant over yellow color alleles at all three loci. Sequence related amplified polymorphism (SRAP) was used for the development of molecular markers co-segregating with the seed coat color genes. A SRAP marker (SA12BG18388) tightly linked to one of the black/brown seed coat color genes was identified in the F₂ and backcross populations. This marker was found to be anchored on linkage group A9/N9 of the A-genome of B. napus. This SRAP marker was converted into sequence-characterized amplification region (SCAR) markers using chromosome-walking technology. A second SRAP marker (SA7BG29245), very close to another black/brown seed coat color gene, was identified from a high density genetic map developed in our laboratory using primer walking from an anchoring marker. The marker was located on linkage group C3/N13 of the C-genome of B. napus. This marker also co-segregated with the black/brown seed coat color gene in B. rapa. Based on the sequence information of the flanking sequences, 24 single nucleotide polymorphisms (SNPs) were identified between the yellow seeded and black/brown seeded lines. SNP detection and genotyping clearly differentiated the black/brown seeded plants from dark/light/yellow-seeded plants and also differentiated between homozygous (Y2Y2) and heterozygous (Y2y2) black/brown seeded plants. A total of 768 SRAP primer pair combinations were screened in dark/light yellow seed coat color plants and a close marker (DC1GA27197) linked to the dark/light yellow seed coat color gene was developed. These three markers linked to the three different yellow seed coat color genes in B. napus can be used to screen for yellow seeded lines in canola/rapeseed breeding programs.     

辣椒31个优良自交系的亲本类群分析

以包含我国重要尖椒品种的亲本材料在内的31份优良自交系为材料, 利用SRAP标记和基因型值分析技术开展了辣椒自交系间遗传差异的分析与类群划分研究。结果表明: 在30个引物组合中, 27个引物组合可以 在自交系间扩增出多态性条带, 共扩增出310个多态性条带, 平均每个引物组合产生11.5个多态性条带, 显示出SRAP技术具有较强的分析效率; 基于SRAP标记和Yule相似系数对这些自交系进行的聚类分析中, 可以基本区分辣椒的2个变种(C. annuum var. grossum和C. annuum var. longum), 而且可以反映出自交系间的亲缘及系谱关系; 在相似系数为0.67处, 可将这31个自交系分为4个类群; 基于基因型值和标准Euclidean距离对这些自交系进行的聚类分析可成功地将辣椒的两个变种完全区分; 在遗传距离约4.5处, 可将这31个自交系分为4个类群; 自交系间基于SRAP标记与基因型值的遗传距离存在一定的相关性。