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。     

与“全红”瓯江彩鲤体色相关的SRAP及SCAR分子标记

利用相关序列扩增多态性(Sequence Related Amplified Polymorphism,SRAP)技术分析"全红"和"粉玉"瓯江彩鲤,筛选与瓯江彩鲤体色相关的分子遗传标记。从88个SRAP引物组合筛选出的12个引物组合共获得扩增条带104个,并筛选出1个SRAP特异扩增带,即"全红"瓯江彩鲤家系SR2,7173 bp带。该条SRAP特异扩增条带经回收、克隆和测序,并将测序结果进行BLAST分析,发现该片段在GenBank中与斑马鱼的POl多蛋白基因和尿红素基因有较高的同源性。根据序列信息分别设计了4对正、反向引物(22—26 bp)。用4对引物分别在"全红"瓯江彩鲤F2和"粉玉"瓯江彩鲤F2群体中进行PCR扩增,仅发现SC-3(154 bp)能够在"全红"瓯江彩鲤群体中特异扩增,而且在"粉玉"瓯江彩鲤F2群体中未出现此扩增带。采用大样本对该SC-3标记进行验证,结果发现,在"全红"瓯江彩鲤群体中呈现阳性,而在"粉玉"瓯江彩鲤群体中为阴性,可以区分这两种群体。因此SC-3标记可以作为"全红"瓯江彩鲤群体一个重要的分子遗传特征指标,为进一步进行分子标记辅助育种奠定了基础。     

起始密码子区域相关序列多态性的开发及在苜蓿遗传多样性上的应用

目前广泛使用的基于PCR基础的分子标记多为扩增非编码区域,或是随机基因组中扩增,在QTL定位中得到的位点一般与目标性状基因距离较远,我们开发了一个新的基于启动子序列目的基因型分子标记技术——启动子区域相关序列多态性（SCRP）,试图使标记能够更为准确的反映不同品种的遗传基础。它利用启动子位置保守一致序列 （“Kozak”序列） 作为其上游引物,利用内含子富含“AATT”的特性,作为核心序列设计下游引物,上下游引物均为18bp,引物间通过组合配对的方式作为扩增引物对。设计了14条上游引物和10条下游引物,共140对引物组合,对34个苜蓿品种进行扩增,研究了34个苜蓿的遗传多样性。每个PCR反应产生3~16个50~2000bp的条带,结果表明该标记简单、可靠、具有较高多态性,并且扩增区域为一种目的基因型分子标记,在种质资源研究中具有重要价值。     

番茄耐低温相关基因的SRAP标记筛选

以番茄耐低温和不耐低温基因组DNA为材料进行SRAP分析,共筛选了225对SRAP引物,其中27对引物在两池之间表现差异,经测序只有Me2Em5扩增出与番茄耐低温相关的差异性片段,大小约为273bp,该片段仅在耐低温植株中稳定扩增。经Blast分析比对,该片段与已报道的PEG和低温诱导后在沙冬青幼苗中表达基因的cDNA片段同源。根据差异片段序列设计特异引物,将M2E5—273标记成功转化为更稳定的SCAR标记。     

SCAR标记技术鉴别榆黄蘑品种

为了建立一套基于DNA分子标记技术快速鉴定榆黄蘑菌株的有效方法,本研究通过对生产上常用的16个榆黄蘑菌株进行SRAP多态性分析,从榆黄蘑2号菌株中扩增获得了一个片段长为537bp的特异片段,将其克隆、测序并设计引物,成功转化为稳定的SCAR标记。试验结果表明,利用该特异SCAR标记,能在1d时间内准确鉴定出榆黄蘑2号菌株。由此可见,SCAR分子标记是一种快速、稳定、准确鉴定榆黄蘑菌株的新方法。     

RAPD及SRAP两种分子标记技术对苦瓜杂交种纯度的鉴定分析

以F1代苦瓜杂交种如玉11号及其亲本为材料,利用RAPD及SRAP两种分子标记技术对这3种苦瓜基因组DNA进行比较分析,以获得该杂交种及其亲本（或母本）差异目的基因片段。经过多次对该3种苦瓜叶片DNA提取,PCR扩增及其PCR产物的琼脂糖凝胶电泳分析,在供试的46个RAPD引物及121对SRAP引物中,筛选出1个RAPD引物及1对SRAP引物能区分该苦瓜杂交种及其母本种子,通过进一步验证分析,证明该两种分子标记的特异引物可作为如玉11号苦瓜杂交种子的纯度鉴定之用。     

SCAR标记在黑木耳栽培菌株分类鉴定中的应用

应用序列特异性扩增区域（SCAR）标记技术分析50株黑木耳栽培菌株的遗传多样性。在SRAP标记分析过程中发现1条1 200 bp的特异性条带,经回收克隆测序转为SCAR标记。根据序列设计出1对特异性引物,经PCR可以扩增出1 000 bp大小的片段,说明成功构建出了＂黑931＂的指纹图谱。     

甘蓝品种'争春'和'寒光2号'的DNA指纹图谱构建

用SDS法提取甘蓝（Brassfca oleraceavat．capitata）品种‘争春’、‘寒光2号’及其各自亲本的基因组DNA,通过SRAP、RAPD两种分子标记方法,构建其DNA指纹图谱,用于种子纯度鉴定。利用30对SRAP引物组合和200个RAPD随机引物,以各品种及其亲本的基因组DNA为模板组进行筛选,结果显示：多数SRAP引物组合对模板组的扩增带型一致,少数组合扩增出差异,但未能找到具有互补差异的引物组合;通过RAPD标记方法筛选出能鉴定2个品种纯度的引物分别为S42、S103、S193和S42、S89、S151,其中引物S42对2组材料均能扩增出特异的RAPD指纹图谱,并将RAPD指纹图谱转变为相应的数字指纹。     

菠菜性别相关SRAP分子标记的筛选及分析

菠菜(2n=2x=12)是研究雌雄异株植物性别分化的一种理想材料。本研究利用SRAP (sequence re-lated amplified polymorphism)分子标记方法,采用256对引物组合对菠菜雌、雄基因池进行筛选,后利用聚丙烯酰胺凝胶电泳和琼脂糖凝胶电泳进行检测。结果表明,经过对256对引物组合进行筛选,其中20对引物组合扩增效果较好,共获得47个雌性特异片段和29个雄性特异片段,其多态性达到19.9%;在这20对引物组合中,引物对em11+me14、em10+me10经琼脂糖凝胶电泳验证扩增效果最好,可以扩增出菠菜雄、雌稳定的分子标记。本研究获得的性别特异标记可以用于菠菜幼苗期性别鉴定,同时为菠菜性别相关基因的克隆奠定了基础。     

甘蓝Ogura胞质雄性不育基因的SRAP标记

本研究以甘蓝Oguar胞质雄性不育系CMS451及其保持系为材料,优化甘蓝SRAP体系,并用SRAP标记分析了Oguar胞质不育基因。结果表明:总反应体积为25μL,含有Mg Cl2,2.5 mmol/L;primer,0.2μmol/L;DNA,45ng;NTPs,0.4 mmol/Ld;1.5 U Taq DNA聚合酶,2.5μL10×buffer,其余用dd H2O填充;这个体系适合甘蓝的SRAP标记。利用92对SRAP组合引物和优化的SRAP体系,发现Me6Em8扩增出稳定的特异条带,特其大小在250-500bp之间,对其进行单株验证,用Jionmap ver3.0分析计算出此条带与不育基因的遗传距离为22.548c M。     

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.     

The Drosophila homologue of the dystrophin gene - introns containing promoters are the major contributors to the large size of the gene

We show that the drosophila gene encoding the dystrophin-like protein (DLP) is as complex as the mammalian dystrophin gene. Three 5' promoters and three internal promoters regulate the expression of three full-length and three truncated products, respectively. The existence of this complex gene structure in such evolutionary remote organisms suggests that both types of products have diverse important functions. The promoters of both the DLP gene and the mammalian dystrophin gene are located in very large introns. These introns contribute significantly to the large size of the genes. The possible relevance of the conservation of the large size of introns containing promoters to the regulation of promoter activity is discussed.     

Genetic diversity of endangered Manglietia patungensis assessed by inter simple sequence repeat and sequence-related amplified polymorphism markers

Manglietia patungensis Hu is an endangered plant native to China. Knowledge of its genetic diversity and structure would aid its conservation. This study assessed nine natural populations of M. patungensis using two methods: inter simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) markers. Using 10 ISSR primer pairs, 334 bands were generated, and 10 SRAP primer pairs generated 276 bands. The percent of polymorphic bands (91.32% and 93.48%), Nei's genetic diversity (0.3448 and 0.3323), and Shannon's information index (0.5075 and 0.4935) revealed a high level of genetic diversity at the species level. Total heterozygosity was 0.3439 by ISSR and 0.3281 by SRAP. The mean heterozygosity was 0.2323 by ISSR and 0.2521 by SRAP. The coefficient of genetic differentiation among natural populations was 0.3245 by ISSR and 0.2316 by SRAP. These data indicated higher levels of genetic diversity of M. patungensis within, rather than among, populations. Estimates of gene flow among natural populations were 1.0411 and 1.0589, which implied a certain amount of gene exchange among populations. A Mantel test revealed no significant correlation between genetic and geographic distance. ISSR and SRAP markers are both effective for genetic diversity research in M. Patungensis. Based on these results, conservation of M. patungensis should be performed both in situ and ex situ.     

Genetic diversity assessment of a germplasm collection of Salvia miltiorrhiza Bunge. based on morphology,ISSR and SRAP markers

Salvia miltiorrhiza is one of the most important traditional Chinese medicinal plants for its therapeutic effects. In the present study, morphological traits, ISSR (inter-simple sequence related) and SRAP (sequence-related amplified polymorphism) markers were used to analyze the genetic diversity of 59 S. miltiorrhiza phenotypes. Out of the 100 ISSR primers and 100 SRAP primer combinations screened, 13 ISSRs and 7 SRAPs were exploited to evaluate the level of polymorphism and discriminating capacity. The results showed that the 13 ISSRs generated 190 repeatable amplified bands, of which 177 (93.2%) were polymorphic, with an average of 13.6 polymorphic fragments per primer. The 7 SRAPs produced 286 repeatable amplified bands, of which 266 (93.4%) were polymorphic, with an average of 38.1 polymorphic fragments per primer. Cluster analysis readily separated different morphological accessions, wild and cultivated controls based on morphological traits, ISSR and SRAP markers. The study indicated that morphological traits, ISSR and SRAP markers were reliable and effective for assessing the genetic diversity of phenotypic S. miltiorrhiza accessions. The overall results suggested that the introduction of genetic variation from morphology-based germplasms enlarged the genetic base for the collection, conservation and further breeding program of S. miltiorrhiza germplasm.     

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。     

SRAP分子标记分析西瓜遗传多态性

目的:探讨西瓜遗传多样性和遗传基础。方法:采用SRAP分子标记对西瓜品种D1、D2、D3、H1、H2、H3、M1、M2、M3、m1、m2、m3的多态性进行了分析。结果:每对引物组合产生13~25对比较清晰的扩增带.8对引物组合共产生131条扩增带。平均每对引物组合产生16.375条。8对引物组合共产生多态性带37条,每对引物组合产生3~7条,平均4.625条。每对引物组合产生的多态性带的比例为16.666%~38.464%,平均为28.675%。另外,对银染过程进行了优化。结论:SRAP标记多态性还是较高的,可以适于分析西瓜等遗传差异小的作物。