SNP检测方法的研究进展

作为近年来最有发展潜力的第三代分子标记,单核苷酸多态性(single nucleotide polymorphism,SNP)在遗传分析中得到了广泛应用。目前SNP的检测方法大致可以分为两大类:一大类是以单链构象多态性(SSCP)、变性梯度凝胶电泳(DDGE)、酶切扩增多态性序列(CAPS)、等位基因特异性PCR(allele-specific PCR,AS-PCR)等为代表的以凝胶电泳为基础的传统经典的检测方法。另一大类是以直接测序、DNA芯片、变性高效液相色谱(DHPLC)、质谱检测技术、高分辨率溶解曲线(HRM)等为代表的高通量、自动化程度较高的检测方法。本文综述了两大类SNP检测方法中主要检测技术的原理和应用,并分析在实际应用中各种检测技术的优缺点。     

木薯块根肉质颜色基因CAPS标记的开发与验证

木薯块根肉质颜色是选育木薯品种尤其是食用木薯品种最重要的性状之一。前人研究表明木薯块根肉质的黄色与白色是由八氢番茄红素基因PSY2中的一个单核苷酸多态性(SNP)位点变异决定。本研究根据木薯PSY2的不同等位基因的DNA序列,开发了一个酶切扩增多态性序列(CAPS)标记,可在黄肉木薯中扩增出194 bp+66 bp的条带,在浅黄色木薯中扩增出194 bp+172 bp+66 bp的条带,在白色木薯中扩增出172 bp+66 bp的条带。利用该标记对木薯品种"新选048"的自交系后代单株进行验证,结合测序结果和田间块根肉质颜色,表明该标记的选择准确率高达100%。该CAPS标记的获得对于木薯分子标记辅助育种,加速木薯品种尤其是食用型品种的选育具有重要意义。     

水稻抗褐飞虱基因Bph3特异性分子标记开发及应用

Bph3是一个对褐飞虱具有广谱、持久抗性的主效基因,对水稻抗褐飞虱育种具有重要利用价值,但其至今仍缺乏基因特异性标记,限制了分子辅助育种中对它的高效利用。本研究通过对Bph3不同等位基因编码区进行测序比对,获得两个Bph3特异性SNP位点SNP1(Bph3为T,其余为C)和SNP2(Bph3为C,其余为T)。在等位基因特异性PCR(allele-specific polymerase chain reaction,AS-PCR)方法的基础上,开发两对显性标记特异扩增两个SNP位点来分别鉴定Bph3和其余等位基因型,组合使用两对显性标记即可获得一套鉴定Bph3基因型的共显性标记。使用该套标记对水稻品种基因组DNA进行PCR扩增,鉴定PCR产物条带大小即可区分Bph3不同基因型。利用该方法对Bph3基因进行分子标记辅助选择育种,可快速有效的将其导入目标水稻品种,加快水稻抗褐飞虱品种选育进程。     

水稻Wx复等位基因基于PCR的功能标记开发与利用

直链淀粉含量是影响稻米食味品质的重要因素,主要由蜡质基因(Wx)调控,Wx基因座存在Wxa、Wxb、Wxin、Wxmw等多个复等位基因,通过分子标记辅助选择(marker-assisted selection,MAS)培育中低直链淀粉含量的水稻品种是提高稻米适口性的重要途径。Wx复等位基因的功能由单碱基核苷酸多态性(single nucleotide polymorphisms,SNP)决定,其检测依赖于测序或酶切,存在耗时多、费用昂贵等缺点。为提高选择效率,本文利用Wx复等位基因的SNP差异和四引物扩增受阻突变(tetra-primer ARMS-PCR)原理,进行PCR功能标记的开发。引物设计过程中,针对扩增效率低和非匹配的延伸2个技术难题,提出了调整错配位点的解决方案,成功开发了基于PCR技术的两对功能性标记Waxygt-ARMS2和Waxyac-ARMS2,可以准确区分上述复等位基因型,且具有操作简单、耗费较低的特点。利用新开发标记对辽宁省育成的部分水稻品种进行Wx基因型检测的结果表明,40份辽宁育成品种均携带Wxb基因,遗传基础单一。上述结果为利用Wx基因位点的分子标记培育中低直链淀粉含量的水稻品种,改良辽宁省水稻品种的食味品质提供了技术支撑。     

油菜抗咪唑啉酮类除草剂基因BnALS1R等位基因特异PCR标记的开发与应用

油菜抗咪唑啉酮类除草剂基因BnALS1R是从抗性突变体M9中克隆获得,抗性基因BnALS1R与野生型基因BnALS1存在1处SNP,即乙酰乳酸合酶第638位丝氨酸残基被天冬酰胺酸替代。为获得油菜抗除草剂基因BnALS1R的分子标记,根据该处点突变,结合获得的BnALS3与BnALS1序列,开发30条等位基因特异PCR (allele-specific PCR,AS-PCR)引物,采用筛选出的3条AS-PCR引物在F₂、BC₁和BC₂群体中进行PCR扩增。结果表明,该标记有效检测出群体中存在的3种基因型,其分离比分别为1∶2∶1、1∶1、1∶1,均遵循单基因遗传规律。应用该标记对获得的抗性恢复系进行PCR扩增,结果发现所有抗性恢复系均能扩增出抗性基因BnALS1R目的条带,表明3条标记引物可应用于抗性基因的检测。AS-PCR标记的获得将促进以抗性基因进行油菜抗除草剂分子标记辅助选择育种。     

水稻抗稻瘟病基因Piz-t特异性分子标记开发及应用

Piz-t是一个具有广谱稻瘟病抗性的基因,对于选育稻瘟病抗性品种具有重要的应用价值,但其所在位点多个不同的等位基因限制了对它的筛选及利用。本研究通过将Piz-t不同等位基因及其上下游测序,比对鉴定到一个Piz-t特异性SNP位点(Piz-t为A,其余均为G)。在两对交叉引物PCR(PCR with confronting two-pair primers,PCR-CTPP)方法的基础上加以改进开发了一套鉴定该SNP的标记,利用该标记及待检测水稻品种基因组DNA进行PCR扩增,通过不同PCR产物条带大小将Piz-t与其它等位基因区分开。该方法简便快速、成本低廉,可广泛应用于分子标记辅助选择育种或者水稻种质资源Piz-t基因型鉴定。     

水稻稻瘟病抗性基因Pi25功能性SNP分子标记开发及应用

为了使稻瘟病抗性基因Pi25在水稻分子育种中得到高效和快速利用,本研究通过将Pi25不同等位基因测序及序列比对鉴定到一个Pi25基因内特异SNP位点。使用PCR-CTPP(PCR with confrontingtwo-pair primers)的方法加以改进开发了一套鉴定该SNP的功能性分子标记,利用该标记及待检测水稻品种基因组DNA进行PCR扩增,通过不同PCR产物条带大小将Pi25与其它等位基因区分开,可快速判断待测水稻Pi25的基因型。本方法快速简单、成本低廉,可广泛应用于水稻种质资源Pi25基因型鉴定和分子标记辅助选择育种。     

甜瓜单性花相关基因CmACS-7的分子标记开发与应用

甜瓜(Cucumismelo L.)性型中主要是雄花、两性花同株型(雄全同株,简称两性花),雌雄异花同株型(单性花)比较少见。甜瓜育种工作中为提高制种效率,往往将母本系转育成单性花。传统方法筛选单性花植株耗时长,占地面积大。根据CmACS-7基因序列设计引物进行PCR扩增,甜瓜9份材料均可获得188 bp的片段。多序列比对发现,材料单性花性状与扩增片段的碱基变异相关联,关联位点为限制性内切酶AluⅠ的识别位点。扩增产物进行酶切,甜瓜单性花材料CmACS-7基因的PCR扩增产物均可以被酶切成两条带(160 bp和28 bp),两性花材料CmACS-7基因的PCR扩增产物不能被酶切(188 bp),因此建立CmACS-7基因的CAPS分子标记。利用此标记对58份甜瓜材料进行验证,甜瓜单性花性状与分子标记结果显著相关。基于甜瓜单性花决定基因CmACS-7的CAPS分子标记可用于单性花的选育。     

基于黄麻转录组序列SNP位点的CAPS标记开发与验证

黄麻CAPS分子标记的开发,可以为黄麻遗传多样性分析、种质资源鉴定和分子标记辅助选择等研究提供有效方法。本试验以黄麻179和爱店野生种为材料,采用IlluminaHiSeq4000测序技术进行转录组测序,对其SNP位点进行分析,设计了与木质素合成基因4CL、COMT及转录因子MYB相兲的SNP引物,在此基础上,应用dCAPS Finder2.0软件开发了CAPS标记,幵对其有效性进行了验证。结果表明:(1)组装后的黄麻unigene序列共72,674条,序列总长度为29,705,997 bp,检测到的SNP位点总数为67,567个,平均每440 bp有1个SNP。(2)获得了39对分别与4CL、COMT和MYB相兲的SNP引物,从中筛选获得26对CAPS标记引物,开发成功率为66.7%,其中11对CAPS标记具有多态性,多态性比例为43.2%。(3)开发的CAPS标记能较好地将12份不同类型的黄麻种质区分开来,表明CAPS是适用于黄麻研究的较理想的分子标记方法,为黄麻遗传基础研究提供了可靠工具。     

大豆EST-SNP的挖掘、鉴定及其CAPS标记的开发

采用生物信息学方法将大豆EST序列联配到大豆基因组序列上,挖掘到大豆EST-SNP位点537个。对其靶向基因进行功能注释分析,发现他们主要参与亚细胞定位、蛋白质结合与催化以及代谢等与大豆重要农艺性状形成相关的生物过程。同时开发了简便易行的SNP检测方法,利用EMBOSS软件筛选导致酶切位点改变的EST-SNP,分别以大豆绥农14、合丰25、Acher、Evans、Peking、PI209332、固新野生大豆、科丰1号、南农1138-2的DNA及其混合的DNA为模板,设计引物进行PCR扩增,发现44个PCR产物中有36个测序峰图在预期的EST-SNP位点表现出多态性。酶切分析发现26个PCR产物具有酶切多态性,可以作为CAPS标记。结果表明该EST-SNP挖掘体系及其CAPS标记转化系统具有高效率、低成本等优点,有利于促进大豆的遗传育种研究。     

Biological Activity and Quantification of Suspected Allelochemicals from Alfalfa Plant Parts

Autotoxicity restricts reseeding of alfalfa (Medicago sativa L.) after alfalfa until autotoxic chemical(s) breaks down or is dispersed into external environments. A series of aqueous extracts from leaves, stems, roots and seeds of alfalfa ‘Vernal’ were bioassayed against alfalfa seedlings of the same cultivar to determine their autotoxicity. The highest inhibition was found in the extracts from the leaves. Extracts at 40 g dry tissue l^?1 from alfalfa leaves were 15.4, 17.5 and 28.7 times more toxic to alfalfa root growth than were those from roots, stems and seeds, respectively. A high‐performance liquid chromatography (HPLC) analysis with nine standard compounds showed that the concentrations and compositions of allelopathic compounds depended on the plant parts. In leaf extracts that showed the most inhibitory effect on root growth, the highest amounts of allelochemicals were detected. Among nine phenolic compounds assayed for their phytotoxicity on root growth of alfalfa, coumarin, trans‐cinnamic acid and o‐coumaric acid at 10^?3 m were most inhibitory. The type and amount of causative allelochemicals found in alfalfa plant parts were highly correlated with the results of the bioassay, indicating that the autotoxic effects of alfalfa plant parts significantly differed.     

Changes in Seed Yield and Quality with Maturity in Onion (Allium cepa L., cv. 'Early Cream Gold')

Development of onion (Allium cepa L., cv. ‘Early Cream Gold’) seed under cool climate conditions in Tasmania, Australia occurred over a longer duration than previously reported, but similar patterns of change in yield components were recorded. In contrast to previous studies, umbel moisture content declined from 85 to 67 % over 57 days while seed moisture content decreased from 85 to 31 %. Seed yield continued to increase over the duration of crop development, with increasing seed weight compensating for seed loss resulting from capsule dehiscence in the later stages of maturation. Germination percentage was high and did not vary significantly from 53 to 77 days after full bloom (DAF), but mean germination time declined and uniformity of germination increased significantly over the same time period. The percentage abnormal seedlings declined with later harvest date, resulting in highest seed quality at 77 DAF. The results of this study suggest that the decision to harvest cool climate onion seed crops before capsule dehiscence will result in a loss of potential seed yield and quality.     

Location of a high-lysine gene and the DDT-resistance gene on barley chromosome 7

Summary The high-lysine gene in Risø mutant 1508 conditions an increased lysine content in the endosperm via a changed protein composition, a decreased seed size, and several other characters of the seed. The designation lys3a, lys3b, and lys3c, is proposed for the allelic high-lysine genes in three Risø mutants, nos 1508, 18, and 19. Linkage studies with translocations locate the lys3 locus in the centromere region of chromosome 7. A linkage study involving the loci lys3 and ddt (resistance to DDT) together with the marker loci fs (fragile stem), s (short rachilla hairs), and r (smooth awn) show that the order of the five loci on chromosome 7 from the long to the short chromosome arm is r, s, fs, lys3, ddt. The distance from locus r to locus ddt is about 100 centimorgans.     

Simultaneous Determination of Four Phenolic Acids in Radix Salviae Miltiorrhizae Formula Granules by QAMS

[Objectives]This study aimed to establish a QAMS(quantitative analysis of multi-components by single-marker)method for simultaneous determination of four phenol...     

Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Water Extraction Process of Chinese Herbal Compound Man Gan Ning

[Objectives]To optimize the water extraction process of Chinese Herbal Compound Man Gan Ning and establish a method for its extraction and content determination...     

Present status and future strategy in breeding faba beans (Vicia Faba L.) for resistance to biotic and abiotic stresses

Progress is being made, mainly by ICARDA but also elsewhere, in breeding for resistance to Botrytis, AScochyta, Uromyces, and Orobanche; and some lines have resistance to more than one pathogen. The strategy is to extend multiple resistance but also to seek new and durable forms of resistance. Internationally coordinated programs are needed to maintain the momentum of this work.Tolerance of abiotic stresses leads to types suited to dry or cold environments rather than broad adaptability, but in this cross-pollinated species, the more hybrid vigor expressed by a cultivar, the more it is likely to tolerate various stresses.     

Optimization of Extraction Technology and Determination of Content of Total Phenols of Leaves in Acanthopanax giraldii Harms

[Objectives] To determine the optimum extraction technology for total phenols of leaves in Acanthopanax giraldii Harms.[Methods]The single factor test and ortho...     

Cytoplasmic male sterility,resistance to gall mite and mildew,and season of leafing out in black currants

Summary Cytoplasmic male sterility (cms) is described in the F₁ hybrids Ribes × carrierei (R. glutinosum albidum × R. nigrum) and R. sanguineum × R. nigrum. In backcrosses to R. nigrum, progenies with R. glutinosum cytoplasm were either all male sterile, or segregated for full male fertility (F) and complete (S) and partial (I) male sterility. Ratios of F:I+S suggested that two linked genes controlled cms, F plants being dominant for one (Rf ₁) and recessive for the other (Rf ₂).Segregation for cms in relation to three linded genes, Ce (resistance to the gall mite, Cecidophyopsis ribes), Sph ₃(resistance to American gooseberry mildew, Sphaerotheca mors-uvae) and Lf ₁(one of two dominant additive genes controlling early season leafing out) indicated that Rf ₁and Rf ₂were in this linkage group. The gene order and approximate crossover values appeared to be: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% aabaqaciGacaGaamqadaabaeaafaaakeaacaWGdbGaamyzamaamaaa% baGaaiiiaiaacccacaGGWaGaaiOlaiaacgdacaGG0aGaaiiiaiaacc% caaaGaaiiiaiaacccacaGGGaGaamOuaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaaccdacaGGUaGaaiOmaiaacs% dacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaaaacaWGsbGaamOzaSGa% aGOmaOWaaWaaaeaacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccaaaGaamitaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccacaGGGaaaaiaadofacaWGWbGaamiAaSGa% aG4maaaa!6E4D!\[Ce\underline { 0.14 } Rf1\underline { 0.24 } Rf2\underline { } Lf1\underline { } Sph3\]. Crossover values of 0.36 for Ce-Lf ₁, and 0.15 for Lf ₁-Sph ₃were estimated from the relative mean differences in season of leafing out between seedlings dominant and recessive for Ce and Sph ₃.It is suggested that competitive disadvantage of lf ₁-carrying gametes and/or zygotes at low temperatures may be implicated in the almost invariable deficit of plants dominant for the closely linked mildew resistance allele Sph ₃. Poor performance of lf ₁- (and possibly lf ₂-) carrying gametes and young zygotes during periods of low temperature at flowering might also account for the liability of some late season cultivars and selections to premature fruit drop (running off).     

Screening techniques and sources of resistance to parasitic angiosperms

Parasitic angiosperms cause great losses in many important crops under different climatic conditions and soil types. The most widespread and important parasitic angiosperms belong to the genera Orobanche, Striga, and Cuscuta. The most important economical hosts belong to the Poaceae, Asteraceae, Solanaceae, Cucurbitaceae, and Fabaceae. Although some resistant cultivars have been identified in several crops, great gaps exist in our knowledge of the parasites and the genetic basis of the resistance, as well as the availability of in vitro screening techniques. Screening techniques are based on reactions of the host root or foliage. In vitro or greenhouse screening methods based on the reaction of root and/or foliar tissues are usually superior to field screenings and can be used with many species. To utilize them in plant breeding, it is necessary to demonstrate a strong correlation between in vitro and field data. The correlation should be calculated for every environment in which selection is practiced. Using biochemical analysis as a screening technique has had limited success. The reason seems to be the complex host-parasite interactions which lead to germination, rhizotropism, infection, and growth of the parasite. Germination results from chemicals produced by the host. Resistance is only available in a small group of crops. Resistance has been found in cultivated, primitive and wild forms, depending on the specific host-parasite system. An additional problem is the existence of pathotypes in the parasites. Inheritance of host resistance is usually polygenic and its transfer is slow and tedious. Molecular techniques have yet to be used to locate resistance to parasitic angiosperms. While intensifying the search for genes that control resistance to specific parasitic angiosperms, the best strategy to screen for resistance is to improve the already existing in vitro or greenhouse screening techniques.