SSR分子标记鉴定玉米杂交种纯度的研究及应用综述

从玉米基因组DNA提取、引物的筛选、PCR反应体系优化、部分推广玉米品种纯度鉴定引物信息等方面综述了SSR分子标记在玉米杂交种纯度鉴定方面的研究及应用。     

正交设计优化玉米SSR—PCR反应体系的研究

为建立适宜玉米SSR—PCR的反应体系和扩增程序,利用正交设计L16（4^5）表,对反应体系的模板DNA、dNTPs、Primers、Taq DNA聚合酶的浓度进行4因素4水平优化筛选和扩增程序的优化,确立了最优反应体系和扩增程序。即在10μL体系中,模板DNA20ng,1×PCR buffer,dNTPs62．5pmol／μL,Primers0．25pmol／μL,Taq DNA polymerase0．5U。反应程序为：94℃预变性5min;94℃变性45S,60℃退火45S,72℃延伸1min,32个循环;72℃延伸5min,4℃保存。使用300对SSR引物对重组近交系的两亲本扩增,筛选出条带清晰,有差异的引物94对,用于基因连锁图谱构建和QTL定位。     

利用小麦微卫星引物建立偃麦草Ee染色体组特异SSR标记

选用40对小麦SSR引物对17份偃麦草(Thinopyrum sp．)、2份小麦(Triticum aestivum)材料进行了PCR扩增分析，从中筛选到引物Xgwm325能在不同偃麦草材料中扩增出4条长度分别为1400、440、120和100bp的特异DNA片段，可以作为偃麦草种质的特异SSR标记。利用小麦-二倍体长穗偃麦草(Th.elongatum)异代换系和异附加系对引物Xgwm325进行了扩增鉴定，结果只有100bp左右的片段出现在长穗偃麦草所有E^e组染色体上，该片段可以作为E^e染色体组的特异SSR标记。     

一种改进的玉米SSR标记的PAGE/快速银染检测新方法

聚丙烯酰胺凝胶电泳技术由于其检测灵敏度和分辨率高的特点．特别适合于像SSR这样的小片段扩增产物的检测。银染法是其常用的显影方法之一，不同的银染法操作步骤及采用试剂有一定差异，主要集中在固定液的选择和洗脱次数上。本研究在参考以往各种银染法的基础上，进一步简化操作步骤，仅需染色和显影两步即可进行结果的判读，将其应用于玉米SSR标记的PAGE银染检测，为SSR技术在玉米种质纯度及真伪快速鉴定上的应用提供技术方法。     

小麦AB基因组中一个重复序列的分离、定位与应用

利用102对微卫星引物对5份黑麦（Secale）、4份普通小麦（Triticum aestivum）和1份分枝小黑麦（Triticale）进行SSR分析,引物Xgwm614能在分枝小黑麦中扩增出一个387bp的特异DNA片段（记为FZ387,GenBank登录号为EF179137）,而黑麦未能扩增出。序列比对结果显示该片段与一粒小麦（T. monococcum）（AY485644）和栽培二粒小麦（T. turgidum）（AY494981）A基因组中Gypsy Ty3-LTR反转座子fatima的一部分分别有94%和95%同源性。根据序列同源性比对结果,在FZ387内部设计1对特异引物FaF和FaR。引物Xgwm614F和FaR能在含有A基因组的物种中扩增出约350bp的条带（记为A350）,而其不含A基因组的物种都未扩增出该条带。利用小麦二体和端体代换系材料对其进行定位,结果显示该片段分布在所有A染色体的长臂和断臂上。此外,引物FaF和Xgwm614R能在含有A、B或AB基因组的物种中扩增出约350bp的条带（记为AB350）,而不含AB基因组的材料未扩增出目标条带。利用这两对特异引物对小麦属近缘物种进行PCR扩增,发现只有中国春能够扩增出A350和AB350。序列比对结果和FZ387两侧SSR引物结合区的规律性变化表明该反转座子在进化上可能存在属间多样性和属内相似性。A350和AB350也可以分别作为分子标记检测A染色体和AB染色体。     

落羽杉属树木基因组总DNA的提取及SRAP反应体系的优化

本文针对落羽杉属植物组织中多糖、多酚等次生物质含量高的特点,对其基因组DNA提取方法进行研究,比较了SDS法、CTAB法提取落羽杉属植物基因组DNA的效果,结果表明：CTAB法提取效果较佳。在此基础上,利用正交设计法,对SRAP反应体系中的各个主要影响因子Mg^2＋．dNTP、引物、TaqDNA聚合酶进行了优化筛选,确立了适合落羽杉属植物SRAP-PCR反应的最佳体系,即10μL体系中含有1μL 10×PCR bufier,Mg^2＋2．0mmol／L,dNTP100μmol／L,引物0-3μmol／L,彻DNA聚合酶0．5U和50ng模板DNA。利用该优化体系,通过48对SRAP引物组合对2个落羽杉属植物（落羽杉和墨杉）及4个杂交后代进行SRAP扩增,结果发现,SRAP引物及优化后的反应体系能够有效地用于落羽杉属植物种质资源鉴定及遗传多样性分析等研究。     

提高小麦×玉米杂交产生单倍体胚频率的研究

为了提高小麦×玉米杂交单倍体胚的产生频率，采用了2,4-D中含有不同体积浓度的二甲基亚砜和不同的激素处理组合两种方法诱导小麦×玉米产生单倍体，研究其对得胚率的影响。结果表明，就平均得胚率而言，在二甲基亚砜激素浓度处理中，最佳的二甲基亚砜体积浓度为3.0%（得胚率为17.0%），而且各不同浓度的处理均高于对照（9.7%）；在不同激素配比试验中，2,4-D+3%二甲基亚砜单独处理的得胚率（7.3%）﹥2,4-D两次处理(6.9%)﹥2,4-D与2,4-D+3%二甲基亚砜配合处理(5.5%)﹥2,4-D一次处理(2.9%)。     

猪粪和土壤样品中微生物DNA提取方法的比较

猪粪施于土壤可能会对土壤微生物多样性造成影响，为选用同一种DNA提取方法用于土壤和猪粪微生物DNA的提取，该文采用了化学裂解法和试剂盒法同时从土壤和猪粪样品中提取微生物DNA，并对这两种方法的提取DNA的效果进行了比较。结果表明，试剂盒法不能用于提取土壤中的微生物DNA；可以从猪粪中提取到DNA，PCR扩增能得到目的产物，但重复性不高。化学裂解法提取的土壤微生物DNA浓度高但纯度低，纯化后纯度增加，但DNA有所损失，用于PCR扩增时结果不理想；处理猪粪样品，提取的DNA浓度较低但纯度较高，PCR扩增结果比较理想。由此可见，化学裂解法用来提取猪粪样品中的微生物DNA是可行的，但需寻求更好的土壤样品微生物DNA的提取方法。     

小麦不同品种外植体的农杆菌转化方法的研究

本实验在小麦中对农杆菌介导的大麦黄矮病病毒CP基因进行了转化，选择生产用小麦品种（系），对影响小麦组织条件和转化的主要因素进行研究，并获得了小麦转基因植株，研究表明，增加肌醇浓度有利于小麦愈伤组织的分化，而添加ABA却只对部分品种有效；采用幼胚和幼胚愈伤组织均能获得转基因植株；以茎尖和花药愈伤组织为受体未能获得阳性植株；小麦不同品种对不同农杆菌菌株的反应不同。     

基因枪法转化小麦幼胚瞬时表达

自Vasil et al．（1996）利用基因枪将获得小麦对除草剂Basta具有抗性的再生植株以来，基因枪介导的小麦遗传转化研究发展很快（Altpeter et al．，1996）。在小麦基因枪转化中，优化基因枪转化因素，提高转化效率仍然是基因转化的关键问题。本实验研究了不同金粉用量、质粒DNA浓度及轰击枪数对小麦幼胚瞬时表达的影响，通过GUS染色和细胞压片，分析了gus基因的瞬间表达。     

高能混合粒子场诱发的小麦矮杆突变体的SSR分析

为了研究高能混合粒子场(CR)和γ射线两种不同处理方法诱变小麦产生的具有相同表型的突变体之间的分子差异,选用随机分布于小麦21对染色体上的114对微卫星(SSR)引物,对CR和γ射线处理冬小麦品种ZY9和ZH7获得的矮杆突变体M3代进行SSR分析。结果表明,CR处理产生的矮杆突变体的多态性位点主要分布在染色体2A、2B、2D、3D和5A上,而γ射线处理产生的矮杆突变体的多态性位点主要分布在染色体2A、2B、2D、4A和5A上;与γ射线处理相比,CR处理较容易产生扩增条带的增加和扩增条带长度的差异,不易产生扩增条带的缺失。序列分析表明,CR处理产生的变异主要是碱基的置换和插入,其中碱基T为易发生突变的碱基。CR诱变能够在DNA水平上导致小麦遗传物质变异,其诱变机制不同于γ射线,是一种有效的诱发突变新途径。     

Analysis of genetic diversity in Tunisian durum wheat cultivars and related wild species by SSR and AFLP markers

Thirty-four durum wheat cultivars representing the Tunisian durum (Triticum durum Desf.) wheat collection and seven wild species of wheat relatives (Triticum turgidum L., T. dicoccon Schrank., T. dicoccoides (Körn) Schweinf., T. araraticum Jakubz., T. monococcum L., Aegilops geniculata Roth, and Aegilops ventricosa Tausch) were analysed with amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers. Both marker systems used were able to differentiate durum wheat cultivars from the wild relatives and to specifically fingerprint each of the genotypes studied. However, the two marker systems differed in the amount of detected polymorphisms. The 15 SSR markers were highly polymorphic across all the genotypes. The total number of amplified fragments was 156 and the number of alleles per locus ranged from 3 to 24 with an average of 10.4. Two SSR markers alone, Xwms47 and Xwms268, were sufficient to distinguish all 34 durum wheat genotypes. The five AFLP primer pair combinations analysed yielded a total of 293 bands, of which 31% were polymorphic. The highest polymorphic information content (PIC) value was observed for SSRs (0.68) while the highest marker index (MI) value was for AFLPs (7.16) reflecting the hypervariability of the first and the distinctive nature of the second system. For durum wheat cultivars, the genetic similarity values varied between 31.3 and 81% for AFLPs (with an average of 54.2%), and between 3.6 and 72.7% for SSRs (with an average of 19.9%). The rank correlation between the two marker systems was moderate, with r = 0.57, but highly significant. Based on SSR markers, highest genetic similarity (GS) values were observed within the modern cultivars (37.3%), while the old cultivars showed a low level of GS (19.9%). Moreover, the modern cultivars showed low PIC and MI values. UPGMA Cluster analysis based on the combined AFLP and SSR data separated the wild wheat species from the durum wheat cultivars. The modern cultivars were separated from the old cultivars and form a distinct group.     

Simple method of genomic DNA extraction from Rhizobia in dried nodules of Phaseolus vulgaris for strain differentiation by PCR-based DNA fingerprinting

Repetitive DNA peR fingerprinting of bacterial genomic DNA is a useful tool for typing and differentiation of rhizobial strains. The method was reported to be suitable for strain differentiation of Rhizobia present in individual root nodules of some leguminous plants without the need for isolation and cultivation of the strains, in which rhizobial genomic DNA was extracted directly from each fresh or frozen nodule. We developed a new protocol of rhizobial genomic DNA extraction/purification from dried nodules of Phaseolus vulgaris for generating repetitive DNA peR fingerprints of Rhizobia present in the nodules. The simplified protocol consists of only three major steps, heat extraction of genomic DNA from rhizobial cells prepared from dried nodules, ethanol precipitation of the DNA and Sephadex G-50 column purification of the DNA, and generated fingerprints with good quality for differentiation of Rhizobia strains. The protocol will be useful to examine the nodule occupancy of inoculated rhizobial strains in field experiments.     

小麦抗叶锈病基因Lr2c的SSR标记

选取抗叶锈病基因位于2D染色体上的TcLr2c等7个小麦（Triticum aestivum）近等基因系、感病亲本Thatcher及215株TcLr2c与Thatcher杂交F2代为材料,研究抗叶锈病基因Lr2c SSR分子标记。从筛选的29对位于小麦2D染色体的SSR引物中获得4对能够揭示Lr2c多态性的分子标记,通过215株TcLr2c × Thatcher F2群体验证,结果表明Xgwm261和Xgwm296与Lr2c紧密连锁,其距目的基因的遗传距离分别为1.9和3.6 cM,可用于小麦抗叶锈病分子辅助育种。     

Characterization of Seashore Paspalum (Paspalum vaginatum Swartz) Germplasm by Transferred SSRs from Wheat, Maize and Sorghum

One hundred and thirty SSR markers from wheat, maize and sorghum were screened for the transferability to Paspalum. The transfer rate was 67.5, 49.0 and 66.8% respectively. This would be a very efficient approach for DNA marker development for species which are not well studied molecularly. The polymorphism level for transferred SSR markers was 51.5% within species (Paspalum vaginatum) and 87.1% among Paspalum species. The high level of polymorphism is directly related to the high degree of heterozygosity maintained by its way of reproduction, i.e. self-incompatibility. Forty transferred polymorphic SSR markers were selected and used for characterization and evaluation of seventy-three Paspalum accessions. In total, 209 polymorphic bands were detected from these 40 SSR markers, with an average of five polymorphic bands per marker. The Paspalum accessions clustered into three major groups. Two very similar dendrograms can be generated from either 109 or 209 polymorphic bands. This led us to determine that 18 of the transferred SSR markers were sufficient for genetically differentiating the investigated germplasm accessions. The number of SSR markers required for germplasm characterization and evaluation is discussed. This is the first report of the transfer of SSR markers from major field crops to newly emerged environmental turfgrasses.     

Rapid Identification of HMW Glutenin Subunits from Different Hexaploid Wheat Species by Acidic Capillary Electrophoresis

High molecular weight glutenin subunits (HMW‐GS) from three hexaploid wheat species (AABBDD, 2n=6x=42, Triticum aestivum L., T. spelta L., and T. compactum L.) were separated and identified by acidic capillary electrophoresis (A‐CE) with phosphate‐glycine buffer (pH 2.5) in uncoated fused‐silica capillaries (50 μm, i.d. × 25.5 cm) at 12.5 kV and 40°C. The rapid separations (<15 min) of HMW‐GS with good repeatability (RSD < 2%) were obtained using a fast capillary rising protocol. All 17 HMW‐GS analyzed could be well separated and their relative migration orders were ranked. In particular, the good quality subunit pair 5+10 could be differentiated from poor quality subunit pair 2+12. In addition, the other three allelic pairs of 13+16, 17+18, and 7+8 subunits that were considered to have positive effects on dough properties, as well as three pairs of novel subunits 13+22*, 13*+19*, and 6.1+22.1 detected from spelt and club wheat, can also be readily separated and identified. An additional protein subunit presented in Chinese bread wheat cultivar Jing 411 and club wheat TRI 4445/75, respectively, was detected by both A‐CE and 2‐D gel electrophoresis (A‐PAGE × SDS‐PAGE), for which further identification is needed.     

Soil DNA extraction and assessment of the fate of Mycobacterium chlorophenolicum strain PCP-1 in different soils by 16S ribosomal RNA gene sequence based most-probable-number PCR and immunofluorescence

Since Mycobacterium chlorophenolicum strain PCP-1 is not detectable in soil by selective plating, a specific tracking method was based on the polymerase chain reaction (PCR) using soil DNA as a target. A direct extraction protocol based on bead beating was adapted and used to obtain PCR-amplifiable DNA from five different soils. In one soil, the disruption of cells of PCP-1, of Pseudomonas fluorescens R2f and of Paenibacillus azotofixans P3L5, as well as of the indigenous bacteria increased with increasing bead beating times. After 4.5 min, lysis efficiency was about 90% or more in all cases. Total DNA yields varied between soils, from 2 to 35 μg g^–1. The purification steps needed to obtain amplifiable DNA were different per soil. To detect target DNA specifically in bacterial cells, a new indirect extraction protocol was developed, which efficiently dislodged bacterial cells from the soil matrix, and produced amplifiable DNA with high yield. To detect strain PCP-1 in soil, 16S ribosomal gene-based PCR combined with oligonucleotide hybridization was applied using a most-probable-number (MPN) set-up, whereas immunofluorescence was used for calibration. Strain PCP-1 was detected shortly after introduction into three soils at about the inoculum levels, as evidenced by both approaches. Both the direct and indirect DNA extraction methods yielded similar MPN estimates. The dynamics of M. chlorophenolicum PCP-1 was estimated in two soils over 14 days via MPN-PCR/oligonucleotide probing. PCP-1 showed good survival in both soils, and results obtained by MPN-PCR with directly and indirectly extracted DNA were internally consistent. Immunofluorescence cell enumerations supported the gross stability of PCP-1 in these two as well as in two additional soils. Received: 8 February 1996