甜菜SRAP扩增产物两种不同检测方法的比较

为了研究不同电泳及染色方法对甜菜SRAP扩增产物的影响,以6个甜菜二倍体品系为材料,比较变性聚丙烯酰胺凝胶电泳与非变性聚丙烯酰胺凝胶电泳对SRAP-PCR扩增产物用不同银染方法的检测效果进行了比较分析。结果显示:非变性聚丙烯酰胺凝胶电泳与变性聚丙烯酰胺凝胶电泳的分辨率差异不大,但非变性聚丙烯酰胺凝胶电泳具有灌胶简单易学,显色时间短,节省药品等特点,可以作为今后甜菜SRAP电泳及显色的首选方法。     

43份茶树品种资源遗传多样性的SSR研究

采用SSR分子标记技术对43个茶树品种(系)进行了遗传多态性分析。用37对可扩增引物对43个茶树品种(系)扩增,经8%非变性聚丙烯酰胺凝胶电泳检测,其中有34对引物产生多态性,不同引物扩增带数分布在1～11条之间,扩增片段大小介于150～350bp。应用DPS软件,进行遗传距离和相似性系数计算,43份材料遗传距离的变化范围在0.059～0.820之间,说明供试茶树资源间的遗传变异十分宽广。根据UPGMA法构建聚类树状图,在平均遗传距离水平上,可将43份材料划分为7个类群。     

玉米微卫星PCR产物变性与非变性PAGE-银染法的比较分析

对变性聚丙烯酰胺凝胶电泳和非变性聚丙烯酰胺凝胶电泳进行比较,探讨建立简便、快速、安全、可靠、经济、效率高、毒害低的应用于玉米遗传多样性分析的SSR标记方法。结果表明,用2对SSR引物对6份玉米自交系基因组DNA进行扩增,将扩增产物在变性与非变性聚丙烯酰胺凝胶(PAGE)上电泳,银染后微卫星扩增产物在二者上的电泳结果存在差异。在变性胶中微卫星扩增产物目的条带清晰,易于鉴定;在非变性凝胶中表现有较多的非特异性条带,但目的条带很明亮,容易看出来,不影响实验结果。非变性聚丙烯酰胺凝胶电泳带型清晰准确、结果可靠、经济快速,能真实地揭示玉米自交系间的遗传多样性,是玉米种质类群划分的有效分子标记方法。     

水稻SSR不同检测和分析方法的比较研究

 用15对水稻微卫星引物,对13个不同类型及来源的水稻基因型材料进行了简单序列重复片段长度的多态性(SSLPs)分析,并与不同的检测和分析方法进行了比较。聚丙烯酰胺凝胶电泳无论是放射性自显影分析还是荧光标记的自动测序仪分析都比高密度的琼脂糖电泳(3% MetaPhor agarose)有效。利用荧光标记自动测序系统的SSLPs分析较之传统的32P标记的聚丙烯酰胺凝胶电泳分析,具有以下优点：(1)借助于荧光染料标记的丰富内标,可以区别几个bp之差的SSLPs ;(2)借助于不同的荧光标记可以在同一样孔里分析多个样品;(3)自动给出SSLPs片段的长度;(4)自动的数据输出和分析;(5)避免了放射性同位素有可能造成的环境污染。因此,该方法具有快速、高效的特点。     

甜菜SRAP扩增产物不同检测方法的比对研究

为了研究不同检测方法对甜菜SRAP扩增产物多态性的影响以及甜菜SRAP扩增产物的适宜检测方法,分别采用2%的琼脂糖凝胶电泳以及6%的聚丙烯酰胺凝胶电泳对甜菜SRAP扩增产物进行检测。结果表明,聚丙烯酰胺凝胶虽然检测到的条带数多于琼脂糖凝胶,但特异性条带检测数相差不大,且琼脂糖凝胶一般不显示非特异性条带,使得琼脂糖凝胶读带较聚丙烯酰胺凝胶容易,而且琼脂糖凝胶从制做到检测均不涉及有毒药品,故推荐甜菜SRAP分子标记检测使用琼脂糖凝胶。     

马铃薯SSR标记聚丙烯酰胺凝胶电泳分析

以马铃薯品种早大白、黄麻子、克新13、荷兰15、大西洋为试验材料,提取基因组DNA,进行SSR标记,并将标记产物进行聚丙烯酰胺凝胶电泳,比较不同电泳参数(聚丙烯酰胺凝胶的浓度、电泳电压、染色方法)对电泳结果的影响。确定了适合马铃薯SSR标记的电泳检测体系,即聚丙烯酰胺凝胶的浓度为12%,电泳电压为170V,染色方法为溴化乙锭染色。     

一种高效省本的非变性聚丙烯酰胺凝胶电泳银染法的建立——以水稻为例

非变性聚丙烯酰胺凝胶电泳银染技术已广泛用于DNA片段检测。介绍一种本实验室改进的高效省本的非变性聚丙烯酰胺凝胶电泳银染方法,该法将固定和染色两步骤合并,整个银染过程只需16 min即可完成。同时,该法无需使用无水乙醇和冰乙酸,减少了硝酸银、氢氧化钠及甲醛的使用量。     

水稻同工酶聚丙烯酰胺凝胶电泳方法探索

 对同工酶聚丙烯酰胺凝胶电泳方法进行了探索，在同工酶的提取、电泳、染色等几个方面做了尝试。建立了一套适合水稻的同工酶聚丙烯酰胺凝胶电泳分析方法。分析了８种同工酶，１８个位点。发现4个新的位点Amp-y,Amp-x,Mal-2, Mal-3。所分析位点绝大多数表现出较强的多态性。     

利用SSR标记技术检测玉米杂交种纯度

对玉米子粒DNA提取、SSR扩增片段检测方法等进行了研究,并以4个利用常规种子贮藏蛋白质电泳技术难以鉴定纯度的玉米杂交种及相应自交系和9对玉米SSR位点引物为材料,分别筛选出适合这些不同杂交种纯度鉴定的SSR位点引物,建立了一套利用SSR标记进行玉米杂交种纯度鉴定的技术规程.该程序包括从粉碎干种子提取DNA和利用非变性聚丙烯酰胺凝胶电泳分离SSR扩增片段,对仪器设备要求较低、简单、快速,易于推广。     

应用清蛋白PAGE技术进行玉米自交系类群划分的初步研究

利用改进的聚丙烯酰胺凝胶电泳技术,对12个玉米自交系子粒清蛋白进行分析,结果共分离出24条谱带,其中公共谱带4条,占16.7%,多态性谱带20条,占83.3%,平均每个自交系分离出1.7条多态性谱带.通过电泳谱带进行聚类,可将12个自交系分为4大类:第Ⅰ类为478、8112、冀815和冀53;第Ⅱ类为黄早4、502、冀35、京404、81515和52106;第Ⅲ类为丹340;第Ⅳ类为Mo17.聚类结果与已知系谱来源基本吻合,表明改进的聚丙烯酰胺凝胶电泳技术在玉米自交系类群划分上的应用是基本可行的.     

SSR荧光标记毛细管电泳检测法在水稻DNA指纹鉴定中的应用

 以16个杂交水稻不育系、恢复系及组合为材料，初步建立了水稻品种SSR荧光标记毛细管电泳检测方法。与常规凝胶电泳检测方法相比，荧光标记毛细管电泳检测方法可以读出目标DNA片段的准确大小，检测数据更为精确，检测效率更高。常规凝胶电泳检测方法验证表明，利用SSR荧光标记毛细管电泳检测方法进行水稻品种DNA指纹鉴定，方法可行、结果可靠。     

An applied fingerprinting system for cultivated potato using simple sequence repeats

The ability to quickly and accurately identify potato (Solanum tuberosum L.) clones is important to potato-breeding programs, seed and commercial potato growers, and marketing and utilization of potato cultivars. Since 1990, the Michigan State University Potato Breeding and Genetics Program has used an isozyme-based fingerprinting system to identify potato cultivars. Isozyme analysis is an economical and effective means of discriminating potato clones; however, isozyme analysis requires fresh, healthy tuber or leaf tissue. DNA-based fingerprinting using simple sequence repeats (SSRs or microsatellites) has been shown to discriminate between potato clones. The objective of this study was to identify the most useful SSR primer pairs that accurately and efficiently distinguish clones for an applied fingerprinting system of cultivated potato. SSR primer pairs with high polymorphism were selected from previous tetraploid potato studies. DNA isolated from 17 potato clones representing round-white, russet, and red market classes were visualized on both polyacrylamide (PAGE) and agarose gel systems. Polymorphism was observed in all 18 primer combinations on PAGE and 14 using agarose gel electrophoresis. All 17 cultivars were discriminated on PAGE with various combinations of two primer pairs: STIIKA using STACCAS3, STIN-HWI, or STM0031; and STACCAS3 using STGBSS1, POTM1-2, STM1104, or STM0031. The combination of STM0019, STM0031, STGBSS1, and POTM1-2 was able to differentiate all 17 clones using agarose gel electrophoresis. PAGE was determined to be the preferred system for variety identification, but agarose gel electrophoresis can be used to differentiate lines when specific varietal comparisons are needed. In addition, five different DNA source tissue types were evaluated (fresh foliar, freeze-dried foliar, fresh tuber, freeze-dried tuber epidermis, and freeze-dried tuber tissue). Amplification products were similar for all five tissue sources used for DNA isolation. This ability to isolate DNA from freeze-dried tissue will allow cultivar identification when fresh tissue is not available. The SSR primer pairs presented here can be used as a practical fingerprinting system for cultivated potato identification.     

Genetic Diversity of Tropical Hybrid Rice Germplasm Measured by Molecular Markers

Investigation of genetic diversity and relationships among breeding lines is of great importance to facilitate parent selection in hybrid rice breeding programs.In this study,we characterized 168 hybrid rice parents from International Rice Research Institute with 207 simple sequence repeat (SSR) and 353 single nucleotide polymorphism (SNP) markers.A total of 1 267 SSR and 706 SNP alleles were detected with the averages of 6.1 (SSR) and 2.0 (SNP) alleles per locus respectively across all lines.Based on the genetic distances estimated from the SSR and SNP markers separately and combined,the unrooted neighbor-joining cluster and STRUCTURE analyses consistently separated the 168 hybrid rice parents into two major groups:B-line and R-line,which is consistent with known parent pedigree information.The genetic distance matrices derived from the SSR and SNP genotyping were highly correlated (r=0.81,P < 0.001),indicating that both of the SSR and SNP markers have distinguishable power to detect polymorphism and are appropriate for genetic diversity analysis among tropical hybrid rice parents.A subset of 60 SSR markers were also chosen by the Core Hunter with 368 alleles,and the cluster analysis based on the total and subset of SSR markers highly corresponded at r=0.91 (P < 0.001),suggesting that fewer SSR markers can be used to classify and evaluate genetic diversity among parental lines.     

利用遗传同质的近等基因对研究水稻抗稻瘟病基因的产物

 应用经典遗传学方法构建了多个只有抗感基因之差，其他遗传背景相似的抗稻瘟病近等基因对， 它们的形态性状比较和过氧化物酶同工酶、过氧化物酶等电聚焦电泳带模式以及蛋白质双向电泳的图谱模式都说明每一组近等基因对有很高的同质程度，近等基因对不接种病原菌的双向电泳图谱中，pI 7.5～8.5， 分子量30～45 kDa区域的蛋白质亚基和稻瘟病的发生发展关系甚密，称该区为稻瘟病有关的蛋白质特异区，接种病原菌5 d后，近等基因对的双向电泳图谱中，蛋白质亚基变化同样发生在该特异区内。     

琼脂糖凝胶电泳在水稻EcoTILLING技术中的应用

 为构建低成本的基于琼脂糖凝胶电泳的EcoTILLING技术体系，用于水稻及其他生物自然群体变异的检测，对基于琼脂糖凝胶的EcoTILLING技术进行了优化。结果表明，从芹菜中提取的CEL Ⅰ核酸酶粗提物结合琼脂糖凝胶电泳检测技术，可以成功地检测到目的基因位点的突变。利用该技术，在云南地方水稻品种大吊糯中检测到2 个SNPs。     

水稻花粉总蛋白质双向凝胶电泳方法建立及应用

 采用石英砂加液氮研磨方法破碎花粉，然后用两种不同的方法提取水稻花粉总蛋白质，之后采用固相pH梯度等电聚焦/SDS PAGE 双向凝胶电泳对红莲型细胞质雄性不育水稻单核期花粉总蛋白质进行了分离。通过银染显色， PDQuest 2DE软件可识别约1 000~1 500个蛋白质点。其中TCA 丙酮提取法更适合提取花粉总蛋白质进行双向凝胶电泳分离，并可获得分辨率和重复性较好的双向电泳图谱。对红莲型细胞质雄性不育水稻的不育系单核期和二核期花粉总蛋白进行了双向电泳分离，通过比较两个时期花粉蛋白质电泳图谱后发现，二核期的花粉蛋白质较单核期花粉蛋白质数目减少，并且部分蛋白质表达量下降。     

Rapid Construction of a High-Density Rice Linkage Map by High Efficiency Genome Scanning （HEGS） System

High-density linkage maps are essential tools for genome analysis of various biological traits. Our developed compact multi-gel system, HEGS (high efficiency genome scanning) is a high-throughput and high-cost-performance electrophoresis apparatus. Using this system, a high-density (average interval 2.3 cM) map with 1 065 AFLP and 63 SSR markers was constructed from recombinant inbred lines of a japonica and indica hybrid in just two months of electrophoreses by a single person. More than 50% of the mapped AFLP markers were commonly polymorphic for several combinations between japonica and indica rice and 15% were applicable for genetically closer crosses between upland and lowland types of japonica rice. This system can be used for rapid analyses of all kinds of markers.