16份石榴RAPD扩增产物的两种电泳方法检测及其序列特征

本实验以16个石榴品种为实验材料,筛选出10个重复性及多态性均较好的引物进行RAPD分析。分别采用琼脂糖凝胶以及聚丙烯酰胺凝胶(PAGE)电泳检测方法对PCR扩增结果进行检测并对其结果进行比较,结果显示,两种电泳方式均能得到较为清晰的扩增条带,且两种电泳方式获得的条带总数及多态性条带数均有所不同,琼脂糖凝胶电泳方法共检测出76条带,其中有43条为多态性谱带,多态性比率为56.4%;而在PAGE电泳方法共检测出123条谱带,多态性谱带数为87条,多态性比率为70.95%。PAGE电泳方法检测出的条带数约为琼脂糖凝胶电泳方法检测出条带数的1.5倍。基于两种电泳方法所得RAPD标记的多态性位点,利用NYSYS软件计算遗传相似系数,并构建遗传关系聚类图,分析结果显示,石榴遗传多样性丰富,两种电泳方法所得聚类结果大致相同,可以利用RAPD分子标记及两种电泳检测方法对不同数量的石榴进行分子水平的品种鉴定和遗传多样性的分析。同时通过对来自几个引物随机挑选的17个片段进行克隆,测序结果显示17个片段都是对应引物的RAPD扩增产物,其中有3条是编码蛋白的基因片段,表明了RAPD不仅扩增基因组上的非编码蛋白序列,同时也可以扩增编码蛋白的基因片段,这为更好地认识RAPD技术的实质以及促进石榴产业的发展提供了理论依据。     

抗风桐(Pisonia grandis)的生态生物学特征

对收集于广西桂林的17份野生毛葡萄种质和24份栽培葡萄种质,分别使用12条ISSR和12条SCoT引物进行了遗传多样性和亲缘关系检测。结果表明：两种分子标记均能产生较丰富的多态性片段,可有效应用于葡萄的遗传多样性检测,但在聚类分析结果上表现出一定的差异性,SCoT分子标记能更好地区分野生种质和栽培品种,说明SCoT分子标记在葡萄遗传多样性检测和系统进化研究上可能更有优势。从SCoT聚类结果上看,广西植物研究所收集的3个野生毛葡萄种质zws1、zws2和zws3相对其它野生种质而言,更偏向于与栽培种质聚为一类,说明这一类野生毛葡萄可能是这些栽培品种的原始亲本来源之一。不同的野生种质聚为多个类群,并表现出明显的地域特性,但遗传距离相对较远,说明桂林野生毛葡萄资源具有丰富的遗传变异。栽培品种没有明显的聚类特点,可能因为所选用的栽培品种的地域代表性并不是很强,也可能是因为栽培品种在不断的人工杂交选育过程中,遗传背景趋向一致,遗传多样性降低。该研究证明SCoT分子标记在葡萄遗传多样性研究上具有一定的优势。该研究结果为桂林毛葡萄资源的保护、利用和品种选育提供了理论依据,也为葡萄的系统进化研究提供了参考。     

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。     

41份葡萄种质遗传多样性的ISSR和SCoT对比分析

对收集于广西桂林的17份野生毛葡萄种质和24份栽培葡萄种质,分别使用12条ISSR和12条SCoT引物进行了遗传多样性和亲缘关系检测。结果表明:两种分子标记均能产生较丰富的多态性片段,可有效应用于葡萄的遗传多样性检测,但在聚类分析结果上表现出一定的差异性,SCoT分子标记能更好地区分野生种质和栽培品种,说明SCoT分子标记在葡萄遗传多样性检测和系统进化研究上可能更有优势。从SCoT聚类结果上看,广西植物研究所收集的3个野生毛葡萄种质zws1、zws2和zws3相对其它野生种质而言,更偏向于与栽培种质聚为一类,说明这一类野生毛葡萄可能是这些栽培品种的原始亲本来源之一。不同的野生种质聚为多个类群,并表现出明显的地域特性,但遗传距离相对较远,说明桂林野生毛葡萄资源具有丰富的遗传变异。栽培品种没有明显的聚类特点,可能因为所选用的栽培品种的地域代表性并不是很强,也可能是因为栽培品种在不断的人工杂交选育过程中,遗传背景趋向一致,遗传多样性降低。该研究证明SCoT分子标记在葡萄遗传多样性研究上具有一定的优势。该研究结果为桂林毛葡萄资源的保护、利用和品种选育提供了理论依据,也为葡萄的系统进化研究提供了参考。     

IGS2-RFLP、SCoT和ISSR在白灵侧耳遗传多样性分析中的比较研究

应用IGS2-RFLP、SCoT和ISSR标记分析新疆5个采集地点的28个白灵侧耳野生样本的遗传多样性,比较这3种分子标记在白灵侧耳种质资源遗传多样性研究中的效用。结果显示,IGS2-RFLP的3个内切酶、5个SCoT引物、5个ISSR引物分别检测到42、59和77条多态性条带,多态性比率分别为91.3%、92.4%和92.8%。3种标记检测出的有效等位基因数(ne)和位点平均的预期杂合度(Hep)没有显著性差异。表明3种标记都适宜作为遗传学标记对白灵侧耳野生种质进行多样性分析。多态性检测效率最高的标记为ISSR,E=15.4,Ai=23.4,其次为IGS2-RFLP,E=14.0,Ai=22.4,SCoT则为最低,E=11.8,Ai=18.2。3种标记中,SCoT和ISSR标记的聚类结果极其相似,且均能较为准确地反映样本的地理来源,虽然二者的聚类图不完全相同。IGS2-RFLP的标记效率较高,准确性和可重复性最好,可用于菌株标准图谱的制作,更适用于品种权保护中的菌株鉴定鉴别;SCoT和ISSR标记的多态性高,信息量大,评价范围广,则更适用于白灵侧耳种质资源的遗传多样性研究。     

一种新的DNA分子标记技术——起始密码子-微卫星扩增多态性

综合SCoT和ISSR分子标记技术开发了一种既能将标记位点与表达序列紧密联系,又具有相对较高的多态性的新的分子标记技术——起始密码子一微卫星扩增多态性（start codon-simple sequence repeat, SC-SSR）。SC—SSR标记是基于PCR的目的基因标记系统．上游引物用SCoT标记引物,瞄准基因区域,下游引物用ISSR标记引物,上下游引物间可自由组配。引物设计原则同SCoT标记和ISSR标记。使用50℃的退火温度,保证了扩增结果的稳定性。PCR结果采用琼脂糖凝胶电冰和聚丙烯酰胺凝胶电泳检测。SC—SSR分子标记结合了ISSR标记和SCoT标记的优点,具有操作简单、成本低廉、多态性丰富、重复性好、引物设计简单且通用性良好、同时与表达序列紧密连锁等诸多优点,可用于种质资源的鉴定评价、遗传图谱的构建、重要性状基因标记、gDNA与cDNA指纹分析乃至图位克隆等方面。     

优化AFLP阳性带回收克隆技术

通过将AFLP聚丙烯酰胺变性胶切小、适当曝光过量、以聚丙烯酰胺凝胶电泳代替琼脂糖电泳检测克隆片段大小的方法 ,增加了回收克隆AFLP阳性带的准确性。该方法对于从变性和非变性大page胶上回收克隆目的片段具有普遍意义。     

棉花遗传多样性SCoT和SRAP标记的研究及比较分析

利用SCoT和SRAP两种分子标记技术对30份彩色棉与白色棉种质资源,进行遗传多样性研究。用29对SRAP引物组合和26个SCoT引物分别对供试棉花的基因组DNA进行扩增。SCoT引物共扩增出163条带,多态性比率为61.96%,遗传相似系数GS值变化范围为0.5405～0.9972。SRAP引物组合共扩增条带1067条,多态性比率仅为14.1%,遗传相似系数GS值变化范围为0.5415～0.9109。两种标记系统得到了相似但并不完全相同的聚类图,2种标记方法间存在显著相关性(r=0.5518,P<0.05)。结果表明,SRAP与SCoT标记均适用于棉花种质的遗传多样性分析,且SCoT的标记指数MI高于SRAP标记,为SCoT这种新兴的标记技术在棉花育种中的应用提供了重要的依据。     

新疆枸杞种质资源遗传多样性分析及DNA指纹图谱构建

利用SCoT分子标记对新疆枸杞种质资源进行遗传多样性分析和DNA指纹图谱构建,为杂交育种和种质鉴定提供理论依据。结果显示:9条SCoT引物扩增出条带256条,其中219条为多态性条带,多态性比率达85.62%,多态性信息含量(PIC)值变化范围在0.77~0.91之间,平均值为0.85,观测等位基因数(Na)、有效等位基因数(Ne)、Nei's基因多样性指数(H)和Shannon信息指数(I)的平均值分别为1.8562、1.4350、0.2611、0.3989,聚类分析表明,遗传相似系数变化范围在0.5938~0.8398之间,在遗传相似系数为0.66和0.71处,可将30份材料分别分为2大类和4个亚类,主坐标分析结果和聚类结果基本一致,同时利用5条多态性SCoT引物构建了30份材料的DNA指纹图谱。新疆枸杞种质资源遗传多样性水平较高,且SCoT分子标记适于新疆枸杞种质资源遗传多样性分析和DNA指纹图谱构建,该研究结果为新疆枸杞种质资源评价、鉴定和新品种选育奠定了基础。     

RFLP和AFLP分析白菜型油菜和甘蓝型油菜遗传多样性及其在油菜改良中的应用价值

采用RFLP和AFLP标记对来自中国和欧美的7份甘蓝型油菜和22份白菜型油菜进行了遗传多样性分析。在这29份材料中,166个酶-探针组合和2对AFLP引物共检测到1477个RFLP标记和183个AFLP标记。RFLP数据显示以拟南芥EST克隆作探针比用油菜基因组克隆做探针能检测到更多的多态性位点,且采用EcoR Ⅰ或BamH Ⅰ酶切比HindⅢ酶切多态性好,白菜型油菜和甘蓝型油菜中基因的拷贝数平均都为3个左右。UPGMA聚类分析表明中国白菜型油菜的遗传多样性比甘蓝型油菜和欧美白菜型油菜丰富,欧美甘蓝型油菜与欧美白菜型油菜聚为一类,而与中国甘蓝型油菜差异更大。中国白菜型油菜丰富的遗传多样性为中国甘蓝型油菜的改良提供了宝贵的资源,揭示了利用白菜型油菜A基因组和甘蓝型油菜A基因组间亚基因组杂种优势的可能性。     

Toward unraveling the structure of Brassica rapa genome

Genomic research in any organism encompasses understanding structure of the target genome and genes, their function, and evolution. Brassica rapa , which is phylogenetically related to Arabidopsis thaliana , is an important species with respect to its uses as vegetable, oil, and fodder. The availability of suitable genetic and genomic resources is a prerequisite to undertake genomic research in B. rapa . We have developed reference mapping populations of Chinese cabbage ( B. rapa ssp. pekinensis ) comprising 78 doubled haploid lines and over 250 recombinant inbred lines. Two Bacterial Artificial Chromosome (BAC) libraries, generated by restriction enzymes Hin dIII (KBrH) and Bam HI (KBrB), comprise 56 592 and 50 688 clones, respectively. We have also constructed 22 cDNA libraries from different plant tissues consisting of 104 914 clones with an average length of 575 bp. Initial BAC-end sequence analysis of 1473 clones of the KBrH library led us to understand the structure of B. rapa genome with respect to extent of genic sequences and their annotation, and relative abundance of different types of repetitive DNAs. Full-length sequence analysis of BAC clones revealed extensive triplication of B. rapa DNA segments coupled with variable gene losses within the segments. The formulation of the 'Multinational Brassica Genome Project' has laid the foundation to sequence the complete genome of B. rapa ssp. pekinensis by the international Brassica research community. It has been proposed to undertake BAC-to-BAC sequencing of genetically mapped seed BACs. In recent years, development of bioinformatics tools in Brassica has given a boost to structural genomics research in Brassica species. The research undertaken with the availability of various genomic resources in the public domain has added to our understanding of the structure of B. rapa .     

Expression, mapping, and genetic variability of Brassica napus disease resistance gene analogues.

Numerous sequences analogous to resistance (R) genes exist in plant genomes and could be involved in resistance traits. The aim of this study was to identify a large number of Brassica napus sequences related to R genes and also to test the adequacy of specific PCR-based tools for studying them. Different consensus primers were compared for their efficiency in amplifying resistance-gene analogues (RGAs) related to the nucleotide-binding-site subgroup of R genes. Specific primers were subsequently designed to fine-study the different RGAs and we tested their efficiency in three species related to B. napus: Brassica oleracea, Brassica rapa, and Arabidopsis thaliana. Forty-four B. napus RGAs were identified. Among 29 examined, at least one-third were expressed. Eighteen RGAs were mapped on 10 of the 19 B. napus linkage groups. The high variability within these sequences permitted discrimination of each genotype within a B. napus collection. The RGA-specific primers amplified RGAs in the B. oleracea and B. rapa genomes, but the sequences appear to be poorly conserved in A. thaliana. Specific RGA primers are a precise tool for studying known-sequence RGAs. These sequences represent interesting markers that could be correlated with resistance traits in B. napus or related Brassica genomes.     

Denaturing gradient gel electrophoresis analysis of bacterial community profiles in the rhizosphere of <Emphasis Type="Italic">cry1AC-carrying Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis>

The effect of genetically modified (GM) Brassica rapa subsp. pekinensis (Chinese cabbage) expressing Bt toxin gene (cry1AC) to the rhizosphere bacterial community was examined using the denaturing gradient gel electrophoresis (DGGE) fingerprinting method. From the visual comparison of the DGGE profiles, there were no significant differences between the profiles of Bt and control rhizosphere in both Suwon and Yesan samples. From the sequence analysis of the individual bands, Sphingomonas sp. of Alphaproteobacteria and several actinobacterial members were identified as the main bacterial taxa in both Suwon and Yesan samples. In the multiple correspondence analysis, no clear separation between Bt and control rhizosphere was seen in both Suwon and Yesan datasets. The profiles of bulk soils were separated from those of rhizosphere. The DGGE fingerprinting analyses indicated that Bt crops did not significantly alter the genetic composition of rhizosphere bacterial communities.     

An apparent discrepancy between chain length and electrophoretic mobility of restriction fragments: a case of human mitochondrial DNA

Summary DNA sequence analysis and electrophoresis in denaturing gel revealed that a 60 base pair insertion which had been previously postulated on the basis of native polyacrylamide gel electrophoresis of mitochondrial DNA from Japanese (Horai and Matsunaga 1986) did not exist at all. Unusual behavior of certain restriction fragments in native polyacrylamide gels apparently resulted in what appeared to be an insertion. Further study revealed that this behavior is most likely due to secondary structures of the fragments. The results of the present study suggest that adequate care should be taken when assessing molecular weights of restriction fragments by native polyacrylamide gel electrophoresis.     

Investigation of the origin and transmission of linear mitochondrial plasmid based on phylogenetic analysis in Japanese rapeseed varieties.

A linear mitochondrial plasmid is present in some varieties of rapeseed. To elucidate its origin and transmission the author investigated types of mitochondrial genome and the presence of plasmid in 78 rapeseed varieties and landraces in Japan and carried out a comparative analysis using the breeding history of Japanese rapeseed varieties. The mitochondrial genome of rapeseed was classified roughly into 2 types, type I (nap) and type II (cam). Type II rapeseed mitochondria closely resembles that of Brassica rapa, which is a related species of rapeseed. In this study, the author found that all varieties with type II mitochondria originated from interspecific crosses between rapeseed (B. napus) and B. rapa. This indicates that type II cytoplasm was introduced to rapeseed through a breeding program. The presence of plasmid was limited to B. rapa landraces and rapeseed varieties that arose by interspecific crosses between B. napus and B. rapa. The results suggest that mitochondrial plasmid is of B. rapa origin and that it has been introduced into rapeseed by interspecific crosses in a modern breeding program, as in the case of the mitochondrial genome. Phylogenetic study of Japanese rapeseed varieties suggests the participation not of the mitochondrial genome but, rather, the nuclear genome for the perpetuation of plasmid in progeny varieties.     

Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. sylvestris.

The spectrum of resistance to isolates of Leptosphaeria maculans and the map location of a new blackleg resistance gene found in the canola cultivar Brassica napus 'Surpass 400' are described. Two blackleg resistance genes, LepR1 and LepR2, from B. rapa subsp. sylvestris and introgressed in B. napus were identified previously. 'Surpass 400' also has blackleg resistance introgressed from B. rapa subsp. sylvestris. Using 31 diverse isolates of L. maculans, the disease reaction of 'Surpass 400' was compared with those of the resistant breeding lines AD9 (which contains LepR1), AD49 (which contains LepR2), and MC1-8 (which contains both LepR1 and LepR2). The disease reaction on 'Surpass 400' was different from those observed on AD9 and MC1-8, indicating that 'Surpass 400' carries neither LepR1 nor both LepR1 and LepR2 in combination. Disease reactions of 'Surpass 400' to most of the isolates tested were indistinguishable from those of AD49, which suggested 'Surpass 400' might contain LepR2 or a similar resistance gene. Classical genetic analysis of F1 and BC1 plants showed that a dominant allele conferred resistance to isolates of L. maculans in 'Surpass 400'. The resistance gene, which mapped to B. napus linkage group N10 in an interval of 2.9 cM flanked by microsatellite markers sR12281a and sN2428Rb and 11.7 cM below LepR2, was designated LepR3. A 9 cM region of the B. napus genome containing LepR3 was found to be syntenic with a segment of Arabidopsis chromosome 5.     

Structural and functional comparative mapping between the Brassica A genomes in allotetraploid Brassica napus and diploid Brassica rapa

Brassica napus (AACC genome) is an important oilseed crop that was formed by the fusion of the diploids B. rapa (AA) and B. oleracea (CC). The complete genomic sequence of the Brassica A genome will be available soon from the B. rapa genome sequencing project, but it is not clear how informative the A genome sequence in B. rapa (A(r)) will be for predicting the structure and function of the A subgenome in the allotetraploid Brassica species B. napus (A(n)). In this paper, we report the results of structural and functional comparative mapping between the A subgenomes of B. napus and B. rapa based on genetic maps that were anchored with bacterial artificial chromosomes (BACs)-sequence of B. rapa. We identified segmental conservation that represented by syntenic blocks in over one third of the A genome; meanwhile, comparative mapping of quantitative trait loci for seed quality traits identified a dozen homologous regions with conserved function in the A genome of the two species. However, several genomic rearrangement events, such as inversions, intra- and inter-chromosomal translocations, were also observed, covering totally at least 5% of the A genome, between allotetraploid B. napus and diploid B. rapa. Based on these results, the A genomes of B. rapa and B. napus are mostly functionally conserved, but caution will be necessary in applying the full sequence data from B. rapa to the B. napus as a result of genomic rearrangements in the A genome between the two species.     

Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species

We report the identification and characterization of the major repeats in the centromeric and peri-centromeric heterochromatin of Brassica rapa. The analysis involved the characterization of 88 629 bacterial artificial chromosomes (BAC) end sequences and the complete sequences of two BAC clones. We identified centromere-specific retrotransposons of Brassica (CRB) and various peri-centromere-specific retrotransposons (PCRBr). Three copies of the CRB were identified in one BAC clone as nested insertions within a tandem array of 24 copies of a 176 bp centromeric repeat, CentBr. A complex mosaic structure consisting of nine PCRBr elements and large blocks of 238 bp degenerate tandem repeats (TR238) were found in or near a derivative of 5S-25S rDNA sequences. The chromosomal positions of selected repeats were determined using in situ hybridization. These revealed that CRB is a major component of all centromeres in three diploid Brassica species and their allotetraploid relatives. However, CentBr was not detected in the most distantly related of the diploid species analyzed, B. nigra. PCRBr and TR238 were found to be major components in the peri-centromeric heterochromatin blocks of four chromosomes of B. rapa. These repetitive elements were not identified in B. oleracea or B. nigra, indicating that they are A-genome-specific. GenBank accession numbers: KBrH001P13 (AC 166739); KBrH015B20 (AC 166740); end sequences of KBrH BAC library (CW 978640 - CW 988843); end sequences of KBrS BAC library (DU 826965 - DU 835595); end sequences of KBrB BAC library (DX 010661 - DX 083363).