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1.
We have constructed a soybean (Glycine max (L.) Merrill) bacterial artificial chromosome (BAC) library from green leaf protoplasts of the cultivar, Misuzudaizu. The library contains 53,760 clones with an average insert size of 116 kb. About 2.9% chloroplast DNA origin was revealed by PCR and colony hybridization. Apart from 2.8% clones having no insert, this library represents 5.2 genome equivalents. With this genome coverage, the probability of having any DNA sequence represented in the library is higher than 99.5%. Three-dimensional pools of the BAC library in combination with the use of a high efficiency genome scanning (HEGS) electrophoresis facilitate rapid and efficient PCR-based screening. An average of five positive clones were identified after screening the BAC library with SSR and STS markers. BAC-end walking was performed for three SSR associated BACs. This library will provide a good resource for positional cloning of agronomically and biologically important QTL genes that Misuzudaizu possesses.  相似文献   

2.
Summary We established a chromosome specific DNA library of the Aegilops markgrafii chromosome B. Eight microdissected chromosomes B obtained from a monosomic T. aestivum-Aegilops markgrafii addition line were PCR-amplified and the DNA was cloned in Escherichia coli DH5. Clones were characterized by dot blot hybridization with total Ae. markgrafii DNA. 62% of clones represented repetitive sequences and 38% low or single copy sequences. The estimated length of excised inserts varied between less than 200 bp and more than 500 bp. The average size of inserts was 310 bp.Abbreviations bp base pairs - DOP-PCR degenerated oligonucleotide primed polymerase chain reaction  相似文献   

3.
The development of soybean varieties that lack the β-conglycinin α′ subunit is an attractive goal because the β-conglycinin α′ subunit exerts a negative influence on nutrition and tofu gelation, and is also a major allergen. We sought to develop a co-dominant DNA marker for the β-conglycinin α′ subunit (Cgy-1) gene for use in marker-assisted selection (MAS). We identified a deleted sequence responsible for the null allele of Cgy-1 in a soybean variety lacking the β-conglycinin α′ subunit known as ‘Keburi’. The deletion spanned 12,998 bp and included Cgy-1 and its incomplete tandem duplicate on chromosome 10. Based on the Cgy-1 sequence from both the Williams 82 soybean reference sequence and the Keburi variety, a set of three allele-specific primers was designed for multiplex PCR assay. These primers enabled allelic discrimination by the sizes of PCR products. This amplified two distinct DNA fragments of 913 and 649 bp in Williams 82 and Keburi, respectively. The practicality of the developed co-dominant marker for Cgy-1 was also confirmed by amplification in five other soybean varieties including three wild types and two mutants. The heterozygosity of the F1 plants at the Cgy-1 locus was ascertained using our novel co-dominant marker. This PCR-based co-dominant marker is capable of detecting the presence or absence of β-conglycinin α′ subunit for soybean marker assisted breeding system.  相似文献   

4.
A bacterial artificial chromosome (BAC) library was constructed using the sunflower (Helianthus annuus L.) restorer line RHA325, which carries the restorer gene Rf1 and the Pl2-gene conferring resistance to downy mildew. High molecular weight DNA was prepared from nuclei using leaf material from two-week old seedlings. The library was constructed using the HindIII site of pBeloBAC11. The current BAC library comprises 104,736 clones. The insert size of the clones varied between 20 and 270 kb, with an average insert size of 60 kb. The whole 1.9× sunflower BAC library was spotted in duplicate on four high-density filters, each carrying 55,296 clones. The content of organellar DNA, which was estimated by colony hybridisation against the mitochondrial probe coxI and the chloroplast probe rbcL, proved to be less than 0.03 and 0.1%, respectively. BAC pools, allowing PCR-based screening, were made and used to identify positive BAC clones for the markers OP-K13_454, closely linked to the restorer gene Rf1. The PCR-based screening was verified by the results obtained for this marker by colony hybridisation.  相似文献   

5.
黏类小麦细胞质雄性不育系mtDNA消减文库的构建   总被引：1，自引：0，他引：1  
本研究利用抑制性消减杂交技术,构建了黏类小麦细胞质雄性不育线粒体DNA的消减文库。分别提取相同细胞质背景下的不育和可育等基因系线粒体基因组DNA(mtDNA),用RsaⅠ酶切成大小不等的片段,并各自与不同的接头连接,连续经过两次消减杂交和两次PCR扩增,将PCR产物与克隆载体连接,转化为大肠杆菌感受态细胞DH5α,经蓝白斑筛选后,再用PCR方法插入片段筛选出阳性重组质粒,构建差异DNA消减文库。在构建的不育mtDNA文库中,将SSH文库的全部扩增产物与SSH文库中的单条扩增条带分别进行胶回收和克隆转化,分别挑取54个和6个阳性克隆进行测序分析和相关功能初步比对。结果表明,不育mtDNA的SSH文库差减杂交效率较高,质量较好;回收单条扩增条带分别进行克隆测序的结果准确率较高;对测序序列进行BLASTx比对及功能注释分析发现,约90%的序列来源于线粒体基因nad1和nad5的非编码区。  相似文献   

6.
Summary Fluorescent in situ hybridization (FISH) of DNA to plant chromosomes has proved to be a powerful cytogenetic tool. The value of fluorescent in situ hybridization of total genomic DNA (GISH) of related species is demonstrated in the determination of wheat/alien chromosome pairing in hybrids. Its use for assessing the relative merits of the various genes that affect chromosome pairing is also shown.The ability of GISH to identify the presence in wheat of whole alien chromosomes or alien chromosome segments is illustrated. The potential of FISH for detecting repeated DNA sequences, low copy sequences and single copy genes is discussed.Abbreviations FISH fluorescent in situ hybridization - GISH genomic in situ hybridization - PRINS primer-induced in situ hybridization  相似文献   

7.
A genome specific DNA sequence that detects Secale africanum chromatin incorporated into wheat was developed in this study. Random amplified polymorphic DNA (RAPD) analysis was used to search for genome specific DNA sequences of S. africanum in lines, R111, “mianyang11” (MY11) and wheat-rye 1RS/1BL translocations R25 and R57. A high copy rye-specific DNA segment pSaD15940 of the S. africanum genome was obtained. The sequence of pSaD15 did not show any significant homology to other reported sequences in databases and it is therefore a new repetitive sequence of Secale. PCR primers were designed for pSaD15940, which amplify a clear 887 bp fragment in S. africanum but not in any wheat. The primers also amplified an 887 bp fragment in other accessions of rye, Chinese Spring-Imperial rye chromosome additions and a diverse range of material carrying different rye chromosomes or chromosomal segments. In situ hybridization showed that probe pSaD15940 was specifically hybridized throughout all rye chromosomes arms except for the terminal regions. The advantage of the rye-specific probe developed herein compared to those of previous reports is that it has been shown to be widely applicable to other Secale species. The probe will be useful as a molecular marker for the introgression of S. africanum and other rye chromosome segments into the wheat genome.  相似文献   

8.
Incidence and molecular markers of 2n pollen in Populus tomentosa Carr.   总被引：2，自引：0，他引：2  
Microscopic examination, amplified fragment length polymorphism (AFLP) and SCAR (sequence-characterized amplified region) molecular markers were employed to determine the incidence of 2n pollen (unreduced pollen) in Chinese white poplar (Populus tomentosa Carr.) and to identify related molecular markers. The presence of a parallel and tripolar spindle at metaphase II and the absence of cytokinesis at telophase II were found to be determining factors in 2n pollen formation. A group of 298 clones that originated from their indigenous areas were investigated for the production of 2n pollen based on pollen size differences, both within a clone and between n and 2n pollen. Pollen grains were collected from 224 of the clones, six of which were subsequently determined to produce only normal pollen; the remainder produced 2n pollen at different frequencies (0.6–21.9%). The frequency at which 2n pollen was produced was significantly and highly significantly different among and within indigenous populations, respectively. Clones produced by the six normal and twenty-two 2n pollen clones were selected for AFLP analysis. Following an initial screening with 55 primer combinations, the E50-M38 (CAT/ACT) primer was identified: it generated a PCR fragment (246 bp) from the normal clones, but not from the 2n pollen producers. In addition, the E31-M50 (AAA/CAT)-amplified DNA fragment (204 bp) was present in 2n pollen producers, and absent in normal clones. These two discriminating AFLP markers were developed into easily detectable SCAR (sequence characterized amplified region) markers which can be used in combination with previously developed AFLP markers to distinguish between normal and 2n pollen clones.  相似文献   

9.
 采用前低渗、酶解、后低渗和盖片轻压相结合的方法,建立了激光法分离二倍体亚洲棉石系亚1号单条染色体技术。单染色体经去蛋白、酶切和PCR扩增后,产物以Southern杂交、简单重复序列(Simple sequence repeats, SSR)引物扩增和荧光原位杂交技术(Fluorescence in situ hybridization, FISH)进行验证,构建了棉花单条染色体文库。克隆片段长度在150~1000 bp,平均为550 bp;文库包含1.38×105个克隆,覆盖染色体长度1倍左右,空载率为1%,滴度为1.3×106 pfu·mL-1,单一拷贝和低拷贝比例达到59%以上,为该条染色体分子标记的筛选、重要基因克隆和定位、遗传图谱的饱和奠定基础。  相似文献   

10.
Chromosome morphology was studied in diploid cultivars of Tulipa fosteriana and T. gesneriana (2n = 2x = 24) and triploid Darwin hybrids (2n = 3x = 36) developed from interspecific crosses of T. gesneriana and T. fosteriana. Chromosomes were arranged in the karyotype according to decreasing total length. Based on our karyotypic analysis, we propose that median chromosomes may serve as markers for diploid genotypes. Discriminant analysis with respect to total chromosome length and short arm length showed a significant difference between the size of the larger median chromosomes of T. gesneriana and T. fosteriana Comparison of median chromosome length in Darwin hybrid tulips showed that two larger chromosomes and one smaller chromosome were derived from T. gesneriana and T. fosteriana, respectively. This finding was clearly and unambiguously confirmed by simultaneous hybridization of differentially labeled genomic probes of T. fosteriana and T. gesneriana to metaphase chromosomes of the triploid cultivar ‘Yellow Dover’, thereby enabling us to distinguish between the 24 chromosomes derived from T. gesneriana and 12 chromosomes derived from T. fosteriana. Thus, genomic in situ hybridization and median chromosome analyses can be useful to identify the genome constitution of triploid Darwin hybrid tulips. In addition, their hybridity was readily verified by flow cytometry using vegetative tissue of Darwin hybrid tulips. Our results clarify the process of Tulipa cultivar formation and will be useful for interspecific hybridization breeding.The first and second author have contributed equally to this paper  相似文献   

11.
高纤维强力棉花种质系苏远7235 BAC文库的构建   总被引：1，自引：3，他引：1  
苏远7235是我国利用异常棉等多个野生种创造的高纤维强力棉花种质系,是开展棉花纤维品质研究的重要材料。本研究以pIndigoBAC-5(HindIII-cloning ready)为载体,构建了苏远7235的细菌人工染色体(Bacterial Artificial Chromosome,BAC)文库,该文库包含30336个BAC克隆。分析结果表明,重组克隆苏远7235 DNA插入片段为50-140 kb,平均120 kb,空载率2.1%,89.6%的克隆插入片段大于100 kb。  相似文献   

12.
In this study, Tulipa fosteriana was found to contain 45S rDNA repeat units of 9.7 and 9.5 kb, in which at least 7 types of 45S rDNAs were identified by restriction site analysis. For 5S rDNA, repeat units ranging from 364 bp to 396 bp were identified. The diploid cultivars (2n = 2x = 24) ‘Christmas Dream’ and ‘Queen of Night,’ representing the horticultural group T. gesneriana, and ‘Red Emperor’, belonging to T. fosteriana, were compared cytogenetically using cloned 5S and 45S rDNAs. Fluorescence in situ hybridization (FISH) analysis identified many rDNA sites located on each chromosome in the diploid genomes. For example, we identified 71 sites of 5S rDNA and 10 sites of 45S rDNA in ‘Red Emperor’. Additionally, FISH analyses enabled construction of karyotypes for these cultivars. Karyotype comparison of T. gesneriana cultivars showed conservation of repetitive rDNA unit positioning. A clear difference in chromosome size and signal pattern was observed between T. gesneriana and T. fosteriana cultivars. Here we demonstrate the unique nature of the highly repeated 5S rDNA units in these Tulipa species and the usefulness of FISH karyotyping with cloned 5S and 45S rDNAs to clearly distinguish between chromosomes from T. gesneriana and T. fosteriana. Hitoshi Mizuochi and Agnieszka Marasek contributed equally to this paper  相似文献   

13.
Z. S. Lin    D. H. Huang    L. P. Du    X. G. Ye    Z. Y. Xin 《Plant Breeding》2006,125(2):114-119
Among the regenerated plants derived from immature hybrid embryos of wheat–Thinopyrum intermedium disomic addition line Z6 × common wheat variety ‘Zhong8601’, a plant with a telocentric chromosome and barley yellow dwarf virus (BYDV) resistance was obtained. The telocentric chromosome paired with an entire Thinopyrum chromosome to form a heteromorphic bivalent at meiotic metaphase I. Genomic in situ hybridization showed that the telosome originated from Th. intermedium. Two ditelosomic additions and one disomic substitution were identified among the offspring of the plant. Two random amplified polymorphic DNA molecular markers were identified among 150 random primers used to detect the different arms of the alien chromosome. These might be useful for developing translocation lines with BYDV resistance.  相似文献   

14.
Bacterial artificial chromosome (BAC) libraries with large DNA fragment inserts have rapidly become the preferred choice for physical mapping. BAC-derived microsatellite or simple sequence repeats (SSRs) markers facilitate the integration of physical maps with genetic maps. The objective of this research was to identify chromosome locations of the BAC-derived SSR markers in tetraploid cotton. A total of 192 SSR primer pairs were derived from BAC clones of an Upland cotton genetic standard line TM-1 (Gossypium hirsutum L.). Metaphor agarose gel electrophoresis results revealed 76 and 59 polymorphic markers between TM-1 and 3–79 (G. barbadense) or G. tomentosum, respectively. Using deletion analysis method, we assigned 39 markers out of the 192 primer pairs to 17 different chromosomes or chromosome arms. Among them, 19 and 17 markers were localized to A-subgenomes (chromosome 1–13) and D-subgenomes (chromosome 14–26), respectively. The subgenome status for the remaining three markers remained unclear due to their two potential chromosome locations achieved by tertiary monosomic stocks deletion analysis. Chromosomal assignment of these BAC-derived SSR markers will help in integrating physical and cotton genetic linkage maps and thus facilitate positional candidate gene cloning, comparative genome analysis, and the coordination of chromosome-based genome sequencing project in cotton. Disclaimer: Mention of trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by USDA, ARS and does not imply its approval to the exclusion of other products or vendors that may also be suitable. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.  相似文献   

15.
Summary Fluorescent in situ hybridization (FISH) has been used to assess the occurrence and frequency of wheat-alien chromosome pairing in a wheat/Thinopyrum bessarabicum hybrid and in wheat/rye hybrids with different levels of chromosome pairing by examining pollen mother cells at metaphase I of meiosis. The use of FISH to identify the presence and size of alien chromatin in a wheat background is also demonstrated.The value of FISH as an aid to the introgression of alien genetic variation into wheat is discussed.Abbreviations FISH fluorescent in situ hybridization - GISH genomic in situ hybridization - PRINS primer-induced in situ hybridization  相似文献   

16.
目的序列长片段PCR产物可作为FISH技术的有效探针进行分子细胞遗传学研究。然而,传统PCR技术对于5 kb以上的长片段进行有效扩增很难。合适的反应条件及反应体系是进行长片段PCR有效扩增的必要前提。为了获得目的序列长片段PCR产物以用于FISH研究,根据大白菜A03染色体顶端无重复序列区段设计了80对长片段PCR引物,从基因组DNA模板质量、d NTPs浓度以及退火温度和延伸时间方面对PCR技术体系进行了优化。试验证明,选用幼苗嫩叶的基因组DNA和LA Taq DNA聚合酶可以提高长片段PCR引物的扩增质量和扩增效率;确定了适合5~15 kb长片段PCR的反应体系为20μL:50 ng/μL模板DNA 2μL,2.5 mmol/L d NTPs 1.6μL,10μmol/μL正反引物各1μL,10×LA PCR BufferⅡ(含Mg2+)2μL,5 U/μL LA Taq酶0.2μL;反应条件为98℃变性15 s;58~64℃退火10 s,68℃延伸5 min,35个循环;68℃延伸10 min,4℃保存。在大白菜基因组中成功获得了60对5~15 kb的扩增片段。为在大白菜粗线期染色体上开展长片段PCR-FISH技术研究及在近缘种间开展比较染色体涂染揭示进化关系奠定了理论基础。  相似文献   

17.
G. Mandolino    S. De  Marco  V. Faeti    M. Bagatta    A. Carboni  P. Ranalli 《Plant Breeding》1996,115(6):439-444
The restriction and amplification patterns of potato genomic DNA of eight different cultivars and breeding clones has been studied. The analysis was carried out by polyacrylamide high resolution gels, and subsequent blotting and hybridization to potato DNA probes, and by agarose gel electrophoresis of the amplification products obtained with a variety of random primers. Fingerprinting of the genotypes was possible using two enzyme-probe combinations (Rsal-GP35 and Rsal-CP6) and three random primers (OPA4, OPA19 and OPG12). Based on the same techniques, a number of plants from the cvs. Monalisa and Spunta originated from the sprouting of in vitro-induced tubers (vitrotubers) were analysed to test the reproducibility of RFLP and PCR patterns. No variations were found with RFLP analysis. Some different RAPD patterns were seen, showing putatively vitrotuber-specific variations.  相似文献   

18.
Total DNA from three putative cytoplasmic male sterile (CMS) progenies derived from crosses between the wild species Cajanus sericeus and the cultivated species Cajanus cajan, five C. cajan, one accession of C. sericeus and two genetic male sterile lines of pigeonpea were compared for their RFLP patterns using maize mitochondrial DNA (mtDNA) specific probes. Three putative cytoplasmic male sterile (CMS) progenies from the multiple cross genome transfer of pigeonpea lines (CMS 7–1, CMS 12–3, and CMS 33–1) showed hybridization patterns identical to that of C. sericeus when DNA was digested with EcoRI and HindIII and probed with maize mtDNA clones. The results suggested that these putative CMS progenies have the mitochondria of the female wild species parent. The hybridization patterns of the three male parental lines used in the development of the CMS progenies were similar in all the restriction enzyme-probe combinations except HindIII-atp6. The genetic male sterile lines, MS Prabhat and QMS 1 differed from each other in their hybridization pattern. The genomic DNA hybridization pattern of HindIII digested DNA from ICPL 87 differed from the other pigeonpea lines when probed with the maize mtDNA clones. The cluster analysis of the hybridization data suggested the occurrence of variation in the mitochondrial genome even among the cultivated species. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

19.
SCoT结合克隆测序鉴别湖南甜橙变异类型   总被引：6，自引：1，他引：5  
本试验对SCoT反应体系进行优化,并筛选合适的引物,然后对24份试材进行SCoT标记,获得的目的片段克隆测序,通过测序结果探讨其遗传变异;结果表明,甜橙SCoT标记的20 μL优化反应体系为：DNA模板80 ng,Mg2+浓度1.6 mmol/L,dNTPs浓度0.3 mmol/L,引物浓度0.2 μmol/L,Taq DNA聚合酶用量1.6 U,扩增产物在100~2000 bp之间,扩增条带明亮清晰,反应体系具有良好的稳定性和可重复性。24份试材的测序片段的大小为1090~1091 bp,一致性达到99.84%,存在单碱基的缺失与替换,BLAST结果显示,该序列的编码蛋白质与核糖体蛋白S3的N端同源性高;利用单碱基变异可以区分其中的12个甜橙变异株系和‘安江香柚’,其他变异株系还需要结合其它的分子标记技术来进行鉴别,有待进一步研究。  相似文献   

20.
雷蒙德氏棉基因组草图的完成为棉花的基础研究奠定基础,然而该基因组草图仍需要后续的补充和完善。利用雷蒙德氏棉基因组草图信息,分别在6条较长染色体的端部选取了4 kb的序列。通过生物信息学分析这些序列在基因组中的拷贝情况,发现2号染色体一端的序列Chr2D属于单拷贝序列,且其内部没有重复序列。随后以该段序列为探针,对雷蒙德氏棉有丝分裂中期染色体进行荧光原位杂交,结果显示在1号染色体和4号染色体的一端有明显的杂交信号,信号大小远远大于4 kb序列所能产生信号的大小,而信号的强度则比正常的杂交信号暗弱。试验结果说明该4 kb序列Chr2D可能在1号染色体和4号染色体的1个端部有较高的拷贝数,而信号强度的暗弱则说明该序列在染色体上的分布方式可能为散漫分布。杂交信号在1号染色体和4号染色体上的相似性说明,这2对染色体可能有一定的亲缘关系。该结果将对雷蒙德氏棉基因组草图的补充完善有帮助。  相似文献   

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