Preparation of megabase‐size DNA from plant nuclei

Zhang, Hong‐Bin ‐B; Zhao, Xinping; Ding, Xiaoling; Paterson, Andrew H.; Wing, Rod A.

doi:10.1046/j.1365-313x.1995.07010175.x

Cited by 266 publications

(204 citation statements)

References 0 publications

Supporting

Mentioning

199

Contrasting

Order By: Relevance

“…SMRT PacBio sequencing. Fifty micrograms of high-molecular-weight Oropetium gDNA was extracted using a modified nuclei preparation method 30 followed by an additional high-salt phenol-chloroform purification to minimize contamination. A 20-kb insert SMRTbell library was generated using a 15 kb lower-end size selection protocol on the BluePippin (Sage Science).…”

Section: Methodsmentioning

confidence: 99%

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

VanBuren

Bryant

Edger

et al. 2015

Nature

288

271

View full text Add to dashboard Cite

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly 1 . The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE) 2 . Here we report the whole-genome sequencing and assembly of the desiccationtolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.The genomes of Arabidopsis 3 , rice 4 , poplar, grape and Sorghum 5 were first sequenced using high-quality and reiterative Sanger-based approaches producing a series of 'gold standard' reference genomes. The advent of next-generation sequencing (NGS) technologies reduced costs of sequencing substantially, which has enabled sequencing of over 100 plant genomes 1 . The quality of plant genome assemblies depends on genome size, ploidy, heterozygosity and sequence coverage, but most NGS-based genomes have on the order of tens of thousands of short contigs distributed in thousands of scaffolds. The short read lengths of NGS, inherent biases and non-random sequencing errors have resulted in highly fragmented draft genome assemblies that are not complete, which means they are missing biologically meaningful sequences including entire genes, regulatory regions, transposable elements, centromeres, telomeres and haplotype-specific structural variations. It is becoming clear from ENCODE projects that complete genomes are needed to better understand the importance of the non-coding regions of genomes 2 .More than 40% of calories consumed by humans are derived from grasses, and the grass family (Poaceae) is arguably the most important plant family with regard to global food security 6 . The size and complexity of most grass genomes has challenged progress in gene discovery and comparative genomics, although draft genomes are now available for most agriculturally important grasses 1 . The largest genome assemblies, such as maize (2,300 megabases (Mb)) 7 , barley (5,100 Mb) 8 and wheat (hexaploid, 1...

show abstract

Section: Methodsmentioning

confidence: 99%

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

VanBuren

Bryant

Edger

et al. 2015

Nature

288

271

View full text Add to dashboard Cite

show abstract

“…Nuclei were isolated from leaves according to Zhang et al (1995). Briefly, leaves were grounded in liquid nitrogen and nuclei were isolated, rinsed five times in washing buffer, resuspended in 2-3 mL in extraction buffer without β-mercaptoethanol, and embedded in 1.5% low-melting-point agarose plugs.…”

Section: High-molecular-weight (Hmw) Dna Isolationmentioning

confidence: 99%

“…Agarose plugs were incubated for 48 h in lysis buffer, and stored in TE buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8.0) at 4°C. All buffers were prepared according to Zhang et al (1995). HMW DNA integrity was tested by pulsed-field gel electrophoresis (PFGE) using a CHEF DRII apparatus (Bio-Rad) at 200 V, with a 1-40 s pulse, for 20 h at 12°C in 0.5× TBE buffer (0.09 M Tris-borate, 0.09 M boric acid, 0.002 M EDTA).…”

Section: High-molecular-weight (Hmw) Dna Isolationmentioning

confidence: 99%

“…Together with vector preparation, quality and quantity of partially digested HMW genomic DNA are the most critical factors in the construction of BAC libraries (Frijters et al 1997;Wang et al 1995;Zhang et al 1995). Zhang et al (1995) suggested that leaf tissues can be homogenized either by blending with a kitchen blender or by grinding in liquid nitrogen with a mortar and pestle.…”

Section: Crucial Steps In the Construction Of The Librarymentioning

confidence: 99%

“…Zhang et al (1995) suggested that leaf tissues can be homogenized either by blending with a kitchen blender or by grinding in liquid nitrogen with a mortar and pestle. Both methods were compared here (data not shown) and the second one resulted in a lower proportion of degraded DNA in T. monococcum.…”

Section: Crucial Steps In the Construction Of The Librarymentioning

confidence: 99%

See 2 more Smart Citations

Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat

Lijavetzky

Muzzi

Wicker³

et al. 1999

Genome

117

View full text Add to dashboard Cite

A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276 480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720 384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.Key words: bacterial artificial chromosome, BAC library, Triticum monococcum, wheat.Résumé : Une banque génomique du génome A du blé a été produite dans des chromosomes bactériens artificiels (BAC). L'accession DV92 du Triticum monococcum a été choisie pour ces fins car il s'agit d'une lignée diploïde qui est cultivée et d'un des parents employés dans la construction de cartes génétiques saturées. De l'ADN de grand poids moléculaire a été extrait de noyaux des feuilles de ce génotype. Cet ADN a été partiellement digéré avec l'enzyme de restriction HindIII, assujetti à une double sélection en fonction de la taille, électroélué et cloné dans le vecteur BAC pINDIGO451. La banque comprend 276,480 clones dont les inserts ont une taille moyenne de 115 kb. En excluant 1,33 % de clones vides et 0,14 % de clones contenant de l'ADN chloroplastique, cette banque offre une couverture génomique équivalent à 5,6 génomes. Avec une telle couverture, la probabilité qu'une séquence y soit représentée excède 99,6 %. Les clones ont été disposés dans 720 boîtes à 384 puits et placés sur 15 membranes à haute densité. Ces membranes ont été criblées avec des clones à copie unique ou à faible nombre de copies et cinq clones BAC ont été choisis pour des analyses détaillées. Comme la plupart des extrémités de BAC du T. monococcum comprennent de l'ADN répété, une modification a été apportée à la méthode classique d'isolement des séquences terminales afin de sélectionner des séquences à faible nombre de copies pour les fins de marche chromosomique.

show abstract

Construction and Application of Genomic DNA Libraries

Kim¹,

Yang²,

Kudrna³

et al. 2004

Handbook of Plant Biotechnology

View full text Add to dashboard Cite

Crop failures, pathogenic outbreaks and famine are serious problems facing society. Understanding an organism's genome will help provide the genetic tools needed to solve these complex problems in a shorter time and with less effort. To efficiently study an organism's genome, it can be partitioned into a permanent and stable collection of DNA fragments, called a library. Such libraries provide convenient access to a genome for both laboratory and breeding applications. Genomic libraries can be used as substrates to physically map and sequence entire genomes, clone agriculturally important genes and to investigate gene expression patterns. Further, genomic libraries also provide powerful tools and resources for evaluating germplasm conservation stocks and biological diversity. The ongoing explosion of genetic data and molecular clone resources has opened new scientific possibilities to researchers venturing across all avenues of applied science. In this chapter we will present the details for small and large insert library construction and their applications.

show abstract

Preparation of megabase‐size DNA from plant nuclei

Cited by 266 publications

References 0 publications

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat

Construction and Application of Genomic DNA Libraries

Contact Info

Product

Resources

About