Fosmid Library Construction and Initial Analysis of End Sequences in Female Half-smooth Tongue Sole (Cynoglossus semilaevis)

Wang, Xubo; Zhang, Quanqi; Sun, Xiaohua; Chen, Yanjie; Zhai, Teng; Zhuang, Wei; Qi, Jie; Wang, Zhigang

doi:10.1007/s10126-008-9137-2

Cited by 20 publications

(6 citation statements)

References 23 publications

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“…The electrophoretic patterns of each clone digested with HindI and EcoRI were identical from days 1 to 6, confirming that the T. mairei formid clone inserts were stable after longterm culture. This result is similar to the studies conducted in other species (Zhang et al 2007;Wang et al 2009). Therefore, as a substitute for cosmid, fosmid vector would continue to be used in genomic studies.…”

Section: Stability Of the Clonessupporting

confidence: 92%

The first insight into the Taxus genome via fosmid library construction and end sequencing

2011

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Taxus mairei is a critically endangered and commercially important cultured medicinal gymnosperm in China and forms an important medicinal resource, but the research of its genome is absent. In this study, we constructed a T. mairei fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of one million clones with an average insert size of about 39 kb, amounting to 3.9 genome equivalents. Fosmid stability assays indicate that T. mairei DNA was stable during propagation in the fosmid system. End sequencing of both 5 0 and 3 0 ends of 968 individual clones generated 1,923 sequences after trimming, with an average sequence length of 839 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 560 (29.1%) significant hits (E \ e -5 ). Repetitive sequences analysis revealed that 20.8% of end sequences are repetitive elements, which were composed of retroelements, DNA transposons, satellites, simple repeats, and low complexity sequences. The distribution pattern of various repeat types was found to be more similar to the gymnosperm Pinus and Picea than to the monocot and dicot. The satellites of T. mairei were significantly longer than those of P. taeda and P. glauca. The tetra-nucleotide repeats of T. mairei were much longer than those of P. glauca and P. taeda. The fosmid library and the fosmid end sequences, for the first time, will serve as a useful resource for large-scale genome sequencing, physical mapping, SSR marker development and positional cloning, and provide a better understanding of the Taxus genome.

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Section: Stability Of the Clonessupporting

confidence: 92%

The first insight into the Taxus genome via fosmid library construction and end sequencing

2011

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“…To achieve this goal, the creation of a single copy genomic fosmid library coupled with high-throughput pyrosequencing were selected as appropriate strategies. Fosmid libraries yield large insert sizes, have high stability and reduced susceptibility to aberrent recombination, thereby ensuring maximum genomic sequence representation [24], [25]. By randomly shearing DNA fragments, these libraries also retain a wider selection of sequences than those based on traditional restriction digestion [26].…”

Section: Resultsmentioning

confidence: 99%

Tandem Quadruplication of HMA4 in the Zinc (Zn) and Cadmium (Cd) Hyperaccumulator Noccaea caerulescens

et al. 2011

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Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea.A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS™ clones with insert sizes ∼20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs.Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter β-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue.This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.

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“…However, little was known about the genetic base of its development, growth and immunity, etc. (Sun et al, 2008;Liao et al, 2009;Wang et al, 2009;Yu et al, 2009a,b). Here we reported the cloning of MHC class II B gene and proved that two distinct B genes existed in half-smooth tongue sole.…”

Section: Introductionmentioning

confidence: 99%

Isolation, polymorphism and expression study of two distinct major histocompatibility complex class II B genes from half‐smooth tongue sole (Cynoglossus semilaevis)

Sun

et al. 2010

Int J Immunogenetics

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Major histocompatibility complex (MHC) class II antigens are important in vertebrate immune system, which present peptides to CD4(+) T cells. In the present study, cDNAs encoding MHC class II B gene were isolated from the cDNA library of half-smooth tongue sole (Cynoglossus semilaevis), and the full length cDNA sequences were got by rapid amplification of cDNA ends polymerase chain reaction. The polymorphism of its open reading frame, 3' untranslated region and intron 1 was studied. Nineteen class II B alleles were identified from nine individuals and clustered into two groups, designated as Cyse-DAB and Cyse-DBB. The deduced amino acid sequences among Cyse-DAB and Cyse-DBB alleles shared identities from 94.0% to 99.6% and 92.4% to 99.6%, respectively, while the identities between Cyse-DAB and Cyse-DBB genes varied from 85.1% to 92.0%. Three Cyse-DAB alleles and one Cyse-DBB allele were observed in each of two individuals, and three Cyse-DBB alleles and one Cyse-DAB allele in another individual, which indicated at least two loci existed in each gene. Two different 3' UTR sequences were identified and one belonged to Cyse-DAB, the other belonged to Cyse-DBB. Both five Cyse-DAB and five Cyse-DBB intron 1 sequences were identified from genomic sequences, among which two sequences of each gene were identified in a single individual, which suggested the existence of at least two Cyse-DAB and two Cyse-DBB loci. Both the two genes'-specific tissue and developmental stage expression were studied by reverse-transcription polymerase chain reaction, which showed that the two genes had similar expression patterns in tissue study with high expression in spleen and kidney, low expression in liver, gill and ovary, moderate expression in brain, heart, intestine and testis. While in developmental analysis, Cyse-DBB had higher expression than Cyse-DAB in early developmental stages, which indicated that the two genes might have different functions in those stages. Therefore, in half-smooth tongue sole, two different MHC class II B genes exist and could differentiate from each other whether by sequence analysis or by developmental stage expression study.

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Fosmid Library Construction and Initial Analysis of End Sequences in Female Half-smooth Tongue Sole (Cynoglossus semilaevis)

Cited by 20 publications

References 23 publications

The first insight into the Taxus genome via fosmid library construction and end sequencing

The first insight into the Taxus genome via fosmid library construction and end sequencing

Tandem Quadruplication of HMA4 in the Zinc (Zn) and Cadmium (Cd) Hyperaccumulator Noccaea caerulescens

Isolation, polymorphism and expression study of two distinct major histocompatibility complex class II B genes from half‐smooth tongue sole (Cynoglossus semilaevis)

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