Discrimination of Repetitive Sequences Polymorphism in Secale cereale by Genomic In Situ Hybridization-Banding

Genomic in situ hybridization banding （GISH-banding）, a technique slightly modified from conventional GISH, was used to probe the Chinese native rye （Secale cereale L.） DNA, and enabled us to visualize the individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat （Triticum aestivum L.） background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S. cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattern of Secale vavilovii was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromaUn is discussed.

Segmental Duplications Are Common in Rice Genome

Segmental duplications on rice (Oryza sativa L.) chromosomes 8, 9, 11, and 12 were studied by examining the distributions of sequences resolved by 13 probes detecting multiple copies of DNA sequences. Four of the hybridization bands detected by a repetitive sequence probe, rTRS, were mapped to the ends of all the four chromosomes. Two or three of the bands detected by each of the other 12 probes were also mapped to different chromosomes. The bands detected by the same probe usually occurred in similar locations of different chromosomes. Loci detected by different DNA probes were often similarly arranged on different chromosomes. Chromosomes 8 and 9 showed colinearity of marker loci arrangement indicating a possible common origin. A segment on chromosome 9 was also very similar to the previously reported duplicated fragments on the ends of chromosomes 11 and 12 which were also detected in this study, indicating a likely common origin. Moreover, the various degrees of distributional similarity of the segments suggest a complex relationship among the chromosomes in the evolution of the rice genome. These results support the proposition that chromosome duplication and diversification may be a mechanism for the origin and evolution of the chromosomes in the rice genome.

BAC end sequencing of Pacific white shrimp Litopenaeus vannamei: a glimpse into the genome of Penaeid shrimp

Little is known about the genome of Pacific white shrimp (Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 paired- ends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for this species.

Repetitive DNA Sequences from the X Chromosome of the Spiny Eel (Mastacembelus aculeatus)

To investigate the characters of repetitive DNA sequence in the sex chromosomes of the spiny eel （Mastacembelus aculeatus）, the X chromosomal library was screened and a family of repetitive sequence, consisting of Ma 1-Ma 6, was isolated. The fluorescence in situ hybridization （FISH） result confirmed that Ma 1- Ma 5 dispersed over sex chromosomes and all autosomes, whereas, Ma 6 is sex chromosome-specific and distributed only on the C-band positive regions of X chromosome, and Ma 6 maybe the main components of the heterochromatic regions of X chromosome. This study provides additional information about the evolution of sex chromosomes in lower vertebrates such as fish.

Integrating the physical and genetic map of bread wheat facilitates the detection of chromosomal rearrangements

The bread wheat genome harbors a high content of repetitive DNA,which is amenable to detection and characterization using fluorescence in situ hybridization(FISH)karyotyping.An integrated genetic map was derived from a recombinant inbred population bred from a cross between a synthetic hexaploid wheat and a commercial Chinese bread wheat cultivar,based on 28 variable FISH sites and>150000 single nucleotide polymorphism(SNP)loci.The majority(20/28)of the variable FISH sites were physically located within a chromosomal region consistent with the genetic location inferred from that of their co-segregating SNP loci.The eight exceptions reflected the presence of either a translocation(1 R/1 B,1 A/7 A)or a presumptive intra-chromosomal inversion(4 A).For eight out of the nine FISH sites detected on the Chinese Spring(CS)karyotype,there was a good match with the reference genome sequence,indicating that the most recent assembly has dealt well with the problem of placing tandem repeats.The integrated genetic map produced for wheat is informative as to the location of blocks of tandemly repeated DNA and can aid in improving the quality of the genome sequence assembly in regions surrounding these blocks.

Telomerase activity analysis of esophageal carcinoma using microdissection-TRAP assay

OBJECTIVES: To investigate telomerase activity in esophageal squamous cell carcinoma (SCC) and its preneoplasia lesions, and to study the relationships between telomerase activity and cancer differentiation, cancer invasiveness, and lymphatic metastasis. METHODS: Telomerase activity in esophageal SCC tissues, adjacent dysplasia tissues and normal epithelia from the surgical edge were assessed by microdissection-TRAP (telomeric repeat amplification protocol)-silver staining assay. RESULTS: Telomerase activity was detected in 37 (82.2%) of 45 esophageal tumors, 23 (79.3%) of 29 dysplasias, and 2 (5%) of 40 normal epithelia. There was a significant difference in activity between dysplasia and normal epithelium, as well as between tumor and normal epithelium. Twenty-six (92.9%) of 28 tumors with lymphatic metastasis had detectable telomerase activity compared to 11 (64.7%) of 17 non-lymphatic metastasis tumors. These relationships were statistically significant (P

Identification of Highly Repetitive Enhancers with Long-range Regulation Potential in Barley via STARR-seq

Enhancers are DNA sequences that can strengthen transcription initiation.However,the global identification of plant enhancers is complicated due to uncertainty in the distance and orientation of enhancers,especially in species with large genomes.In this study,we performed self-transcribing active regulatory region sequencing(STARR-seq)for the first time to identify enhancers across the barley genome.A total of 7323 enhancers were successfully identified,and among 45 randomly selected enhancers,over 75%were effective as validated by a dual-luciferase reporter assay system in the lower epidermis of tobacco leaves.Interestingly,up to 53.5%of the barley enhancers were repetitive sequences,especially transposable elements(TEs),thus reinforcing the vital role of repetitive enhancers in gene expression.Both the common active mark H3K4me3 and repressive mark H3K27me3 were abundant among the barley STARR-seq enhancers.In addition,the functional range of barley STARR-seq enhancers seemed much broader than that of rice or maize and extended to±100 kb of the gene body,and this finding was consistent with the high expression levels of genes in the genome.This study specifically depicts the unique features of barley enhancers and provides available barley enhancers for further utilization.

Gene deletion and carrier detection in the family of Becker muscular dystrophy by short tandem repeat sequence polymorphism

Objective To type haplotypes among the patients, carriers and normal offspring in a family of males with Becker muscular dystrophy in one generation by allelic fragment length polymorphism analysis. Methods Deletion analysis of the patients were performed using multiplex polymerase chain reaction (PCR) of amplification with 9 dystrophin exon primers. Intragenic short tandem repeat (STR) sequence (STR44, STR45, STR49 and STR50) were amplified by PCR to analyse allelic fragment length polymorphisms in the members of the family. Results The deletions of exons 17, 19 and 45, as well as deletions of allelic fragments at the loci of STR44 and STR45 were determined in the patients. Hemizygosity at those two loci were detected and carrier status ascertained in the mother of the patients. The normal haplotypes were typed in the sister of the patients. Conclusion The method of STR sequence polymorphism analysis can determine haplotypes at normal status or at risk status. It would be used in prenatal diagnosis and carrier detection in the families of Duchenne and Becker muscular dystrophy.

De novo characterization of the root transcriptome of a traditional Chinese medicinal plant Polygonum cuspidatum

Various active components have been extracted from the root of Polygonum cuspidatum. However, the genetic basis for their activity is virtually unknown. In this study, 25600002 short reads （2.3 Gb） of P. cuspidatum root transcriptome were obtained via lllumina HiSeq 2000 sequencing. A total of 86418 urtigenes were assembled de novo and annotated. Twelve, 18, 60 and 54 unigenes were respectively mapped to the mevalonic acid （MVA）, methyl-D-erythritol 4-phosphate （MEP）, shikimate and resveratrol biosynthesis pathways, suggesting that they are involved in the biosynthesis of pharmaceutically important anthra- quinone and resveratrol. Eighteen potential UDP-glycosyltransferase unigenes were identified as the candidates most likely to be involved in the biosynthesis of glycosides of secondary metabolites. Identification of relevant genes could be important in eventually increasing the yields of the medicinally useful constituents of the P. cuspidatum root. From the previously published transcriptome data of 19 non-model plant taxa, 1127 shared orthologs were identified and characterized. This information will be very useful for future functional, phylogenetic and evolutionary studies of these plants.

Heterozygosity of Knob-Associated Tandem Repeats and Knob Instability in Mitotic Chromosomes of Zea （Zea mays L. and Z. diploperennis Iltis Doebley）

Knobs are blocks of heterochromatin present on chromosomes of maize （Zea mays L.） and its relatives that have effects on the frequency of genetic recombination, as well as on chromosome behavior. Knob heterozygosity and instability in six maize inbred lines and one Z. diploperennis Iltis Doebley line were investigated using the fluorescence in situ hybridization （FISH） technique with knob-associated tandem repeats （180 bp and 350 bp （TR- 1）） as probes. Signals of seven heterozygous knobs containing 180- bp repeats and of one heterozygous knob containing TR- 1 were captured in chromosomes of all materials tested according to the results of FISH, which demonstrates that the 180-bp repeat is the main contributor to knob heterozygosity compared with the TR- 1 element. In addition, one target cell with two TR- 1 signals on one homolog of chromosome 2L, which was different from the normal cells in the maize inbred line GB57, was observed, suggesting knob duplication and an instability phenomenon in the maize genome.