Detection of Differentiation Among BB, CC and EE Genomes in the Genus Oryza by Two-probe Genomic in situ Hybridization (GISH)

The genus Oryza consists of two cultivated species (O. sativa L. and O. glaberrima Steud.) and approximately 20 wild relative species widely distributed in the pan-tropics. These species have been classified into four complexes following the Vaughan's taxonomic system([1]). The O. officinalis complex is the largest complex in the genus, which includes ten species, having BE, CC, on, and EE genomes in the diploids as well as BBCC and CCDD genomes in the tetraploids. The relationships among the BE, CC, and EE genomes still remain unclear, although previous studies have indicated certain affinities of these genomes([2-4]). Genomic in situ hybridization (GISH) is a powerful technique to detect the relationships among the related genomes at chromosome and DNA levels. The objective of the present study was to investigate the relationships among the BE, CC and EE genomes in the genus Oryza by the two-probe GISH.

Establishment of a Multi-color Genomic in situ Hybridization Technique to Simultaneously Discriminate the Three Interspecific Hybrid Genomes in Gossypium

To identify alien chromosomes in recipient progenies and to analyze genome components in polyploidy, a genomic in situ hybridization （GISH） technique that is suitable for cotton was developed using increased stringency conditions. The increased stringency conditions were a combination of the four factors in the following optimized state： 100：1 ratio of blocking DNA to probe, 60% formamide wash solution, 43 ℃ temperature wash and a 13 min wash. Under these specific conditions using gDNA from Gossypium sturtianum （C1 C1 ） as a probe, strong hybridization signals were only observed on chromosomes from the C1 genome in somatic cells of the hybrid F1 （G. hirsutum x G. sturtianum） （AtDtC1）. Therefore, GISH was able to discriminate parental chromosomes in the hybrid. Further, we developed a multi-color GISH to simultaneously discriminate the three genomes of the above hybrid. The results repeatedly displayed the three genomes, At, Dt, and C1, and each set of chromosomes with a unique color, making them easy to identify. The power of the multi-color GISH was proven by analysis of the hexaploid hybrid F1 （G. hirsutum x G. australe） （AtAtDtDtG2G2）. We believe that the powerful multi-color GISH technique could be applied extensively to analyze the genome component in polyploidy and to identify alien chromosomes in the recipient progenies.

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

The genomic structures of Oryza sativa （A genome） and O. meyeriana （G genome） were comparatively studied using bicolor genomic in situ hybridization （GISH）. GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa （A-A）, O. sativa-O, meyeriana （A-G） and O. meyeriana-O, meyeriana （G-G）, were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.

Comparative Study on the Genomes Between Oryza alta and Oryza latifolia Based on C-Genome

[Objective] Genomic in situ hybridization (GISH) was used to study the relationship between the two CCDD genomes of Oryza alta and Oryza latifolia. [Method] Total DNA of Oryza officinalis (C-genome) was used as a probe for genomic in situ hybridization on metaphase chromosomes from Oryza alta and Oryza latifolia, respectively. [Result] Under certain post-hybridization washing stringencies, C- and D-genome could be distinguished in CCDD genome type; there were huge differences in some CC chromosomes of Oryza alta, Oryza latifolia, and Oryza officinalis. The genome of Oryza latifolia was more original. [Conclusion] Comparative analysis of the Oryza species with identical genome type may facilitate to elucidate the possible approaches to plant genome evolution and species evolution.

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modem lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the FI hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic （LA） hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that： at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell （PMC）, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva- lents were disjoined and most univalents were divided. Both the disjoined bivalents （half-bivalents） and the divided univalents （sister chromatids） moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the Fl LA hybrids and the variation of their BCx progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.

Comparative genomic hybridization analysis of genetic aberrations associated with development of esophageal squamous cell carcinoma in Henan, China

AIM: To characterize cytogenetic alterations in esophageal squamous cell carcinoma (ESCC) and its metastasis. METHODS: A total of 37 cases of primary ESCC and 15 pairs of primary ESCC tumors and their matched metastatic lymph nodes cases were enrolled from Linzhou, the high incidence area for ESCC in Henan, northern China. The comparative genomic hybridization (CGH) was applied to determine the chromosomal aberrations on the DNA extracted from the frozen ESCC and metastatic lymph node samples from these patients. RESULTS: CGH showed chromosomal aberrations in all the cases. In 37 cases of primary ESCC, chromosomal profile of DNA copy number was characterized by frequently detected gains at 8q (29/37, 78%), 3q (24/37, 65%), 5p (19/37, 51%); and frequently detected losses at 3p (21/37, 57%), 8p and 9q (14/37, 38%). In 15 pairs of primary ESCC tumors and their matched metastatic lymph node cases, the majority of the chromosomal aberrations in both primary tumor and metastatic lymph node lesions were consistent with the primary ESCC cases, but new candidate regions of interest were also detected. The most significant finding is the gains of chromosome 6p with a minimum high-level amplification region at 6p12-6q12 in 7 metastatic lymph nodes butonly in 2 corresponding primary tumors (P = 0.05) and 20p with a minimum high-level amplification region at 20p12 in 11 metastatic lymph nodes but only in 5 corresponding primary tumors (P < 0.05). Another interesting finding is the loss of chromosome 10p and 10q in 8 and 7 metastatic lymph nodes but only in 2 corresponding primary tumors (P < 0.05). CONCLUSION: Using the CGH technique to detect chromosomal aberrations in both the primary tumor and its metastatic lymph nodes of ESCC, gains of 8q, 3q and 5p and loss of 3p, 8p, 9q and 13q were specifically implicated in ESCC in Linzhou population. Gains of 6p and 20p and loss of 10pq may contribute to the lymph node metastasis of ESCC. These findings suggest that the gains and losses of chromosomal regions may contain ESCC-related oncogenes and tumor suppressor genes and provide important theoretic information for identifying and cloning novel ESCC-related oncogenes and tumor suppressor genes.

Identification of Intergeneric Hybrid Plants Between Oryza sativa and O.minuta via GISH and RAPD

To transfer desirable resistance traits from O. minuta to O. sativa, intergeneric hybrid plants between O. sativa (AA, 2n=2X=24) and O. minuta (BBCC, 2n=4X=48) were produced by embryo rescue after sexual cross. Morphological observation and chromosome counts indicated their hybrid status (ABC, 2n=3X=36). Genomic in situ hybridization (GISH) was further applied to confirm the parentage of the chromosomes of F 1 hybrids. Chromosomes of O. minuta and O. sativa were distinguishable in the hybrids in different fluorescence colors. GISH indicated that A and BC chromosomes were not randomly assembled in a cell. RAPD profiles unequivocally revealed their hybrids with double parent patterns. The results of blast tests showed that the hybrids had obtained disease resistance from O. minuta, and had a level of susceptibility between the parents.

Identification and cell wall analysis of interspecific hybrids between Oryza sativa and Oryza ridleyi

Oryza ridleyi is an allotetraploid wild species with the HHJJ genome, and Oryza sativa is a diploid cultivated rice that has the AA genome. Although the wide hybrid between the two species is difficult to obtain, we overcome this difficulty by young embryo rescue. An obvious heterosis was primarily found for the plant height, tillering ability, vegetative vigor, etc. However, the hybrid panicle and culm traits were found to resemble that of the wild rice parent, O. ridleyi, for the long awns, exoteric purple stigma, grain shattering, dispersed panicles, and culm mechanical strength. Genomic in situ hybridization （GISH） analysis was subsequently performed on the mitotic metaphase chromosome of the root tips, and we determined that the hybrid is an allotriploid with 36 chromosomes and its genomic constitution is AHJ. Chemical analyses conducted on the culm of O. sativa, O. ridleyi, and their interspecific hybrids showed that major changes occurred in the xylose, glucose, and arabinose concentrations, which are correlated with the specific hemicellulose polymer and cellulose components that are important in the primary cell walls of green plants. Meanwhile, the culm anatomical analyses indicated that additional large vascular bundles and an extra sclerenchyma cell layer were found in O. ridleyi. Additionally, further thickening of the secondary cell walls of the cortical fiber sclerenchyma cells and the phloem companion cells was discovered in O. ridleyi and in the interspecific hybrids. These results imply that there may be a potential link between culm mechanical strength and culm anatomical structure.

Association between chromosomal aberration of COX8C and tethered spinal cord syndrome:array-based comparative genomic hybridization analysis

Copy number variations have been found in patients with neural tube abnormalities.In this study,we performed genome-wide screening using high-resolution array-based comparative genomic hybridization in three children with tethered spinal cord syndrome and two healthy parents.Of eight copy number variations,four were non-polymorphic.These non-polymorphic copy number variations were associated with Angelman and Prader-Willi syndromes,and microcephaly.Gene function enrichment analysis revealed that COX8 C,a gene associated with metabolic disorders of the nervous system,was located in the copy number variation region of Patient 1.Our results indicate that array-based comparative genomic hybridization can be used to diagnose tethered spinal cord syndrome.Our results may help determine the pathogenesis of tethered spinal cord syndrome and prevent occurrence of this disease.

Molecular Cytogenetic Analysis of Spontaneous Interspecific Hybrid Between Oryza sativa and Oryza minuta

Genomic in situ hybridization (GISH) is a powerful tool to characterize parental chromosomes in interspecific hybrids, including the behaviour of autosynapsis and chromosome pairing. It was used to distinguish the chromosomes of Oryza sativa from wild species in a spontaneous interspecific hybrid and to investigate the chromosome pairing at metaphase I in meiosis of the hybrid in this study. The hybrid was a triploid with 36 chromosomes according to the chromosome number investigated in mitosis of root tips. During metaphase I of meiosis in the hybrid, less chromosome pairing was observed and most of the chromosomes existed as univalent. Based on GISH and FISH (Fluorescent in situ hybridization) analyses, the chromosomes of the hybrid were composed of genomes A, B and C. Thus, it was believed that the hybrid was the result of natural hybridization between cultivated rice and wild species O. minuta which was planted in experimental fields.

Detection of chromosomal imbalance in oligodendroglial tumors by comparative genomic hybridization

Objective To investigate the relationship between genomic DNA imbalance in oligodendroglial tumors and its different classification. Methods 16 oligodendrogliomas and 17 anaplastic oligodendrogliomas were investigated by comparative genomic hybridization on Paraffin-Embedded tissue samples,and the chromosomal genomic DNA imbalances were analyzed. Results Chromosome DNA imbalance rates in oligodendrogliomas

Methyl-CpG-Binding protein 2 duplication syndrome in a Chinese patient:A case report and review of the literature

BACKGROUND Chromosomal Xq28 region duplication encompassing methyl-CpG-binding protein 2(MECP2)results in an identifiable phenotype and global developmental delay known as MECP2 duplication syndrome(MDS).This syndrome has a wide range of clinical manifestations,including abnormalities in appearance,neurodevelopment,and gastrointestinal motility;recurrent infections;and spasticity.Here,we report a case of confirmed MDS at our institution.CASE SUMMARY A 12-year-old Chinese boy presented with intellectual disability(poor intellectual[reasoning,judgment,abstract thinking,and learning]and adaptive[lack of communication and absent social skills,apraxia,and ataxia]functioning)and dysmorphism.He had no history of recurrent infections,seizures,or bowel dysfunction,which is different from that in reported cases.Microarray comparative genomic hybridization confirmed MECP2 duplication in the patient and his mother who is a carrier.The duplication size was the same in the patient and his mother.No prophylactic antibiotic or anti-seizure therapy was offered to the patient or his mother before or after the consultation.CONCLUSION MDS is rare and has various clinical presentations.Clinical suspicion is critical in patients presenting with developmental delays.

Identification of Wheat-Barley 2H Alien Substitution Lines

The genetic constitution of fifteen materials derived from the cross wheat (Triticum aestivum L. cv. 'Chinese Spring') X barley (Hordeum vulgare L. cv. 'Betzes') was analyzed, and six disomic alien substitution lines were screened by GISH. The chromosome configurations in pollen mother cells at meiotic metaphase I (PMCs M I) of F, from each disomic substitution line respectively crossed with double ditelocentric lines 2A, 2B and 2D of 'Chinese Spring' were observed, and a set of wheat-barley disomic alien substitution lines 2H(A), 2H(B) and 2H(D) were obtained. The RFLP analysis with the probe psr131 on the short arm of wheat homeologous group 2 combining with four restriction enzymes were carried out. The results indicated that the probe psr131 could be used as molecular marker to tag the barley chromosome 2H. The barley chromosome 2H had good genetic compensation ability for wheat chromosomes 2B and 2D in vitality and other agronomic characters. The result of testing seed was that the wheat appearance starch quality had been changed from the half-farinaceous of 'Chinese Spring' to the half-cutin of substitution lines by transferring the barley chromosome 2H to wheat.

Primary Identification of Alien Chromatin in T911289,a Maintainer of Wheat Male Sterile Line with Cytoplasm of Aegilops kotschyi

The genomic composition of 1911289, a wheat ( Tritium aestivum L.) maintainer of K-CMS, was examined by several methods, such as genomic in situ hybridization (GISH), biochemical marking, and DNA molecular marking. The results got by GISH and PCR amplification of dispersed rye-specific repetitive DNA sequence suggested that the alien chromatin in T911289 derived from rye. Specifically PCR amplification of the rye-specific microsatellite primers (SCM9) and seed storage protein analysis indicated that the alien chromatin in T911289 had developed from the short arm of 1R chromosome of rye (1RS). PCR amplification by using microsatellite primers locating on 1BS and seed storage protein analysis also revealed that 1911289 had lost the arm of 1BS or a small distal segment of it. We conclude that T911289 is a heterogeneous population which displays two distinct different types of translocation, i.e. the Robertsonian translocation and small segment translocation. The Robertsonian translocation type observed in our study is different from the 1BL/1RS translocation which is widely used in wheat production; it may be a novel and complex translocation form. Though the linkage between the desirable agronomic traits and the deleterious genes expressed as sticky dough has not got broken in T911289, the recovery of small segment translocation will still benefit the genetic study of wheat and rye.

Identification of Molecular Markers for the Thinopyrum intermedium Chromosome 2Ai-2 with Resistance to Barley Yellow Dwarf Virus

The wheat_ Thinopyrum intermedium addition lines Z1,Z2 contain a pair of Th. intermedium chromosomes 2Ai_2 carrying the gene with resistance to barley yellow dwarf virus (BYDV). Genomic in situ hybridization (GISH) was used to analyze the chromosome constitution of Z1,Z2 by using genomic DNA probes from Th. intermedium and Pseudoroegneria strigosa . The results showed that the chromosome constitution of either Z1 or Z2 composes of 42 wheat chromosomes and two Th. intermedium chromosomes (2Ai_2). The 2Ai_2 chromosome is St_E intercalary translocation, in which the E genomic chromosome segment translocated into the middle region of the long arm of chromosome belonging to St genome. With the genomic DNA probe of Ps. strigosa , the GISH pattern specific to the 2Ai_2 chromosome may be used as a molecular cytogenetic marker. A detailed RFLP analysis on Z1, Z2 and their parents was carried out by using 12 probes on the wheat group 2 chromosomes. Twenty RFLP markers specific to the 2Ai_2 chromosome were identified. Two RAPD markers of OPR16 -350 and OPH09 -1580 , specific to the 2Ai_2 chromosome, were identified from 280 RAPD primers. These molecular markers could be used to assisted_select translocation lines with small segment of the 2Ai_2 chromosome and provide tools to localize the BYDV resistance.

Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences

The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.

Cytological and Molecular Identification of Alien Chromatin in Giant Spike Wheat Germplasm

Alien chromosomes of twelve giant spike wheat germplasm lines were identified by C-banding, genomic in situ hybridization (GISH), sequence characterized amplified region (SCAR), and random amplified polymorphic DNA (RAPD). All lines showed a chromosome number of 2n = 42, five of them carried both a pair of wheat-rye (Triticum aestivum-Secale cereal) 1BL/1RS translocation chromosomes and a pair of Agropyron intermedium (Ai) chromosomes, three carried a pair of Ai chromosomes only, three others carried a pair of 1BL/1RS chromosomes only, and one carried neither 1BL/1BS nor Ai chromosome. Further identification revealed that the identical Ai chromosome in these germplasm lines substituted the chromosome 2D of common wheat (T aestivum L.), designated as 2Ai. The genetic implication and further utilization of 2Ai in wheat improvement were also discussed.

Location and Analysis of Introgressed Segments in the Parthenogenetic Progenies of Zea mays×Z. diploperennis by GISH

用来自玉米 (ZeamaysL .)与二倍体多年生类玉米 (Z .diploperennisIltis,DoebleyandGuzm偄ｎ)杂交的孤雌生殖后代同一抗病个体的 4个株系进行了基因组原位杂交 ,用改进的杂交技术获得了近 10 0 %的检出率 ,每一检出片段在同源染色体两成员和每两个姊妹染色单体上均有清晰的信号。

Genetic Relationships Among Five Basic Genomes St, E, A, B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

The St and E are two important basic genomes in the perennial tribe Triticeae （Poaceae）. They exist in many perennial species and are very closely related to the A, B and D genomes of bread wheat （Triticum aestivum L.）. Genomic Southern hybridization and genomic in situ hybridization （GISH） were used to analyze the genomic relationships between the two genomes （St and E） and the three basic genomes （A, B and D） of T. aestivum. The semi-quantitative analysis of the Southern hybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, and relatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion. St and E are the two basic genomes of Thinopyrum ponticum （StStE^eE^bE^x） and Th. intermedium （StE^eE^b）, two perennial species successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of the spontaneous wheat-Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genome and rarely in the B genome. This would develop further use of alien species for wheat improvement, especially those containing St or E in their genome components.

Copy number changes of target genes in chromosome 3q25.3-qter of esophageal squamous cell carcinoma: TP63is amplified in early carcinogenesis but down-regulated as disease progressed

AIM: By using comparative genomic hybridization, gain of 3q was found in 45-86% cases of esophageal squamous cell carcinoma (EC-SCC). Chromosome 3q25.3-qter is the minimal common region with several oncogenes found within this region. However, amplification patterns of these genes in EC-SCC have never been reported. The possible association of copy number changes of these genes with pathologic characteristics is still not clear. METHODS: Real-time quantitative PCR (Q-PCR) was performed to analyze the copy number changes of 13 candidate genes within this region in 60 primary tumors of EC-SCC, and possible association of copy number changes with pathologic characteristics was analyzed by statistics. Immunohistochemistry (IHC) study was also performed on another set of 111 primary tumors of EC-SCC to verify the association between TP63 expression change and lymph node metastasis status. RESULTS: The average copy numbers (±SE) per haploid genome of individual genes in 60 samples were (from centromere to telomere): SSR3: 4.19 (±0.69); CCNL1: 5.24 (±0.67); SMC4L1: 2.01 (±0.16); EVI1: 2.02 (±0.12); hTERC. 5.28 (±0.54); SKIL 2.71 (±0.14); EIF5A2. 1.95 (±0.12); ECT2: 9.18 (±1.68); PIK3CA: 8.13 (±1.17); EIF4G1: 1.07 (±0.05); 557: 3.07 (±0.25); TP63: 2.51 (±0.22); TFRC. 2.42 (±0.19). Four clusters of amplification were found: SSR3 and CCLN1 at 3q25.31; hTERC and SKIL at 3q26.2; ECT2 and PIK3CA at 3q26.31-q26.32; and 55T, TP63 and TFRC at 3q27.3-q29. Patients with lymph node metastasis had significantly lower copy number of TP63 in the primary tumor than those without lymph node metastasis. IHC study on tissue arrays also showed that patients with lymph node metastasis have significantly lower TP63 staining score in the primary tumor than those without lymph node metastasis. CONCLUSION: This study showed that different amplification patterns were seen among different genes within 3q25.3-qter in EC-SCC, and several novel candidate oncogenes (SSR3, SMC4L1, ECT2, and SST) were identified. TP63 is amplified in early stage of EC-SCC carcinogenesis but down-regulated in advanced stage of disease.