Southern Hybridization Patterns Research Articles

Molecular diagnosis to discriminate pathogen and apathogen species of the hybrid Verticillium longisporum on the oilseed crop Brassica napus.

The cruciferous fungal pathogen Verticillium longisporum represents an allodiploid hybrid with long spores and almost double the amount of nuclear DNA compared to other Verticillium species. V. longisporum evolved at least three times by hybridization. In Europe, virulent A1xD1 and avirulent A1xD3 hybrids were isolated from the oilseed crop Brassica napus. Parental A1 or D1 species are yet unknown whereas the D3 lineage represents Verticillium dahliae. Eleven V. longisporum isolates from Europe or California corresponding to hybrids A1xD1 or A1xD3 were compared. A single characteristic type of nuclear ribosomal DNA could be assigned to each hybrid lineage. The two avirulent A1xD3 isolates carried exclusively D3 ribosomal DNA (rDNA) which corresponds to V. dahliae. The rDNA of all nine A1xD1 isolates is identical but distinct from D3 and presumably originates from A1. Both hybrid lineages carry two distinct isogene pairs of four conserved regulatory genes corresponding to either A1 or D1/D3. D1 and D3 paralogues differ in several single nucleotide polymorphisms. Southern hybridization patterns confirmed differences between the A1 and D1/D3 isogenes and resulted in similar patterns for D1 and D3. Distinct signatures of the Verticillium transcription activator (VTA)2 regulatory isogene pair allow identification of V. longisporum hybrids by a single polymerase chain reaction and the separation from haploid species as V. dahliae or Verticillium albo-atrum. The combination between VTA2 signature and rDNA type identification represents an attractive diagnostic tool to discriminate allodiploid from haploid Verticillia and to distinguish between A1xD1 and A1xD3 hybrids which differ in their virulence towards B. napus.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-012-4530-1) contains supplementary material, which is available to authorized users.

Characterization of Inago1 and Inago2 retrotransposons in Magnaporthe oryzae

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.

Evidence for the Possible Existence of a Remnant L-gulono-gamma-lactone Oxidase (GULO) Gene in a Teleost Genome

DNA fragments related to the cloudy catshark Scyliorhinus torazame L-gulono-gamma-lactone oxidase (GULO) cDNA were detected in a distant fish species. Although the Southern hybridization pattern was more distinct in species with active GULO, DNA fragments related to the GULO gene were also discovered in the common carp Cyprinus carpio. Additionally, in the common carp, inter-individual variation of the hybridization pattern was observed. Regular screening of available teleost fish gene libraries did not reveal GULO related DNA sequences.

Hereditary Behavior of bar Gene Cassette is Complex in Rice Mediated by Particle Bombardment

Particle bombardment transformation using minimal gene cassette (containing the promoter, open reading frame and terminator) is the novel trend in plant genetic transformation, and its use helps to alleviate the undesirable effects of plasmid vector backbone sequences on transgenic plants. In the present article, studies related to the hereditary behavior of bar gene cassette in T 1 to T 3 generations of the transgenic rice ( Oryza sativa L.) lines transformed by particle bombardment have been discussed. The selectable marker bar gene cassette that integrated with the rice genome had multiple copies and showed complex segregation behaviors including the presence of ‘false homozygotes’, with abnormal segregation ratios ranging from 35:1 to 144:1 (Basta-resistant: sensitive plants) in their progenies. In five out of ten original transgenic lines, bar gene can be stably transmitted as a dominant gene to self-pollinated T 2 progeny. The homozygotes were obtained in three transgenic lines in T 1 generation regardless of the multiple-copy integration patterns of bar gene. Southern blotting analysis showed that multiple copies of bar gene cassette were linked, which formed transgene arrays in the host rice genome. The authors also observed stable transmission of integration patterns of bar gene cassette, as obtained from Southern blotting analysis, in the regularly segregated transgenic rice lines and loss of gene in an irregularly segregated transgenic line. The segregation behavior varied among the transgenic progenies that exhibited similar Southern hybridization patterns of bar gene. On the basis of these results, the multiple-copy integration, gene lost, and gene expression interaction were the major reasons for the complex segregation behaviors of bar gene cassette in transgenic rice plants.

Molecular characterization and isolation of the F/f gene for femaleness in cucumber (Cucumis sativus L.)

The biological processes leading to sex expression in plants are of tremendous practical significance for fruit production of many agricultural and horticultural crops. Sex-expression studies in cucumber showed that the different sex types are determined by three major genes: M/m, F/f and A/a. The M/m gene in the dominant condition suppresses stamina development and thus leads to female flowers. The F/f gene in the dominant condition shifts the monoecious sex pattern downwards and promotes femaleness by causing a higher level of ethylene in the plant. To investigate the molecular character of the gene F/f, we used nearly isogenic gynoecious ( MMFF) and monoecious ( MMff) lines (NIL) produced by our own backcross programme. Our investigations confirmed the result of other groups that an additional genomic ACC synthase (key enzyme of ethylene biosynthesis) sequence ( CsACS1G) should exist in gynoecious genotypes. A linkage was also verified between the F/f locus and the CsACS1G sequence with our plant material. After the exploration of different Southern hybridization patterns originating from different CsACS1 probes, a restriction map of the CsACS1 locus was constructed. By using this restriction map, the duplication of the CsACS1 gene and following mutation of the CsACS1G gene could be explained. The promoter regions of the genes CsACS1G and CsACS1 were amplified in a splinkerette PCR and sequenced. An exclusive amplification of the new isolated sequence ( CsACS1G) in gynoecious ( MMFF) and sub-gynoecious ( MMFf) genotypes confirmed that the isolated gene is the dominant F allele.

Novel germline hMSH2 genomic deletion and somatic hMSH2 mutations in a hereditary nonpolyposis colorectal cancer family

Germline and somatic mutations of the hMSH2 gene were determined in a Japanese hereditary nonpolyposis colorectal cancer (HNPCC) family fulfilling the Amsterdam criteria. PCR-SSCP-sequencing of genomic DNA detected a somatic hMSH2 mutation of an A deletion at codon 227–229 in a duodenal carcinoma and a somatic hMSH2 mutation of an A insertion at codon 21 in a gastric carcinoma from affected family members, both carcinomas exhibiting high microsatellite instability. However, no germline hMSH2 mutation was detected by the PCR-SSCP-sequencing method. Genomic DNA was then analyzed by Southern blot hybridization using three hMSH2 cDNA probes (probe A involving exons 1–5, probe B involving exons 4–11 and probe C involving exons 9–16) after digestion by restriction enzymes, EcoRI, HindIII and NsiI. The NsiI digest of DNA from normal tissues of affected members exhibited an aberrant 8.6 kb restriction fragment, in addition to the normal 10.6 kb fragment, when hybridized to probes A and B. This suggested the presence of a heterozygous 2 kb genomic deletion encompassing exon 4, 5 or 6. RT-PCR-sequencing revealed that the deleted region encompassed exon 5. This novel genomic deletion of the hMSH2 gene was confirmed to be pathogenic, and the Southern hybridization pattern was applied to the pre-symptomatic diagnosis.

Inheritance of transgenes in transgenic tall fescue (Festuca arundinacea schreb.)

Tall fescue (Festuca arundinacea Schreb.) is the most important forage species worldwide of the Festuca genus. Single genotype-derived embryogenic suspension cultures were established from tall fescue cultivar Kentucky-31, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric β-glucuronidase (gusA) gene was co-transformed with hph. Transgenic plants were recovered after microprojectile bombardment of suspension cells and subsequent selection in the presence of a high concentration of hygromycin. Fertile transgenic plants were obtained after vernalization under field conditions. T1 and T2 progenies were obtained after reciprocal crosses between transgenic and untransformed control plants. PCR and Southern hybridization analyses revealed a 1∶1 segregation ratio for both transgenes in the T1 and T2 generations. Southern hybridization patterns were identical for T0, T1, and T2 plants. The results demonstrated for the first time the stable meiotic transmission of transgenes following Mendelian rules in transgenic tall fescue.

Molecular cloning and cytochemical analysis of exopolygalacturonase from carrot.

Exopolygalacturonase (exo-PGase, EC 3.2.1.67) attacks the non-reducing terminus of the polygalacturonic acid in pectic molecules, releasing galacturonic acid. We cloned the cDNA of exo-PGase purified from cell homogenates of suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells. The nucleotide sequence of the cDNA (1.4 kb) contains an open reading frame that encodes a 391-amino-acid polypeptide. Sequence homology research showed 97.9% identity to the glycoprotein EP4 obtained from cultured carrot cells and 49.3% identity to the ENOD8 gene product of alfalfa ( Medicago sativa). However, no significant similarity was found to known PGases. The Southern hybridization pattern indicated that this exo-PGase protein is a member of a small-sized gene family. Predominant expression of the exo-PGase gene was detected by in situ hybridization and immunohistochemistry in the root apical meristem and in the elongation region, but not in the root cap. A cross-immunoresponse with anti-exo-PGase also occurred in the root nodule meristem of alfalfa. These results suggest that this exo-PGase plays a role in the degradation of pectic molecules during root development.

Large deletions in chloroplast DNA of rice calli after long-term culture

Alteration in chloroplast DNA (ctDNA) in long-term cultures of calli derived from rice seeds were investigated using a Bam HI-1 fragment of rice ctDNA as a probe. Among 10 callus lines cultured for 36 months, two lines (T36-1 and T36-2) were found to have large deletions (76 kb and 63 kb, respectively) in the chloroplast genome, while none of the same callus lines cultured for 6 months showed any ctDNA alteration. When T36-1 and T36-2 were cultured on MS maintenance medium for an additional six months (becoming T42-1 and T42-2, respectively), the ctDNA of T42-2 underwent a further deletion of 0.5 kb. Maps of deleted ctDNA molecules were constructed using fifteen hybridization probes. Based on the restriction maps of the deleted ctDNAs, the T36-1 and T42-1 callus lines lost two large inverted repeats, together with the intervening small single copy region. The T36-2 and T42-2 callus lines lost the rbcL gene and most of the large single copy region. The only region that these two ctDNA molecules have in common is an approximately 18 kb region that includes various psb and trn genes. These results suggest that this region of the rice chloroplast genome contains at least one functional replication origin of rice ctDNA. Regenerated plantlets were obtained from long-term cultured calli of T36-2, indicating that the deletion of a part of the ctDNA did not affect shoot differentiation from the calli. Southern hybridization patterns of the regenerated plantlets were almost the same as those of the callus lines. The mechanisms responsible for deletion of ctDNA in the callus cells are discussed.

Increased sulfur amino acids in soybean plants overexpressing the maize 15 kDa zein protein

Four transgenic soybean [Glycine max (L.) Merrill] lines were generated containing the maize 15 kDa zein protein gene using somatic embryogenic protocols. The zein gene was inserted behind the β-phaseolin promoter for seed-specific expression. All four lines represent separate transformation events as they were generated in different experiments at different locations. Two of the transformation events produced multiple plants, and these produced identical Southern hybridization patterns (UKY/Z1, UKY/Z2 and UKY/Z3 from the first; and OSU/Z4, OSU/Z8 and OSU/Z10 from the second). Molecular characterization revealed that multiple copies of the zein gene were present in all of the transgenic lines. Immunoblot analysis confirmed the accumulation of the zein protein product in the seeds of the UKY/Z1, UKY/Z2, UKY/Z3, OSU/Z4, OSU/Z8 and OSU/Z10 transgenic lines. However, there was no accumulation of zein protein in the UGA/Z1 line and Northern analysis confirmed that the zein transgene was silenced in this line. It was not possible to analyze the zein expression in the seeds of the UKY/Z4 line, as it was sterile. Amino acid analysis of the UKY and OSU lines confirmed that there was a 12–20% increase in methionine, and 15–35% increase in cysteine content in these lines compared to the control. There were no consistent changes in the content of the other amino acids in the transgenic lines. These results suggest that while the increase in methionine content in these lines is modest, it is possible to increase the methionine content without adversely affecting the protein composition of soybean.

The perennial ryegrass endophyte Neotyphodium lolii genetically transformed with the green fluorescent protein gene (gfp) and visualization in the host plant

The green fluorescent protein gene was inserted into isolates of the endophyte Neotyphodium lolii from perennial ryegrass Lolium perenne using the method with polyethylene glycol treatment of protoplasts. The transformation rate was 3.2 × 10 −6 of the original number of protoplasts 5 weeks after the transformation. Freshly isolated mycelium from the host plant was used in the transformation experiments, since difficulties of infecting the host plant with older isolates were observed. Isolates were identified using the universal primed (UP) PCR fingerprinting method. The transformed mycelium readily expressed the green fluorescent protein gene. Fluorescence was observed in the cytoplasm of the mycelium cells in pure culture, both before infection and after re-isolation from the host plant. The fluorescent hyphae were easily detected in the host plant. No disturbing autofluorescence from the host plant leaf sheaths or from the wild type isolate was observed. The importance of mitotic stability is discussed, based on the transformed phenotype and southern hybridization pattern, since the Neotyphodium lolii transformants lost 1-2 copies of the integrated gene after re-isolation from the host plant without losing their GFP phenotype. This is the first report on transformation of a Neotyphodium sp. with a gfp gene. In the future, the green fluorescent protein may serve as a tool for studying gene expression and protein localization in the endophyte/host plant interaction using heterologous fusion proteins.

Cytosine Methylation Changes in Rice Centromeric and Telomeric Sequences Induced by Foreign DNA Introgression

Cytosine methylation changes (hyper- or hypomethylation) in centromeric and telomeric sequences were observed in all three studied rice introgression lines containing DNA from wild rice, Zizania latifolia Griseb. The changed genomic Southern hybridization patterns were complex and non-concordant between a pair of isoschizomers (HpaII/MspI) digests, indicating methylation modifications at both the inner and outer cytosines of the CCGG sites. The changed patterns were inherited through generations. Possible mechanism for the methylation changes and their potential implications for the phenotypic variation and genome organization are discussed.

Isolation and characterization of two novel retrotransposons of the Ty1-copia group in oat genomes.

Two repetitive sequences, As32 and As22, of 826 and 742 bp, respectively, were isolated from Avena strigosa (As genome). Databank searches revealed their high homology to different segments of the family of Ty1-copia retrotransposons. Southern hybridization showed them to be present in diploid and polyploid oat species. Polymerase chain reaction with primers designed to amplify the segment between them showed that As32 and As22 sequences are composed of two different Ty1-copia retrotransposons. The segment amplified from the pAs32 insert was 2,264 bp long and contained the entire GAG and AP domains, and more than half of the IN domain. This new element has been designated TAS-1 (transposon, A. strigosa, 1) and appears to contain a long open reading frame that encodes a polypeptide of 625 amino acids. Slot-blot and fluorescence in situ hybridization analyses revealed it to be a component of both A- and D-genome chromosomes. Further, the chromosomes involved in one C-A intergenomic translocation in A. murphyi (AC genomes), one C-D intergenomic translocation in A. byzantina cv. Kanota (ACD genomes), and two C-D intergenomic translocations in A. sativa cv. Extra Klock, were identified. Based on its physical distribution and Southern hybridization pattern, a parental retro-transposon represented by TAS-1 appears to have been active at least twice during the evolution of the genomes in species of Avena.

Retrotransposon activation followed by rapid repression in introgressed rice plants

Plant retrotransposons are largely inactive during normal development, but may be activated by stresses. Both copia-like and gypsy-like retrotransposons of rice were activated by introgression of DNA from the wild species Zizania latifolia Griseb. The copy number increase was associated with cytosine methylation changes of the elements. Activity of the elements was ephemeral, as evidenced by nearly identical genomic Southern hybridization patterns among randomly chosen individuals both within and between generations for a given line, and the absence of transcripts based on Northern analysis. DNA hypermethylation, internal sequence deletion, and possibly other mechanisms are likely responsible for the rapid element repression. Implications of the retroelement dynamics on plant genome evolution are discussed.Key words: epigenetics, DNA methylation, genome evolution, retrotransposons, rice, introgression.

Retrotransposon activation followed by rapid repression in introgressed rice plants

Plant retrotransposons are largely inactive during normal development, but may be activated by stresses. Both copia-like and gypsy-like retrotransposons of rice were activated by introgression of DNA from the wild species Zizania latifolia Griseb. The copy number increase was associated with cytosine methylation changes of the elements. Activity of the elements was ephemeral, as evidenced by nearly identical genomic Southern hybridization patterns among randomly chosen individuals both within and between generations for a given line, and the absence of transcripts based on Northern analysis. DNA hypermethylation, internal sequence deletion, and possibly other mechanisms are likely responsible for the rapid element repression. Implications of the retroelement dynamics on plant genome evolution are discussed.

Chromosomal organization of a sequence related to LTR-like elements of Ty1-copia retrotransposons in Avena species

A repetitive sequence, pAs17, was isolated from Avena strigosa (As genome) and characterized. The insert was 646 bp in length and showed 54% AT content. Databank searches revealed its high homology to the long terminal repeat (LTR) sequences of the specific family of Ty1-copia retrotransposons represented by WIS2-1A and Bare. It was also found to be 70% identical to the LTR domain of the WIS2-1A retroelement of wheat and 67% identical to the Bare-1 retroelement of barley. Southern hybridizations of pAs17 to diploid (A or C genomes), tetraploid (AC genomes), and hexaploid (ACD genomes) oat species revealed that it was absent in the C diploid species. Slot-blot analysis suggested that both diploid and tetraploid oat species contained 1.3 x 10(4) copies, indicating that they are a component of the A-genome chromosomes. The hexaploid species contained 2.4 x 10(4) copies, indicating that they are a component of both A- and D-genome chromosomes. This was confirmed by fluorescent in situ hybridization analyses using pAs17, two ribosomal sequences, and a C-genome specific sequence as probes. Further, the chromosomes involved in three C-A and three C-D intergenomic translocations in Avena murphyi (AC genomes) and Avena sativa cv. Extra Klock (ACD genomes), respectively, were identified. Based on its physical distribution and Southern hybridization patterns, a parental retrotransposon represented by pAs17 appears to have been active at least once during the evolution of the A genome in species of the Avena genus.

Karyotyping of the chromosomes and physical mapping of the 5S rRNA and 18S-26S rRNA gene families in five different species in Capsicum.

Since clear identification of individual chromosomes in Capsicum species was not possible due to the gradient in sizes and similar morphology, in situ hybridization using 5S rDNA and 18S-26S rDNA probes was utilized to karyotype the chromosomes of the five species, Capsicum annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens. The 5S rDNA located on only one pair of chromosomes in all five species. The 5S rDNA carrying chromosome was designated as chromosome 1 by the synteny relationship with tomato. In two species, C. baccatum and C. pubescens, the 5S rDNA was physically linked with a 18S-26S rDNA locus. The number and chromosomal location of 18S-26S rDNA loci were highly variable among the species. The number of loci was as few as two in C. annuum and as many as 14 in C. baccatum. The chromosomal positions of the 18S-26S loci were in the telomere regions in the corresponding chromosomes and some chromosomes showed hybridization signals in this region of both long and short arms. A pair of acrocentric chromosomes carrying the 18S-26S rDNA locus in the satellite region was found in all five species. The in situ and Southern hybridization patterns of the rDNAs were similar between C. annuum and C. chinense. C. frutescens was similar to C. chinense. C. baccatum and C. pubescens were speculated to be less related to the former three Capsicum species. The variable nature of the 18S-26S rDNA loci in the Capsicum species might provide a good model for rDNA evolution.

Mitochondrial genome structure of a cytoplasmic hybrid between tomato and wild potato.

A physical map of the mitochondrial genome was constructed for a male-sterile tomato, MSA1, which had been generated by an asymmetric cell fusion between tomato (Lycopersicon esculentum) and wild potato (Solanum acaule). The entire genomic sequence of the MSA1 mitochondria (450 kb) was represented by five maps. Even if sequence duplications were taken into consideration, at least two linkage groups (maps 1-4 and map 5) were necessary to show the overall genome. The mitochondrial genome structure of MSA1 was also analyzed by comparing the Southern hybridization patterns of MSA1 and its parents (tomato and wild potato). The mitochondrial genome of MSA1 consists of a complex mixture of the parental genomes with at least 11 molecular recombination events.

Somatic hybridization in mint: identification and characterization of Mentha piperita (+) M. spicata hybrid plants

Twenty eight somatic hybrid plants were identified following protoplast fusions between peppermint (Mentha piperita L. cv Black Mitcham), producing high-quality oil, and spearmint (Mentha spicata L. cv Native Spearmint), likewise producing high-quality oil and also possessing resistance to verticillium wilt. Prior to fusion, peppermint protoplasts were subjected to iodoacetic acid to inhibit cell division. Protoplasts of peppermint and spearmint were fused using polyethylene glycol plus DMSO. Fusion products were cultured according to an efficient protoplast-to-plant-cycle protocol developed for peppermint. Using this protocol, iodoacetic acid-treated peppermint protoplasts were not able to divide, whereas untreated spearmint protoplasts had the ability to produce callus but not shoots. Therefore, selection of somatic hybrid calli was based on the presumed capability of hybrid cells to form calli and shoots. Shoots in vitro were initially identified as hybrids using RAPD profiles. Subsequently, observations on morphology, chromosome counts, and Southern-hybridization patterns confirmed their hybrid status. The results of verticillium tests revealed that 18 somatic hybrids were more susceptible than Native Spearmint, while hybrid II-14 had a level of susceptibility intermediate between that of the fusion parents. Oil-analysis of hybrid plants indicated that they all have a GC-profile typical of spearmint oil.

A repetitive DNA sequence common to the different B chromosomes of the genus Brachycome

Dot-like micro B chromosomes of Brachycome dichromosomatica were analysed for their sequence composition. Southern hybridization patterns of a total micro B probe to genomic DNA from plants with and without micro Bs demonstrated that the micro Bs shared sequences with the A chromosomes. In addition to telomere, rDNA and common A and B chromosome sequences, a new B-specific, highly methylated tandem repeat (Bdm29) was detected. After in situ hybridization with Bdm29 the entire micro B chromosome was labelled and clustering of the condensed micro Bs could be observed at interphase. A high number of Bdm29-like sequences were also found in the larger B chromosomes of B. dichromosomatica and in other Bs within the genus Brachycome.