Molecular characterization of UV-treated sugar beet somaclones using RFLP markers

Sugar beet plants regenerated from UV-treated calluses were examined by restriction fragment length polymorphism (RFLP) analysis to determine the extent of somaclonal variation occurring at the DNA level. In total, 50 random sugar beet DNA sequences were used to screen 42 somaclones for genetic alterations. Three polymorphisms were detected among the 7 644 alleles analysed. From these data a mutation frequency of 0.03 ± 0.02% per allele was estimated. This frequency is in agreement with similar studies of somaclonal DNA variation using molecular markers and lies in the upper range of the spontaneous gene mutation frequencies found in plants. The two probegenotype combinations showing independent polymorphisms, were further analysed using the restriction enzymes Bam HI, Eco RI, Eco RV and Hind III. Both polymorphisms are likely to result from structural rearrangements rather than from point mutations. Differences in methylation among 10 of the investigated somaclones were tested for by comparing Hpa II and Msp I generated RFLP patterns. The somaclones showed extensive methylation, but no differences in their degree of methylation. Cytological analysis revealed 34 diploid, 8 tetraploid, but no aneuploid plants.     

Modifications of Mitochondrial DNA Cause Changes in Floral Development in Homeotic-like Mutants of Tobacco

To investigate the influence of mitochondrial genes on stamen development of higher plants, protoplasts from three different, male-sterile tobacco cultivars were fused. The fused cells were cultured individually into calli, from which plants were regenerated. Cybrid plants were obtained that exhibited flowers with recombined biparental male-sterile morphology and with novel male-sterile stamens that differed from any types from sexual or somatic hybridizations described previously. The male-sterile morphologies of these cybrids and their parents support the hypothesis that nuclear-mitochondrial interaction occurs at several stages in tobacco floral development and that several mitochondrial genes are necessary for normal stamen and corolla development. Analysis by restriction endonuclease digestion of mitochondrial DNA of male-sterile cybrids and their parents revealed that the mitochondrial DNA of male-sterile cybrids with parental floral morphology was unchanged when compared with parental mitochondrial DNA. Cybrids that were morphologically similar to one parent's male-sterile phenotype had mitochondrial DNA almost identical to that parent, whereas cybrids with recombined biparental or novel male-sterile phenotypes contained mitochondrial DNA different from both male-sterile parents and from each other. A set of mitochondrial DNA fragments could be correlated with split corollas, a feature found in several tobacco male-sterile cultivars. DNA gel blot analysis using a number of mitochondrial genes confirmed the conclusions based on ethidium bromide staining of mitochondrial DNA restriction digests.     

Telomere looping in P. sativum (common garden pea)

Telomeres vary greatly in size among plants and, in most higher plants, consist of a long array of 5'-TTTAGGG-3'/3'-AAATCCC-5' (TTTAGGG) repeats. Recently, telomeric DNA in human, mouse, oxytricha, and trypanosome chromosomes have been found arranged into loops (t-loops), proposed to sequester the telomere from unwanted repair events and prevent activation of DNA damage checkpoints. We have asked whether t-loops exist in the higher order plant Pisum sativum (garden pea). DNA was isolated from the shoots and root tips of germinating seeds. Analysis of the telomeric restriction fragments showed that DNA hybridizing to a (TTTAGGG)n probe migrated as a smear centering around 25 kb, and direct sequencing verified the repeat to be (TTTAGGG)n. Total DNA in isolated nuclei was photo-cross-linked, and the telomeric restriction fragments were purified by gel filtration. Electron microscopic (EM) analysis revealed DNA molecules arranged as t-loops with a size distribution consistent with that seen by gel electrophoresis. Some molecules had loops as large as 75 kb. These results show that the arrangement of telomeric DNA into loops occurs in higher plants.     

Cot-based sampling of genomes for polymorphic low-copy DNA

DNA polymorphisms are powerful tools for many evolutionary and genomic studies in plants including molecular breeding. Single nucleotide polymorphisms (SNPs) are the most elemental DNA marker for genomic studies, but even with advances in DNA sequencing technology, SNP discovery remains costly and computationally demanding, especially in large genomes that are rich in repetitive DNA such as those of many plants. Here we report a method using DNA renaturation kinetics (Cot techniques), sequencing, and BLAST-based screening to identify low-copy, non-coding DNA sequences that were subsequently found to be relatively rich in polymorphisms. A total of of 63 such fragments isolated from a diploid D genome cotton species (Gossypium raimondii) revealed a higher frequency of polymorphisms than that observed for cotton expressed sequence tags or hypomethylated (PstI-susceptible) genomic DNA. While microsatellite-derived loci show still higher polymorphism rates, they often fall in repetitive elements and their sequence analysis is often complicated by alignment difficulties. The potential applications of Cot-filtered noncoding (CFNC) DNA in development of DNA markers are discussed.     

Polymorphism and Linkage of the αa-Crystallin Gene in t-Haplotypes of the Mouse

Restriction fragment polymorphisms were used to order the alpha A-crystallin locus (Crya-1) relative to other genes in mouse t-chromatin and to investigate the relatedness of alpha-A-crystallin sequences among different t-haplotypes. Analysis of DNA from t-recombinant mice mapped Crya-1 to the K end of the H-2 complex and within the distal inverted region characteristic of t-haplotypes. Hybridization with Crya-1 cDNA revealed three distinct phenotypic groups among the 17 different t-haplotypes studied. A majority (9 of 17) of the t-haplotypes were classified into a novel group (Crya-1t) characterized by restriction fragments apparently unique to t-chromosomes and therefore thought to contain alpha A-crystallin sequences descended from the original t-chromosome. A second group of t-haplotypes had restriction fragment patterns indistinguishable from those observed among many common inbred strains of mice of the Crya-1a type, and a third restriction fragment pattern, observed only in the tw121 haplotype, was indistinguishable from the fragment pattern for C3H/DiSn (Crya-1b) and several other inbred strains of mice. Thus, with respect to sequences around the Crya-1 locus, different t-haplotypes show restriction fragment polymorphisms, some of which are comparable to those found in wild-type chromosomes and provide further evidence for genetic heterogeneity in DNA from the distal region of t-haplotypes.     

Identification of minor DNA variations in rice somaclonal variants

Some somaclonal variants derived from a landrace rice variety, Indrayani, were shown to be high yielding and resistant to multiple diseases in previous analysis carried out in our laboratory. An attempt was made to assess the effect of culturing and regeneration of rice plants on DNA variation at microsatellite loci in R₂ progeny of callus-derived rice plants. Different somaclones of the rice line Indrayani differing in yield and disease response (high, low and no change in yield, as compared to the original genotype) were used as genetic material for these analyses. Analysis of microsatellite loci was accomplished by digesting DNA from regenerated rice somaclones and assaying for polymorphisms at microsatellite loci by in-gel hybridization with synthetic oligonucleotide probes such as (GATA)₄, (CAC)₅ and (TG)₁₀. Specific variation at a PCR-amplified locus containing three internal microsatellite repeats (1E₆) using restriction site fingerprinting was also investigated. The locus-specific amplification of a sequence-tagged microsatellite marker followed by digestion with HinfI and Sau3AI restriction endonucleases showed differences in some somaclonal variants. The technique used in this study enables monitoring of DNA changes in successive generations of somaclonal variants as a measure of DNA variability and possibly to identify the regions which are responsible for specific traits. Received: 7 November 1997 / Revision received: 22 April 1997 / Accepted: 5 June 1998     

Identification of growth hormone DNA polymorphisms which respond to divergent selection for abdominal fat content in chickens

Summary Two strains of meat-type chickens which had been derived from the same genetic base, but were selected for high or low abdominal fat content, respectively, were analyzed for polymorphisms in the growth hormone gene (GH). A total of four DNA polymorphisms were identified, one at a SacI restriction site and three at MspI restriction sites. Restriction mapping indicated that all polymorphisms were in exons and/or introns and not in flanking regions of the gene. The incidence of GH polymorphisms was determined in 20 chickens from each strain and significant differences were observed for two of the four polymorphisms. Analysis by DNA fingerprinting using (CAC)₅ as a probe indicated that the inbreeding coefficient was 0.1 in both strains and that random genetic drift was minimal. Thus, the selection for abdominal fat appears to have affected the frequency of alleles of the growth hormone gene. Whether this is the direct consequence of an altered growth hormone gene on fat metabolism or reflects linkage to an allele of a neighbouring gene remains to be determined.     

Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes.

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues.     

Sensitivity of random amplified polymorphic DNA analysis to detect genetic change in sugarcane during tissue culture

Random amplified polymorphic DNA (RAPD) analysis using 10-mer oligonucleotide primers efficiently differentiated sugarcane cultivars and proved suitable for detecting gross genetic change such as that which can occur in sugarcane subjected to prolonged tissue culture, for example in protoplast-derived callus. However, RAPD analysis was not sufficiently sensitive to detect smaller genetic changes that occur during sugarcane genetic transformation. The length of DNA scored for polymorphism per primer averaged 13.2 kb, or 0.0001% of the typical sugarcane genome size of 1.2 × 10₇ kb (2C). RAPD analysis of sugarcane plants regenerated from embryogenic callus revealed very few polymorphisms, indicating that gross genetic change is infrequent during this tissue culture procedure, although epigenetic effects result in transient morphological changes in regenerated plants. More sensitive variations on the RAPD technique may increase the practicality of DNA-based screening of regenerated plant lines to reveal somaclonal variants.     

Identification of Phytophthora citrophthora with Cloned DNA Probes

Two different DNA fragments, one of 2.9 kilobases and the other of 5.1 kilobases, were cloned from Phytophthora citrophthora and showed no homology with DNA from plants and other related fungi. These DNA probes hybridized with DNA from 12 different P. citrophthora isolates obtained from a variety of hosts but did not hybridize with DNA from 6 P. citrophthora isolates obtained from cacao. Southern blot analysis revealed that the probes contained repetitive DNA, and restriction fragment length polymorphisms were identified among several P. citrophthora isolates. Of the isolates tested, two major groups were observed whose genetic similarity correlated with geographical distribution. One of the DNA probes was used to detect P. citrophthora growing from infected citrus roots incubated on semiselective medium. P. citrophthora was not detected by a hybridization assay of total DNA extracted directly from infected roots.     

Diploid Brassica napus somatic hybrids: characterization of nuclear and organellar DNA

Summary Five somatic hybrids between Brassica campestris and B. oleracea were obtained. Molecular, morphological and cytological information all suggest that the resynthesized B. napus plants were hybrids. All five plants were diploid (2n=38) and had mainly bivalents at meiosis. Seedset was low after selfing but normal after crossing with B. napus. Molecular proof of the hybrid nature of these plants was obtained by hybridization of a rDNA repeat to total DNA. Analysis of chloroplast DNA restriction patterns revealed that all hybrids had chloroplasts identical to the B. oleracea parent. The analysis of mitochondrial DNA indicated that three hybrids had restriction patterns identical to those of B. campestris, and the other two had restriction patterns similar to those of B. oleracea. The 11.3 kb plasmid present in mitochondria of the B. campestris parent was also found in mitochondria of all five hybrids. This suggests that the plasmid from a B. campestris type of mitochondria was transferred into mitochondria of a B. oleracea type.     

Isolation of Y Chromosome-Specific Sequences from Silene Latifolia and Mapping of Male Sex-Determining Genes Using Representational Difference Analysis

The genomic subtraction method representational difference analysis (RDA) was used to identify male-specific restriction fragments in the dioecious plant Silene latifolia. Male-specific restriction fragments are linked to the male sex chromosome (the Y chromosome). Four RDA-derived male-specific restriction fragments were used to identify polymorphisms in a collection of X-ray-generated mutant plants with either hermaphroditic or asexual flowers. Some of the mutants have cytologically detectable deletions in the Y chromosome that were correlated with loss of male-specific restriction fragments. One RDA-derived probe detected a restriction fragment present in all mutants, indicating that each has retained Y chromosomal DNA. The other three probes detected genomic fragments that were linked in a region deleted in some hermaphroditic and some asexual mutants. Based on the mutant phenotypes and the correlation of cytologically visible deletions with loss of male-specific restriction fragments, these markers were assigned to positions on the Y chromosome close to the carpel suppression locus. This RDA mapping also revealed a Y-linked locus, not previously described, which is responsible for early stamen development.     

Differentiation of Erwinia amylovora strains by pulsed-field gel electrophoresis.

Erwinia amylovora strains, isolated from several host plants in various geographic regions during different years, were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the DNA from lysed, agar-embedded cells with rare-cutting restriction enzymes. The banding patterns obtained with enzyme XbaI digests revealed significant differences among strains from different areas. North American strains E9 and Ea-Rb, a Rubus strain, were highly divergent from other E. amylovora strains. French strains were different from central European and English strains. E. amylovora strains from central Europe and New Zealand had identical PFGE patters, as had strains from Egypt, Greece, and Turkey. PFGE of genomic DNA from American and English strains gave rise to dissimilar patterns. Patterns of some American strains resembled those from strains isolated in other parts of the world. The restriction fragment length polymorphisms observed by PFGE analysis can be used to group strains and may give hints about the course of distribution of the plant disease. From the sizes of the restriction fragments obtained, a molecular mass of approximately 4.5 Mb was calculated for the genome of E. amylovora.     

Biological properties of some nitrogen-fixing associative enterobacteria

Enterobacteria isolates from potato tubers were able to fix nitrogen, to protect plants against phytopathogens and to produce phytohormones thus increasing the plant yield. These isolates were previously phenotypically identified as Erwinia carotovora; however, they differed from typical E. carotovora in a number of biological characteristics and were found to be nonphytopathogenic (avirulent) due to the lack of pectate lyase activity. A data matrix, containing 31 strains and 105 biological characteristics was used for computer cluster analysis. The avirulent strains formed a separate cluster more closely related to Klebsiella spp. strains (with a 0.67 level of similarity) than to typical phytopathogenic bacteria of the E. carotovora group (with a 0.48 level of similarity). A phylogenetic analysis based on restriction polymorphisms of an amplified ribosomal DNA spacer region revealed that the avirulent strains studied here were different from all Erwinia, Klebsiella and other enterobacteria species strains. The AP PCR/hybridization technique showed cross homology of amplified DNA of these avirulent strains and a lack of such homology with the DNA from strains of other species. Numerical taxonomy data, rDNA analysis and AP PCR/hybridization assays confirmed that these avirulent bacteria may be regarded as an independent group of enterobacteria.     

Variability in the distribution and composition of insertion sequence-like elements in strains of Mycoplasma fermentans

Mycoplasma fermentans has been reported to be pathogenic for man. All fourteen strains tested contain an insertion sequence-like element (ISLE) which may be present in multiple copies. To determine whether ISLE copies are similarly distributed in different strains of M. fermentans, restriction enzyme digest fragments of genomic DNA from 14 isolates, from a variety of sources, were separated by electrophoresis, blotted and hybridized to a biotin labelled polymerase chain reaction (PCR) amplified fragment of ISLE. A range of patterns was observed suggesting that the element has a tendency to undergo rearrangement within the genome. Analysis of ISLE sequences revealed inter- and intra-strain polymorphisms.     

Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.)

A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials.The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.Abbreviations PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism.     

Detection of Phytoplasma from deferent infected hosts in Jordan based on PCR amplification of the 16S rDNA sequences

Sixteen leaf samples, both healthy and Phytoplasma diseased, were collected from different plants such as grape, peach, almond, tomato, paper, squash, apple and pear in northern Jordan. Extracted DNA from diseased grape, peach, almond, tomato, paper and squash plus from infected periwinkle (Catharanthus roseus) samples were amplified with the Phytoplasma universal 16S rDNA sequences primer pairs. Extracted DNA samples from healthy and diseased apple and pear plants were not amplified with the same primer pairs. All the PCR-amplified DNA samples show a common band with size of 558 bp, indicating Phytoplasma pathogens as a disease-causative agent for grape, peach, almond, tomato, paper and squash plants. The restriction fragment length polymorphisms of Alu1 enzyme for the amplified 16S rDNA sequences shows the same DNA fragment patterns indicating no or a limited diversity among the DNA of the detected Phytoplasma pathogens.     

Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.     

植物DNA甲基化及其研究策略

DNA甲基化是表观遗传学研究的热点问题之一,植物DNA甲基化的研究对植物研究领域的发展有着举足轻重的作用。本文阐述了植物DNA甲基化的相关机制,其中包括RdDM（RNA—dependent DNA methylation）、DNA甲基化与组蛋白修饰以及DNA去甲基化等近几年研究的热点问题：讨论了DNA甲基化在植物发育中的功能（包括基因组防御和调控基因表达）、DNA甲基化与转基因沉默的关系以及其在表观遗传学中的地位。最后就目前国内外研究植物DNA甲基化所采取的常用策略,即高效液相色谱法、亚硫酸盐测序法、甲基化敏感的限制性内切酶结合Southern杂交分析法和MSAP（methylation—sensitive amplified polymorphism）法进行了详尽的介绍和讨论。     

植物DNA甲基化及其研究策略

DNA甲基化是表观遗传学研究的热点问题之一, 植物DNA甲基化的研究对植物研究领域的发展有着举足轻重的作用。本文阐述了植物DNA甲基化的相关机制, 其中包括RdDM(RNA-dependent DNA methylation)、DNA 甲基化与组蛋白修饰 以及DNA 去甲基化等近几年研究的热点问题; 讨论了DNA甲基化在植物发育中的功能(包括基因组防御和调控基因表达)、DNA甲基化与转基因沉默的关系以及其在表观遗传学中的地位。最后就目前国内外研究植物DNA甲基化所采取的常用策略,即高效液相色谱法、亚硫酸盐测序法、甲基化敏感的限制性内切酶结合Southern杂交分析法和MSAP(methylation-sensitive amplified Polymorphism)法进行了详尽的介绍和讨论。