Unidirectional gene conversions in the chloroplast of Chlamydomonas interspecific hybrids

Summary With the goal of studying directly the inheritance and recombination of physically mapped markers on the chloroplast genome, we have recently identified and localized physical differences between the chloroplast DNAs (cpDNAs) of the interfertile algae Chlamydomonas eugametos and C. moewusii. Here we report the inheritance patterns of 24 polymorphic loci mapping throughout the chloroplast genome in hybrids recovered from reciprocal crosses between the two algae. Most polymorphic loci were found to be inherited mainly from the mt ⁺ parent, with no apparent preference for one or the other parental alternatives in reciprocal crosses. Virtually all hybrids, however, inherited exclusively the long alleles of three loci; i.e. an intron in the large subunit ribosomal RNA gene of C. eugametos, a 21 kbp sequence addition in the inverted repeat of the C. moewusii cpDNA and a 5.8 kbp sequence addition in one of the single-copy regions of C. moewusii cpDNA. As these alleles are derived from opposite parental strains, their unidirectional inheritance in hybrids results necessarily from interspecific recombination of cpDNA molecules. We propose that gene conversion events led to the spreading of the long alleles of the three loci.     

Sequence analysis of the junctions between a large inverted repeat and single-copy regions in tobacco chloroplast DNA

Summary Tobacco chloroplast DNA contains a large inverted repeat sequence of 26 kilobase pairs (kbp). The inverted repeat is separated by 20 kbp small single-copy and 90 kbp large single-copy regions. We have cloned four DNA fragments containing each junction between the inverted repeat and the single-copy regions. The sequence analysis revealed the exact edges of the inverted repeat. A putative coding region for a ribosomal protein CS19 was found 4 base pairs (bp) away from the inverted repeat on the left margin of the large single-copy region. A sequence AGGAG, which is complementary to the 3 terminal sequence of tobacco chloroplast 16S rRNA, was found within the inverted repeat. A tRNA^His gene was found 5 bp away from the inverted repeat on the right-hand margin of the large single-copy region.     

Chloroplast DNA inheritance in Populus

Summary The inheritance of chloroplast (cp) DNA was examined in F₁ hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen enzymes and five heterologous cpDNA probes were used to screen restriction fragment polymorphisms among the parents. The mode of cpDNA inheritance was demonstrated in progenies of P. deltoides × P. nigra crosses with 26 restriction fragment polymorphisms of cpDNA differentiating P. deltoides from P. nigra, as revealed by 12 enzyme-probe combinations, and in progenies of P. deltoides × P. maximowiczii crosses with 12 restriction fragment polymorphisms separating P. deltoides from P. maximowiczii, as revealed by 7 restriction enzyme-probe combinations. In all cases, F₁ offspring of P. deltoides × P. nigra and P. deltoides × P. maximowiczii crosses had cpDNA restriction fragments of only their maternal P. deltoides parent. The results clearly demonstrated uniparental-maternal inheritance of the chloroplast genome in interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii. Intraspecific P. deltoides hybrids also had the same cpDNA restriction fragments as their maternal parent. Maternal inheritance of the chloroplast genome in Populus is in agreement with what has been observed for most other angiosperms.     

Paternal plastid DNA can be inherited in lentil

Restriction fragment analysis was used to study the inheritance of chloroplast DNA (cpDNA) in F₁ progeny from crosses between Lens culinaris ssp. orientalis and L. culinaris ssp. culinaris. Twenty-five combinations of 11 restriction enzymes and three heterologous probes from Petunia hybrida cpDNA were used to screen six accessions of L.c. culinaris and one accession of L. c. orientalis for restriction fragment length polymorphisms (RFLPs). No variation in cpDNA was observed within the subspecies L. c. culinaris, but the L. c. orientalis accession was unambiguously distinguished from all six L. c. culinaris accessions by two RFLPs. Of ten F₁ progeny from L. c. orientalis x L. c. culinaris crosses, nine had only maternal cpDNA restriction fragments but one F₁ plant inherited cpDNA fragments from both parents. Nuclear DNA inheritance was biparental in all ten F₁ progeny.     

The use of DNA markers to estimate the extent and nature of genetic variability in Solatium tuberosum cultivars

High levels of DNA polymorphism were detected in 27 Solanum tuberosum cultivars examined. Combinations of at least two c-DNA clones have been identified which in conjunction with EcoRI allow important UK potato cultivars to be characterised by their molecular profiles. The widely grown North American cultivar Russett Burbank was also successfully fingerprinted. Estimates of genetic diversity based on restriction fragment length polymorphism (RFLP) data indicate the important role that wild potato species and exotic germplasm have played in the development of the cultivars studied. A graphical method for simultaneously highlighting similarities and differences between genotypes for individual hybridising fragments is presented. This approach is particularly useful in identifying and recording fragments which are unique to certain genotypes. Two potato cultivars: Fiona and Morag produce unique RFLP profiles when digested with EcoRI and EcoRV and probed with a flax ribosomal DNA sequence. Both Fiona and Morag possess incomplete or partial (quantitative) type resistance to G. pallida which was transferred from S. vernei. The preferential transmission of the r-DN A fragments from S. vernei may indicate that this locus is associated with genetic factors controlling resistance to G. pallida.     

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein

Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.     

The sequence-directed bent DNA detected in the replication origin of Chlamydomonas reinhardtii chloroplast DNA is important for the replication function

Summary We demonstrated that the 1055 by restriction fragment containing OriA, a chloroplast DNA replication origin of Chlamydomonas reinhardtii, has electrophoretic anomalies characteristic of bent DNA. A tandem dimer of the region was constructed. Quantitative measurement of the relative gel mobility of a set of permuted fragments was used to extrapolate the approximate position of the bent DNA segment. By analyzing the gel mobility of short, sequenced fragments of the bent DNA region, the putative bending locus was identified. Two A₄ tracts and two A5 tracts were located in the bending locus. Oligonucleotide-directed mutagenesis was then used to disrupt the A tract or the spacing between A tracts and the effect of site-specific mutation on electrophoretic mobility was analyzed. To assess the functional role of the bent DNA region, subclones containing the bending locus, mutated bending locus, and regions flanking the bending locus were constructed. Each subclone was used as template in an in vitro DNA replication system which preferentially initiated DNA replication at OriA. A 224 by subclone with the bending locus positioned in the middle displayed the highest replication function and was sufficient to initiate DNA replication in vitro. Site-specific mutations or alterations of the A tracts resulted in decreased DNA bending and decreased DNA replication activity.     

用寡核苷酸探针（CAC）5／（GTG）5进行人的DNA指纹分析

在人或动物的基因组中,存在着类似于小卫星DNA的简单衔接重复单位,如(GACA)_4、(GATA)_4、(TCC)_5、(CA)_8和(CAC)_5等,由于这些重复单位在人或动物基因组中出现的数目和频率不同而表现出多态性。本文用人工合成的寡核苷酸探针(CAC)_5/(GTG)_5,调查了北京地区的50名无关个体,经过统计学处理,计算出无关个体的相关机率是3.8×10~(-10);此外,还对2个家系中的11名成员和1对双胞胎进行了检测,其结果显示子代中的杂交带分别来自父亲和母亲;双胞胎的DNA指纹图完全一致。研究结果表明,(CAC)_5/(GTG)_5探针检出的谱带具有高度的个体特异性(同卵双生子除外),并且谱带在亲代与子代间的传递符合孟德尔遗传规律。     

A 21 kilobase-pair deletion/addition difference in the inverted repeat sequence of chloroplast DNA from Chlamydomonas eugametos and C. moewusii

Summary Our recent physical mapping of chloroplast DNA (cpDNA) from Chlamydomonas moewusii, a unicellular green alga which is interfertile with Chlamydomonas eugametos, has revealed a two-fold size difference between the inverted repeat sequences of these algae. With a size of 42 kbp, the inverted repeat of C. moewusii is the largest yet identified in any chloroplast genome. Here we have compared the arrangement of conserved sequences within the two algal inverted repeats by hybridizing cloned restriction fragments representing over 90% of these repeats to Southern blots of cpDNA digests from the two algae. We found that the size difference between the two algal inverted repeats is due to the presence of an extra DNA segment of 21 kilobase pairs (kbp) in C. moewusii. Except for this sequence, the C. moewusii inverted repeat is highly homologous to the entire C. eugametos repeat and the arrangement of conserved sequences in the two repeats is identical. Southern hybridizations with specific gene probes revealed that the conserved sequences include the rDNA region and the genes coding for the large subunit of ribulose 1,5 bisphosphate carboxylase-oxygenase (rbcL) and for the 32 kilodalton thylakoid membrane protein (psbA). With respect to the conserved sequences, the extra 21 kbp DNA segment of C. moewusii lies in the region of psbA, most probably slightly downstream from this gene.     

Inheritance of mitochondrial DNA in lentil (Lens culinaris Medik.)

Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F₁ and F₅ progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F₁ progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris Laird and Eston from L.c. ssp. culinaris ILL5588, and one restriction fragment polymorphism distinguished L.c. ssp. culinaris Laird and Eston from L.c. ssp. orientalis LO4. Twelve of 13 f₁ progeny and all F₅ progeny from the intrasubspecific crosses, and all F₁ progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f₁ plant from an Eston x ILL5588 cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F₁ progeny.NRCC No. 38451     

Gene rearrangements in Chlamydomonas chloroplast DNAs are accounted for by inversions and by the expansion/contraction of the inverted repeat

To gain insight into the mutational events responsible for the extensive variation of chloroplast DNA (cpDNA) within the green algal genus Chlamydomonas, we have investigated the chloroplast gene organization of Chlamydomonas pitschmannii, a close relative of the interfertile species C. eugametos and C. moewusii whose cpDNAs have been well characterized. At 187 kb, the circular cpDNA of C. pitschmannii is the smallest Chlamydomonas cpDNA yet reported; it is 56 and 105 kb smaller than those of its C. eugametos and C. moewusii counterparts, respectively. Despite this substantial size difference, the arrangement of 77 genes on the C. pitschmannii cpDNA displays only three noticeable differences from the organization of the corresponding genes on the collinear C. eugametos and C. moewusii cpDNAs. These changes in gene order are accounted for by the expansion/contraction of the inverted repeat and one or two inversions in a single-copy region. In land plant cpDNAs, these kinds of events are also responsible for gene rearrangements. The large size difference between the C. pitschmannii and C. eugametos/C. moewusii cpDNAs is mainly attributed to multiple events of deletions/additions as opposed to the usually observed expansion/contraction of the inverted repeat in land plant cpDNAs. We also found that the mitochondrial genome of C. pitschmannii is a circular DNA molecule of 16.5 kb which is 5.5 and 7.5 kb smaller than its C. moewusii and C. eugametos counterparts, respectively.     

Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm-2a region of chromosome 9 in tomato

Summary A method has been developed which allows the isolation of very high molecular weight DNA (>2 million bp) from leaf protoplasts of tomato (Lycopersicon esculentum). The DNA isolated in this manner was digested in agarose with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The size range of the reslting fragments was determined by hybridization to a number of single copy clones and the suitability of these enzymes for the mapping of large DNA fragments was evaluated. Furthermore, five genetically tightly linked single copy clones have been used to begin the construction of a physical map in a region of the genome containing the Tm-2a gene which confers resistance to tobacco mosaic virus. Two of the five clones were found to be on the same 560 kb SalI fragment and therefore are no further apart than that distance. The remaining three markers are distributed over at least 3 million bp, so that the total minimum physical distance of that cluster is at least 4 million bp. The results are discussed with respect to correlations between recombination frequencies and physical distance as well as physical mapping large regions of a complex plant genome like tomato.     

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region

Using a fluorescence-based PCR-SSCP (single-strand conformation polymorphism), we verified imperfectibility in the paternal inheritance of chloroplast DNA (cpDNA) in Chamaecyparis obtusa (Cupressaceae) controlled crosses. An intraspecific sequence polymorphism of the intergenic spacer region between the trnD and trnY genes was utilized as a molecular marker. Of 361 progenies, in which the cpDNA haplotypes of their female and male parents were different, 352 (97.5%) possessed the same haplotypes as their male parents, and nine (2.5%) the same haplotypes as their female parents. The parentage of the nine progenies with female parental types was diagnosed using DNA fingerprinting based on fluorescence-based RAPD profiles. Their parentage showed convincing evidence of the low frequency of maternal inheritance. Moreover, heteroplasmy was observed in the open-pollinated seeds collected in a seed orchard. The confirmation of maternal plastid transmission in the full-sib families and the observation of heteroplasmy in seeds reveal that the paternal inheritance of cpDNA is not an exclusive phenomenon and that the mode of its inheritance is biparental in C. obtusa. Received: 15 April 2000 / Accepted: 13 July 2000     

Intraspecific sequence variation of chloroplast DNA among the component species of evergreen broad-leaved forests in Japan

For the purpose of phylogeographic study of lucidophyllous (evergreen broad-leaved) forests in Japan, we surveyed intraspecific chloroplast DNA (cpDNA) variation in 41 component species of such forests. Intraspecific cpDNA variations were detected in 14 species. In 15 species and one species group, 16 non-coding cpDNA regions were examined to find intraspecific sequence variation. The extent of variation in these regions was compared. The largest amount of intraspecific variation was detected in the rps16 region. A relatively large amount of intraspecific variation was detected in the petD-rpoA, rpl16, and trnL-F regions. It is suggested that these regions of cpDNA would be useful for detecting intraspecific variation in plant species, and could provide valuable information for various research purposes.     

Intraspecific sequence variation of chloroplast DNA among the component species of deciduous broad-leaved forests in Japan

To select appropriate plant materials for a phylogeography of deciduous broad-leaved forests in Japan, we surveyed intraspecific chloroplast DNA variation in 34 species found in these forests. A relatively large number of intraspecific cpDNA variations were detected in ten species: Carpinus japonica (nucleotide diversity π=0.00083), C. laxiflora (π=0.00221), Magnolia obovata (π=0.00134), Lindera triloba (π=0.00255), L. obtusiloba (π=0.00289), Pourthiaea villosa var. leavis (π=0.00263), Acer japonicum (π=0.00170), A. micranthum (π=0.00237), Euonymus oxyphyllus (π=0.00322) and Styrax obassia (π=0.00100).     

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Abstract Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases Eco RI or Hae III. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus . Digestion with Eco RI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae . Digestion of genomic and mitochondrial DNA with Hae III showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium .     

Phylogenetic analysis of tnpR. genes in mercury resistant soil bacteria: the relationship between DNA sequencing and RFLP typing approaches

Abstract The diversity of resolvase ( tnpR ) genes carried by a number of mercury resistant soil bacteria has been investigated by DNA sequencing. The resulting DNA sequence information was compared to previously published tnp R. DNA sequences and to previously published restriction fragment length polymorphism (RFLP) data, permitting the relationships between DNA sequencing and RFLP approaches to be studied by the use of phylogenetic trees. DNA maximum likelihood and DNA parsimony were used to construct a variety of phylogenetic trees. DNA sequencing confirmed the validity of RFLP analysis and highlighted the importance of restriction endonuclease choice upon the resulting RFLP patterns and dendrogram topology. The tnp R genes of two previously uncharacterised mercury resistant bacteria, T2–7 and T2–12 were also studied. DNA sequence data placed T2–7 in a previously described gene class, tnp R-D and T2–12 in a new gene class, tnp R-F. The significance of this data with respect to the recombination and evolution events occurring within bacterial populations are discussed.     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.