Six primers for the amplification of three non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA from various plant species. The primers worked for most species tested including algae, bryophytes, pteridophytes, gymnosperms and angiosperms. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology (in supplying markers) and evolution (inter- and probably intraspecific phylogenies) of plants.

Universal primers for amplification of three non-coding regions of chloroplast DNA

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

Methods for identifying species by using short orthologous DNA sequences, known as “DNA barcodes,” have been proposed and initiated to facilitate biodiversity studies, identify juveniles, associate sexes, and enhance forensic analyses. The cytochrome c oxidase 1 sequence, which has been found to be widely applicable in animal barcoding, is not appropriate for most species of plants because of a much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals. We therefore propose the nuclear internal transcribed spacer region and the plastid trnH-psbA intergenic spacer as potentially usable DNA regions for applying barcoding to flowering plants. The internal transcribed spacer is the most commonly sequenced locus used in plant phylogenetic investigations at the species level and shows high levels of interspecific divergence. The trnH-psbA spacer, although short (≈450-bp), is the most variable plastid region in angiosperms and is easily amplified across a broad range of land plants. Comparison of the total plastid genomes of tobacco and deadly nightshade enhanced with trials on widely divergent angiosperm taxa, including closely related species in seven plant families and a group of species sampled from a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53 families), suggest that the sequences in this pair of loci have the potential to discriminate among the largest number of plant species for barcoding purposes.

https://www.pnas.org/content/pnas/102/23/8369.full.pdf

Use of DNA barcodes to identify flowering plants

The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.

Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae).

Publisher Summary This chapter highlights tobacco BY-2 (TBY-2) cell line. TBY-2 derived from the seedlings of N. tabacum L. cv. Bright Yellow 2 grows fast and multiplies 80- to 100-fold in 1 week. After the stationary phase, cells of TBY-2 are transferred to a medium containing aphidicolin for 24 hr and then released from treatment; high synchrony is obtained starting from the S phase. TBY-2 cells are propagated in the modified medium of Linsmaier and Skoog, in which KH2PO4 and thiamine HCl are increased to 370 and 1 mg/liter, respectively, and sucrose and 2,4-D are supplemented to 3% and 0.2 mg/liter, respectively. The preparation of protoplasts from TBY-2 cells, from which the isolation of organelles is easy, has been established. Using the synchrony system, the change in the cell cycle progression of TBY-2 cells successfully followed the change in cytoskeletons. Biochemical and molecular biological studies can also be done on TBY-2 cells, as mass culture of this material is readily feasible.

Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants

The c o m p l e t e n u c i e o t i d e sequence [155 ,844 bp) o f t o b a c c o ( N i c o t i a n a tabecum v a r . B r i g h t y e l l o w 4) c h l o r o p l a s t DNA [ S h i n o z a k i e t e l . 1986) is p r e s e n t e d . The c i r c u l a r DNA [see F ig . 1) i8 i nea r zed by c u t t i n g a t the j u n c t i o n JLA between IR A and LSC JLA is d e s i g n a t e d ze ro and numbered p r o c e e d i n g t owa rds LSC The DNA s t r a n d which codes f o r the l a r g e s u b u n i t of r i b u l o s e l , % b 8phospha te c a r b o x y l a s e is d e s i g n a t e d as A s t r a n d and the comp lemen ta ry s t r a n d as B s t r a n d . The B s t r a n d i8 shown he re . 6enes are boxed . The n o m e n c l a t u r e f o r genes f o l l o w s the p r o p o s a l s of H a l l i c k and B o t t o m l e y [1983, see Tab le 1) . A s t e r i s k s i n d i c a t e s p l i t genes and [C) deno tes genes l o c a t e d on the comp lemen ta ry s t r a n d ( t h e A s t r a n d ) .

The complete nucleotide sequence of the tobacco chloroplast genome

The location and nucleotide sequences of tobacco chloroplast genes for tRNAGlu(UUC), tRNATyr(GUA) and tRNAAsp(GUC) have been determined. These genes lie midway between the genes for alpha and beta/epsilon subunits of H+-ATPase on the large single-copy region of the chloroplast DNA. The gene organization is tRNAGlu - 59bp spacer - tRNATyr - 108bp spacer - tRNAAsp on the same DNA strand. Northern blot hybridization studies revealed that these three tRNA genes are cotranscribed. The transcription initiation site was localized at 24 bp upstream from the tRNAGlu coding region and its termination site at 90 bp downstream from the tRNAAsp coding region by S1 mapping. The tricistronic tRNA precursor is thus calculated to be 512 bases long. Its processing was also studied by S1 mapping.

Structure and cotranscription of tobacco chloroplast genes for tRNAGlu(UUC), tRNATyr(GUA) and tRNAAsp(GUC).

The nucleotide sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC) have been determined. None of these genes contains an intron. One unusual feature is that the tRNAGly contains only two base-pairs (A-U, G-U) in the D stem. These four tRNA genes were located in the known physical map of tobacco chloroplast DNA. Hybridization analysis to chloroplast tRNA revealed that all four tRNA genes are transcribed in vivo.

Locations and sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC): the tRNAGly contains only two base-pairs in the D stem.

T. Kamogashira

Papers

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression.

The complete nucleotide sequence of the tobacco chloroplast genome

Structure and cotranscription of tobacco chloroplast genes for tRNAGlu(UUC), tRNATyr(GUA) and tRNAAsp(GUC).

Locations and sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC): the tRNAGly contains only two base-pairs in the D stem.

The complete nucleotide sequence of the tobacco chloroplast genome. EMBO J 5: 2043-2049