Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

Summary The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome.     

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.     

Highly rearranged and size‐variable chloroplast genomes in conifers II clade (cupressophytes): evolution towards shorter intergenic spacers

Although conifers are of immense ecological and economic value, bioengineering of their chloroplasts remains undeveloped. Understanding the chloroplast genomic organization of conifers can facilitate their bioengineering. Members of the conifer II clade (or cupressophytes) are highly diverse in both morphologic features and chloroplast genomic organization. We compared six cupressophyte chloroplast genomes (cpDNAs) that represent four of the five cupressophyte families, including three genomes that are first reported here (Agathis dammara, Calocedrus formosana and Nageia nagi). The six cupressophyte cpDNAs have lost a pair of large inverted repeats (IRs) and vary greatly in size, organization and tRNA copies. We demonstrate that cupressophyte cpDNAs have evolved towards reduced size, largely due to shrunken intergenic spacers. In cupressophytes, cpDNA rearrangements are capable of extending intergenic spacers, and synonymous mutations are negatively associated with the size and frequency of rearrangements. The variable cpDNA sizes of cupressophytes may have been shaped by mutational burden and genomic rearrangements. On the basis of cpDNA organization, our analyses revealed that in gymnosperms, cpDNA rearrangements are phylogenetically informative, which supports the ‘gnepines’ clade. In addition, removal of a specific IR influences the minimal rearrangements required for the gnepines and cupressophyte clades, whereby Pinaceae favours the removal of IRB but cupressophytes exclusion of IRA. This result strongly suggests that different IR copies have been lost from conifers I and II. Our data help understand the complexity and evolution of cupressophyte cpDNAs.     

A synopsis of <Emphasis Type="Italic">Cohniella</Emphasis> (Orchidaceae,Oncidiinae)

A synopsis of the genus Cohniella is presented with nomenclatural updates with a discusion of diagnostic features and biogeographical data. Thirteen species and one natural hybrid are recognized. Two new species, Cohniella pendula and Cohniella biorbicularis, are described and illustrated. The following four new combinations are proposed: Cohniella binotii, Cohniella brachyphylla, Cohniella cepula, and Cohniella longifolia. Lectotypes are selected for Oncidium subulifolium, Oncidium helicanthum, Oncidium humboldtii, Oncidium jonesianum var. phaeanthum, Oncidium cepula, and Oncidium wittii. We also propose an amended lectotype for Epidendrum cebolleta and several epitypifications and new synonyms. A key to the genera of the Trichocentrum complex is presented (Appendix) as well as keys to the species of Cohniella.     

Vicia faba chloroplast DNA has only one set of ribosomal RNA genes as shown by partial denaturation mapping and R-loop analysis

Summary Broad-bean (Vicia faba) chloroplast DNA (cpDNA) was isolated and characterized. The intact DNA is circular and has a molecular weight of 79.8x 10⁶ dalton. Electron microscopic analysis of self-annealed intact single-strand circles show that it does not have a large double-stranded inverse repeat as seen in spinach chloroplast DNA. Only one ribosomal RNA gene (one set of 16S and 23S rRNA sequences) was found in preparations of R-loops between the Vicia rRNA and cpDNA circles. A restriction enzyme map for SalI and KpnI was derived by comparing the partial denaturation pattern of the fragments with the pattern of the intact circle. The map was confirmed by gel analysis. The ribosomal RNA gene was localized on the SalI fragment 3b by R-loop analysis. SalI fragment 1a although it contains a G-C rich region did not form R-loops with rRNA. Partial denaturation patterns of spinach cpDNA circles and BglI fragments were determined and from this the position of the fragments mapped. This confirmed the reliability of these methods for the arrangement of restriction enzyme fragments along circular molecules. The structures of the two cpDNAs were compared.     

Chloroplast DNA differences between two species of Oenothera subsection Munzia: location in the chloroplast genome and relevance to possible interactions between nuclear and plastid genomes

Summary The chloroplast DNAs (cpDNAs) of Oenothera berteriana and Oe. odorata (subsection Munzia) were examined by restriction endonuclease analysis with Sal I, Pvu II, Kpn I, Pst I, Hind III, and Bam HI. The fragment patterns show that these cpDNAs have all 133 restriction sites in common as well as a lot of individual bands. Nevertheless the cpDNAs of the two species can be distinguished by distinct differences in size between a small number of fragments. The 42 cleavage sites produced by Sal I, Pvu II and Kpn I were mapped on the circular cpDNAs. This was achieved by an approach which combined experimental and mathematical procedures. The overall serial order of the fragments was found to be the same for both cpDNAs. The size differences of individual fragments in the Sal I, Pvu II and Kpn I patterns between Oe. berteriana and Oe. odorata cpDNA are located within five regions scattered along the plastid chromosome. Two of these regions have been localized in the larger and one in the smaller of the two single-copy parts of the cpDNA molecule. The remaining two overlap the borders between the large single-copy and each of the duplicated parts of the molecule. The positions of distinct restriction sites are altered among the two Oenothera plastome DNAs by 0.02–0.4 MDa (30–600 base pairs). These alterations probably result from insertions/deletions.Abbreviations cpDNA chloroplast, plastid DNA - Oe. Oenothera - MDa Megadalton - rRNA, rDNA ribosomal RNA, DNA Dedicated to Professor Berthold Schwemmle, Tübingen, on the occasion of his 60th birthday     

Investigation of chloroplast DNA heteroplasmy in Medicago sativa L. using cultured tissue

Summary Medicago sativa L. cv Regen S is heteroplasmic for chloroplast DNA (cpDNA). Previous analyses of regenerated plants have shown a predominance of one of the cpDNAs which we have designated type A (the other we have designated type B). Studies of the replication of the two cpDNAs in tissue culture were carried out using leaflet expiants with defined cpDNA types and a distinguishing probe. The explants obtained showed a bias toward type A cpDNA during tissue culture. The data suggest that chloroplasts with different DNAs in a common nuclear background can multiply at different rates.     

Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis

Summary Restriction fragment analysis of chloroplast (cp) DNAs from 35 wheat (Triticum) and Aegilops species, including their 42 accessions, was carried out with the use of 13 restriction enzymes to clarify variation in their cpDNAs. Fourteen fragment size mutations (deletions/insertions) and 33 recognition site changes were detected among 209 restriction sites sampled. Based on these results, the 42 accessions of wheat-Aegilops could be classified into 16 chloroplast genome types. Most polyploids and their related diploids showed identical restriction fragment patterns, indicating the conservatism of the chloroplast genome during speciation, and maternal lineages of most polyploids were disclosed. This classification of cpDNAs was principally in agreement with that of the plasma types assigned according to phenotypes arising from nucleus-cytoplasm interactions. These mutations detected by restriction fragment analysis were mapped on the physical map of common wheat cpDNA, which was constructed with 13 restriction endonucleases. Length mutations were more frequently observed in some regions than in others: in a 16.0 kilo base pairs (kbp) of DNA region, including rbcL and petA genes, 6 of 14 length mutations were concentrated. This indicates that hot spot regions exist for deletions/insertions in chloroplast genome. On the other hand, 33 recognition site mutations seemed to be distributed equally throughout the genome, except in the inverted repeat region where only one recognition site change was observed. Base substitution rate (p) of cpDNA was similar to that of other plants, such as Brassica, pea and Lycopersicon, showing constant base substitution rates among related taxa and slow evolution of cpDNA compared with animal mitochondrial DNA. Phylogenetic relationships among Triticum and Aegilops species were discussed, based on the present data.Contributions no. 45 and no. 490 from the Kihara Institute for Biological Research, Yokohama City University and the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, respectively.     

Chloroplast genome organization of bromegrass,Bromus inermis Leyss

Summary A physical map of the Bromus inermis chloroplast genome was constructed using heterologous probes of barley and wheat chloroplast DNA (cpDNA) to locate restriction sites. The map was aligned from data obtained from filter hybridization experiments on single and double enzyme digests. Cleavage sites for the enzymes PstI, SalI, KpnI, XhoI and PvuII were mapped. The chloroplast genome of B. inermis is similar in physical organization to that of other grasses. The circular cpDNA molecule of B. inermis has the typical small (12.8 kbp) and large (81.3 kbp) single-copy regions separated by a pair of inverted repeat (21 kbp) regions. The cpDNA molecule of B. inermis is collinear in sequence to that of wheat, rye, barley and oats. No structural rearrangements or major deletions were observed, indicating that the cpDNA of Bromus is a useful tool in phylogenetic studies.     

CHLOROPLAST DNA RESTRICTION SITE VARIATION AND THE PHYLOGENY OF COREOPSIS SECTION COREOPSIS (ASTERACEAE)

Restriction site variation in chloroplast DNAs (cpDNAs) of Coreopsis section Coreopsis was employed to assess divergence and phylogenetic relationships among the nine species of the section. A total of fourteen restriction site mutations and one length mutation was detected. Cladistic analysis of the cpDNA data produced a phylogeny that is different in several respects from previous hypotheses. CpDNA mutations divide the section into two groups, with the two perennial species C. auriculata and C. pubescens lacking any derived restriction site changes. The other seven species are united by five synapomorphic restriction site mutations and the one length mutation. These seven species fall into three unresolved clades consisting of 1) the remaining three perennial species, C. grandiflora, C. intermedia, and C. lanceolata; 2) three annual species, C. basalis, C. nuecensoides, and C. nuecensis; and 3) the remaining annual, C. wrightii. The cpDNA data suggest that, although the perennial habit is primitive within the section, the annual species of section Coreopsis have likely not originated from an extant perennial species. The estimated proportion of nucleotide differences per site (given as 100p) for the cpDNAs of species in the section ranges from 0.00 to 0.20, which is comparable to or lower than values reported for other congeneric species. The low level of cpDNA divergence is concordant with other data, including cross compatibility, interfertility and allozymes, in suggesting that species of the section are not highly divergent genetically.     

The chloroplast genome of Phalaenopsis aphrodite (Orchidaceae): comparative analysis of evolutionary rate with that of grasses and its phylogenetic implications

Whether the Amborella/Amborella-Nymphaeales or the grass lineage diverged first within the angiosperms has recently been debated. Central to this issue has been focused on the artifacts that might result from sampling only grasses within the monocots. We therefore sequenced the entire chloroplast genome (cpDNA) of Phalaenopsis aphrodite, Taiwan moth orchid. The cpDNA is a circular molecule of 148,964 bp with a comparatively short single-copy region (11,543 bp) due to the unusual loss and truncation/scattered deletion of certain ndh subunits. An open reading frame, orf91, located in the complementary strand of the rrn23 was reported for the first time. A comparison of nucleotide substitutions between P. aphrodite and the grasses indicates that only the plastid expression genes have a strong positive correlation between nonsynonymous (Ka) and synonymous (Ks) substitutions per site, providing evidence for a generation time effect, mainly across these genes. Among the intron-containing protein-coding genes of the sampled monocots, the Ks of the genes are significantly correlated to transitional substitutions of their introns. We compiled a concatenated 61 protein-coding gene alignment for the available 20 cpDNAs of vascular plants and analyzed the data set using Bayesian inference, maximum parsimony, and neighbor-joining (NJ) methods. The analyses yielded robust support for the Amborella/Amborella-Nymphaeales-basal hypothesis and for the orchid and grasses together being a monophyletic group nested within the remaining angiosperms. However, the NJ analysis using Ka, the first two codon positions, or amino acid sequences, respectively, supports the monocots-basal hypothesis. We demonstrated that these conflicting angiosperm phylogenies are most probably linked to the transitional sites at all codon positions, especially at the third one where the strong base-composition bias and saturation effect take place.     

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.     

Structural analysis of chloroplast DNA in Prunus (Rosaceae): evolution, genetic diversity and unequal mutations

In order to understand the evolutionary aspects of the chloroplast DNA (cpDNA) structures in Rosaceous plants, a physical map of peach (Prunus persica cv. Hakuhou) cpDNA was constructed. Fourteen lambda phage clones which covered the entire sequence of the peach cpDNA were digested by restriction enzymes (SalI, XhoI, BamHI, SacI, and PstI) used singly or in combination. The molecular size of peach cpDNA was estimated to be about 152 kb. The gene order and contents were revealed to be equivalent to those of standard type of angiosperms by the localization of 31 genes on the physical map. Eighteen accessions from 14 Prunus species (P. persica, P. mira, P. davidiana, P. cerasis, P. cerasifera, P. domestica, P. insititia, P. spinosa, P. salicina, P. maritima, P. armeniaca, P. mume, P. tomentosa, P. zippeliana, and P. salicifolia) and one interspecific hybrid were used for the structural analysis of cpDNAs. Seventeen mutations (16 recognition site changes and one length mutation) were found in the cpDNA of these 18 accessions by RFLP analysis allowing a classification into 11 genome types. Although the base substitution rate in the recognition site (100p = 0.72) of cpDNA in Prunus was similar to that of other plants, i.e., Triticum–Aegilops, Brassica, and Pisum, it differed from Pyrus (100p = 0.15) in Rosaceae. Seven mutations including one length mutation were densely located within a region of about 9.1 kb which includes psbA and atpA in the left border of a large single-copy region of Prunus cpDNAs. The length mutation was detected only in P. persica and consisted of a 277 bp deletion which occurred in a spacer region between the trnS and trnG genes within the 9.1 kb region. Additional fragment length mutations (insertion/deletion), which were not detected by RFLP analysis, were revealed by PCR and sequence analyses in P. zippeliana and P. salicifolia. All of these length mutations occurred within the 9.1 kb region between psbA and atpA. This region could be an intra-molecular recombinational hotspot in Prunus species.     

木芙蓉三个品种及近缘种的叶绿体基因组比较分析

木芙蓉(Hibiscus mutabilis)栽培历史悠久,是原产中国的古老园林树种和药用植物。为了探讨木芙蓉品种及近缘种的进化特征,厘清木芙蓉品种间及其与近缘种间的亲缘关系,以及探究木芙蓉叶绿体基因组(chloroplast DNA, cpDNA)的遗传方式,该文选择了一个杂交组合中的3个木芙蓉栽培品种(‘单瓣白’‘金秋颂’‘牡丹粉’),用高通量测序平台Illumina NovaSeq对其cpDNA进行首次测序。经组装注释后得到3条完整的cpDNA序列,结合该团队已经完成的近缘种台湾芙蓉(H.taiwanensis)和来自基因库的木槿、朱槿的cpDNA,对木槿属4种及木芙蓉种下的3个品种进行了cpDNA组成和结构特征的比较分析,并完成了其系统发育树重建。结果表明：(1)‘单瓣白’‘金秋颂’‘牡丹粉’的cpDNA序列长度分别为160 880、160 879、160 920 bp,基因数目均为130个,其中蛋白编码基因85个、核糖体RNA 8个和转运RNA 37个。(2)比较分析结果显示,木芙蓉的种下3个品种及其近缘种台湾芙蓉在cpDNA上高度保守,反向重复区(IR)均为26 300 b...     

Molecular Evidence and the Origin and Development of the Domesticated Sunflower (Helianthus annum, Asteraceae)

The domesticated sunflower,Helianthus annuus, is an important economic crop, yet molecular data regarding its evolution are limited. Here we review morphological, geographical, archaeological, and molecular evidence pertaining to its origin and development. New isozyme and chloroplast DNA (cpDNA) evidence is also presented. Morphological, geographical, and archaeological evidence has led to the hypothesis that the domesticated sunflower was derived from a wild/weedy form ofH. annuus possibly in the Midwest. Molecular evidence was concordant with this hypothesis. A high degree of enzymatic and cpDNA sequence similarity was observed between wild and domesticatedH. annuus, and domesticatedH. annuus contained a subset of the alleles and cpDNAs found in wildH. annuus. The extensive polymorphism in the wild plants and the virtual monomorphism in cultivated lines for both isozyme and cpDNA phenotypes further suggest a single origin of the domesticated sunflower from a very limited gene pool. In addition, Native American varieties of the domesticated sunflower were genetically more variable than other cultivated lines, possibly indicating that they gave rise to the other cultivated stocks. Molecular evidence did not, however, allow conclusions as to the exact geographic origin of the domesticated sunflower.     

Chloroplast and mitochondrial DNA composition of triploid and tetraploid somatic hybrids between Lycopersicon esculentum and Solanum tuberosum

The chloroplast (cp) DNA type and mitochondrial (mt) DNA composition of 17 somatic hybrids between a cytoplasmic albino tomato and monoploid potato (A7-hybrids) and 18 somatic hybrids between a nitrate reductase-deficient tomato and monoploid potato (C7-hybrids) were analyzed. Thirteen A7-hybrids and 9 C7-hybrids were triploids (with one potato genome); the other hybrids were tetraploid. As expected, all A7-hybrids contained potato cpDNA. Of the C7-hybrids 7 had tomato cpDNA, 10 had potato cpDNA and 1 hybrid contained both tomato and potato cpDNA. The mtDNA composition of the hybrids was analyzed by hybridization of Southern blots with four mtDNA-specific probes. The mtDNAs in the hybrids had segregated independently from the cpDNAs. Nuclear DNA composition (i.e. one or two potato genomes) did not influence the chloroplast type in the C7-hybrids, nor the mtDNA composition of A7- or C7-hybrids. From the cosegregation of specific mtDNA fragments we inferred that both tomato and potato mtDNAs probably have a coxII gene closely linked to 18S+5S rRNA genes. In tomato, atpA, and in potato, atp6 seems to be linked to these mtDNA genes.     

The complete chloroplast genome of the green algae <Emphasis Type="Italic">Hariotina reticulata</Emphasis> (Scenedesmaceae,Sphaeropleales, Chlorophyta)

In this study, the chloroplast genome of Hariotina reticulata was fully sequenced and compared to other Sphaeropleales chloroplast genomes. It is 210,757 bp larger than most Sphaeropleales cpDNAs. It presents a traditional chloroplast structure, and contains 103 genes, including 68 protein-coding genes, six rRNA genes and 29 tRNA genes. The coding region constitutes of 43% of the whole cpDNA. Eighteen introns are found in 11 genes and six introns are unique for Hariotina. 11 open reading frames are identified among these introns. The synteny between Hariotina and Acutodesmus cpDNAs is in general identical, while within Sphaeropleales order, high variability in cpDNA architecture is indicated by general high DCJ distances. Ankyra judayi exhibits the greatest dissimilarity in gene synteny to the others and share some unique gene clusters with Treubaria triappendiculata. The phylogenomic analyses show that A. judayi is clustered with Treubariaceae species and sister to Chlorophyceae incertae sedis and other Sphaeropleales species. The monophyly of Sphaeropleales is rejected.     

Physical map and gene localization on sunflower (Helianthus annuus) chloroplast DNA: evidence for an inversion of a 23.5-kbp segment in the large single copy region

As a first step in the study of chloroplast genome variability in the genus Helianthus, a physical restriction map of sunflower (Helianthus annuus) chloroplast DNA (cpDNA) has been constructed using restriction endonucleases BamH I, Hind III, Pst I, Pvu II and Sac. I. Sunflower circular DNA contains an inverted repeat structure with the two copies (23 kbp each) separated by a large (86 kbp) and a small (20 kbp) single copy region. Its total length is therefore about 152 kbp. Sunflower cpDNA is essentially colinear with that of tobacco with the exception of an inversion of a 23.5-kbp segment in the large single copy region. Gene localization on the sunflower cpDNA and comparison of the gene map with that from tobacco chloroplasts have revealed that the endpoints of the inversion are located between the trnT and trnE genes on the one hand, and between the trnG and trnS genes on the other hand.Analysis of BamH I restriction fragment patterns of H. annuus, H. occidentalis ssp. plantagineus, H. grossesseratus, H. decapetalus, H. giganteus, H. maximiliani and H. tuberosus cpDNAs suggests that structural variations are present in the genus Helianthus.     

The chloroplast genome of the “basal” angiosperm Calycanthus fertilis – structural and phylogenetic analyses

The nucleotide sequence of the complete chloroplast genome of a basal angiosperm, Calycanthus fertilis, has been determined. The circular 153337 bp long cpDNA is colinear with those of tobacco, Arabidopsis and spinach. A total of 133 predicted genes (115 individual gene species, 18 genes duplicated in the inverted repeats) including 88 potential protein-coding genes (81 gene species), 8 ribosomal RNA genes (4 gene species) and 37 tRNA genes (30 gene species) representing 20 amino acids were identified based on similarity to their homologs from other chloroplast genomes. This is the highest gene number ever registered in an angiosperm plastome. Calycanthus fertilis cpDNA also contains a homolog of the recently discovered mitochondrial ACRS gene. Since no gene transfer from mitochondria to the chloroplast has ever been documented, we investigated the evolutionary affinity of this gene in detail. Phylogenetic analysis of the protein-coding subset of the plastome suggests that the ancient line of Laurales emerged after the split of the angiosperms into monocots and dicots. Calycanthus fertilis Walter var. ferax (Michy.) Rehder is a synonym of C. floridus L. var. glaucus (Willd.) Torr. & A. Gray.Data deposition: The sequence reported in this paper has been deposited in the EMBL database (accession no. AJ428413).