Comparison of the primary structure ofwaxy proteins (granule-bound starch synthase) between polyploid wheats and related diploid species

Thewaxy proteins encoded by the genomes A, B, and D in polyploid wheats and related diploid species were isolated by SDS-PAGE. The N-terminal amino acid sequences of mature proteins and V8 protease-induced fragments were determined. A total of five amino acid substitutions was detected in these sequences, which represent about 10% of the whole sequences of thewaxy proteins. A comparison of these sequences in polyploid wheats with those in related diploid species revealed the following: (i)waxy proteins encoded by the A genome of polyploid wheats were identical to that ofTriticum monococcum, (ii) thewaxy protein encoded by the B genome ofT. turgidum was identical to that ofT. searsii, but differed from those ofT. speltoides andT. longissimum by one amino acid substitution, (iii) thewaxy protein encoded by the B genome ofT. aestivum differed from that encoded by the B genome ofT. turgidum by one amino acid substitution, and (iv) thewaxy protein encoded by the D genome ofT. aestivum was identical to that ofT. tauschii.     

A chromosome-specific sequence common to the B genome of polyploid wheat and Aegilops searsii

A low-copy, non-coding chromosome-specific DNA sequence, isolated from common wheat, was physically mapped to the distal 19% region of the long arm of chromosome 3B (3BL) of common wheat. This sequence, designated WPG118, was then characterized by Southern hybridization, PCR amplification and sequence comparison using a large collection of polyploid wheats and diploid Triticum and Aegilops species. The data show that the sequence exists in all polyploid wheats containing the B genome and absent from those containing the G genome. At the diploid level, it exists only in Ae. searsii, a diploid species of section Sitopsis, and not in other diploids including Ae. speltoides, the closest extant relative to the donor of the B genome of polyploid wheat. This finding may support the hypothesis that the B-genome of polyploid wheat is of a polyphyletic origin, i.e. it is a recombined genome derived from two or more diploid Aegilops species.     

High molecular weight glutenin subunit variation in wild and cultivated einkorn wheats (Triticum spp.,Poaceae)

Variation in high molecular weight (HMW) glutenin subunit composition among wild and cultivated einkorn wheats (2n = 2x = 14, AA) was investigated using one- (SDS-PAGE and urea/SDS-PAGE) and two-dimensional (IEF × SDS-PAGE) electrophoretic analyses. The material comprised 150 accessions ofTriticum urartu, 160 accessions ofT. boeoticum, 24 accessions ofT. boeoticum subsp.thaoudar and 74 accessions of primitive domesticatedT. monococcum from many different germplasm collections. The biochemical characteristics of HMW-glutenin subunits ofT. boeoticum andT. monococcum were highly similar to one another but distinctly different from those ofT. urartu. All the species analysed were characterised by large intraspecific variation and only three HMW-glutenin subunit patterns were identical betweenT. boeoticum andT. monococcum. Consistent with the distinct nature ofT. urartu, all its HMW-glutenin patterns were different from those found inT. boeoticum andT. monococcum. The differences detected between these species might reflect their reproductive isolation and are consistent with recent nomenclatural and biosystematic treatments that recogniseT. urartu as separate species fromT. boeoticum andT. monococcum. The presence of three distinct glutenin components in some accessions of the species studied seems to be evidence for the existence of at least three active genes controlling the synthesis of the HMW-glutenin subunits in the A genome of wild and primitive domesticated diploid wheats. Results indicate also that HMW-glutenin subunits could represent useful markers for the evaluation of genetic variability present in different wild diploid wheat collections and subsequently for their conservation and future utilisation.     

CHy-banding patterns and chromatin organization inAegilops andTriticum species (Poaceae)

The distribution of CHy-banded heterochromatin was studied in the chromosomes ofAegilops longissima, Ae. speltoides, Triticum monococcum, andT. turgidum. Interphase nuclei were measured after Feulgen staining at different thresholds of optical density; the curves so obtained indicated the relationship among the species with respect to the different fractions of the genomic DNA. The karyological and cytophotometric analyses indicate differences betweenAe. speltoides andAe. longissima, the latter species being enriched in heterochromatin. Similar results were demonstrated for the genusTriticum, in whichT. turgidum showed more heterochromatin when compared withT. monococcum. The results suggest that the B genome of the cultivated wheats possesses a type of heterochromatin that resembles the type present inAe. longissima.     

NADP-dependent aromatic alcohol dehydrogenase in polyploid wheats and their diploid relatives. On the origin and phylogeny of polyploid wheats

Summary The three major isoenzymes of the NADP-dependent aromatic alcohol dehydrogenase (ADH-B), distinguished in polyploid wheats by means of polyacrylamide gel electrophoresis, are shown to be coded by homoeoalleles of the locus Adh-2 on short arms of chromosomes of the fifth homoeologous group. Essentially codominant expression of the Adh-2 homoeolleles of composite genomes was observed in young seedlings of hexaploid wheats (T. aestivum s.l.) and tetraploid wheats of the emmer group (T. turgidum s.l.), whereas only the isoenzyme characteristic of the A genome is present in the seedlings of the timopheevii-group tetraploids (T. timopheevii s.str. and T. araraticum).The slowest-moving B³ isoenzyme of polyploid wheats, coded by the homoeoallele of the B genome, is characteristic of the diploid species Aegilops speltoides S.l., including both its awned and awnless forms, but was not encountered in Ae. bicornis, Ae. sharonensis and Ae. longissima. The last two diploids, as well as Ae. tauschii, Ae. caudata, Triticum monococcum s.str., T. boeoticum s.l. (incl. T. thaoudar) and T. urartu all shared a common isoenzyme coinciding electrophoretically with the band B² controlled by the A and D genome homoeoalleles in polyploid wheats. Ae. bicomis is characterized by the slowest isoenzyme, B⁴, not found in wheats and in the other diploid Aegilops species studied.Two electrophoretic variants of ADH-B, B¹ and B², considered to be alloenzymes of the A genome homoeoallele, were observed in T. dicoccoides, T. dicoccon, T. turgidum. s.str. and T. spelta, whereas B² was characteristic of T. timopheevii s.l. and only B¹ was found in the remaining taxa of polyploid wheats. The isoenzyme B¹, not encountered among diploid species, is considered to be a mutational derivative which arose on the tetraploid level from its more ancestral form B² characteristic of diploid wheats.The implication of the ADH-B isoenzyme data to the problems of wheat phylogeny and gene evolution is discussed.     

Heterochromatin differentiation and phylogenetic relationship of the A genomes in diploid and polyploid wheats

Summary Heterochromatin differentiation, including band size, sites, and Giemsa staining intensity, was analyzed by the HKG (HCl-KOH-Giemsa) banding technique in the A genomes of 21 diploid (Triticum urartu, T. boeoticum and T. monococcum), 13 tetraploid (T. araraticum, T. timopheevi, T. dicoccoides and T. turgidum var. Dicoccon, Polonicum), and 7 cultivars of hexaploid (T. aestivum) wheats from different germplasm collections. Among wild and cultivated diploid taxa, heterochromatin was located mainly at centromeric regions, but the size and staining intensity were distinct and some accessions' genomes had interstitial and telomeric bands. Among wild and cultivated polyploid wheats, heterochromatin exhibited bifurcated differentiation. Heterochromatinization occurred in chromosomes 4A^t and 7A^t and in smaller amounts in 2A^t, 3A^t, 5A^t, and 6A^t within the genomes of the tetraploid Timopheevi group (T. araraticum, and T. timopheevi) and vice versa within those of the Emmer group (T. dicoccoides and T. turgidum). Similar divergence patterns occurred among chromosome 4A^a and 7A^a of cultivars of hexaploid wheat (T. aestivum). These dynamic processes could be related to geographic distribution and to natural and artifical selection. Comparison of the A genomes of diploid wheats with those of polyploid wheats shows that the A genomes in existing diploid wheats could not be the direct donors of those in polyploid wheats, but that the extant taxa of diploids and polyploids probably have a common origin and share a common A-genomelike ancestor.Contribution of the College of Agricultural Sciences, Texas Tech Univ. Journal No. T-4-233.     

Electrophoretic survey of seedling esterases in wheats in relation to their phylogeny

Summary Evolutionary and ontogenetic variation of six seedling esterases of independent genetic control is studied in polyploid wheats and their diploid relatives by means of polyacrylamide gel electrophoresis. Four of them are shown to be controlled by homoeoallelic genes in chromosomes of third, sixth and seventh homoeologous groups.The isoesterase electrophoretic data are considered supporting a monophyletic origin of both the primitive tetraploid and the primitive hexaploid wheat from which contemporary taxa of polyploid wheats have emerged polyphyletically and polytopically through recurrent introgressive hybridization and accumulation of mutations. Ancestral diploids belonging or closely related to Triticum boeoticum, T. urartu, Aegilops speltoides and Ae. tauschii ssp. strangulata are genetically the most suitable genome donors of polyploid wheats. Diploids of the Emarginata subsection of the section Sitopsis, Aegilops longissima s.str., Ae. sharonensis, Ae. searsii and Ae. bicornis, are unsuitable for the role of the wheat B genome donors, being all fixed for the esterase B and D electromorphs different from those of tetraploid wheats.     

Detection of 5S rDNA and other repeated DNA on supernumerary B chromosomes ofTriticum species (Poaceae)

The supernumerary B chromosomes ofTriticum speltoides andT. tripsacoides were analyzed in mitotic metaphases of spike primordium cells by C-banding and in situ hybridization (ISH) analyzes. B chromosomes ofT. speltoides have larger telomeric and interstitial C-bands, whereas those ofT. tripsacoides are almost completely devoid of C-bands. A prominent ISH site of rye related DNA sequences (using probe pSc119) was detected on B chromosomes ofT. tripsacoides and only a minor ISH site was observed on theT. speltoides B chromosomes. However, two ISH sites of 5S rRNA loci were detected at a terminal and an interstitial location of theT. speltoides B chromosomes. These sites were absent on B chromosomes ofT. tripsacoides. The results are discussed with respect to the phylogenetic origin of these B chromosomes.     

Transferable bread wheat EST-SSRs can be useful for phylogenetic studies among the Triticeae species

The genetic similarity between 150 accessions, representing 14 diploidand polyploid species of the Triticeae tribe, was investigated following the UPGMA clustering method. Seventy-three common wheat EST-derived SSR markers (EST-SSRs) that were demonstrated to be transferable across several wheat-related species were used. When diploid species only are concerned, all the accessions bearing the same genome were clustered together without ambiguity while the separation between the different sub-species of tetraploid as well as hexaploid wheats was less clear. Dendrograms reconstructed based on data of 16 EST-SSRs mapped on the A genome confirmed that Triticum aestivum and Triticum durum had closer relationships with Triticum urartu than with Triticum monococcum and Triticum boeoticum, supporting the evidence that T. urartu is the A-genome ancestor of polyploid wheats. Similarly, another tree reconstructed based on data of ten EST-SSRs mapped on the B genome showed that Aegilops speltoides had the closest relationship with T. aestivum and T. durum, suggesting that it was the main contributor of the B genome of polyploid wheats. All these results were expected and demonstrate thus that EST-SSR markers are powerful enough for phylogenetic analysis among the Triticeae tribe.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Conservation of rDNA inPolystichum (Dryopteridaceae)

Restriction site variation in the nuclear 18S–25S ribosomal RNA genes (rDNA) was analyzed hierarchically in a species complex in the fern genusPolystichum. Two distinct rDNA repeat types were present in all individuals ofPolystichum examined. No variation was detected among individuals within a population ofP. munitum, among populations ofP. munitum orP. imbricans, or among the six diploid species ofPolystichum from North America, including the circumborealP. lonchitis. The identity of rDNA repeats across all six North American species ofPolystichum may reflect an overall similarity of the nuclear genomes of these species, an observation supported by isozyme data as well. However, this nuclear similarity contrasts sharply with the highly divergent chloroplast genomes of these six species. The conservative nature of the rDNA inPolystichum also is in contrast to the much more variable rDNAs of most angiosperms investigated. Perhaps the tempo and mode of evolution of rDNA in ferns differ from those of angiosperms; however, the data base for fern rDNA is very small. Furthermore, the number of repeat types per individual is consistent with a diploid, rather than polyploid, condition despite the high chromosome number (n = 41) of these plants, although homogenization of multiple, divergent rRNA genes cannot be disproven.     

Polymorphism and inheritance of gliadin polypeptides in T. monococcum L.

Summary More than 80 different gliadin electrophoretic patterns (spectra) have been found in 109 accessions of the diploid wheat Triticum monococcum. Each pattern consists of 15–20 gliadin bands. Some patterns are clearly related and might arise from one another through single mutations in the gliadin-coding loci. From the analysis of 15 grains of each, only 61 accessions were found to be uniform; others consisted of two or more grain variants differing in their gliadin spectrum. An analysis of F₂ grains from three crosses between different accessions showed that groups (blocks) of components are jointly and codominantly inherited. Two independent major Gli loci were established. The close resemblance of the composition of some blocks of T. monococcum to some of those in polyploid wheats indicates that one locus in each T. monococcum genotype is located on chromosome 1A (Gli-A1) and the other on 6A (Gli-A2). However, the blocks of T. monococcum include more bands than corresponding (equivalent) blocks of polyploid wheats. Two out of 275 F₂ grains of the cross k-14244 x k-20409 were found to have gliadin spectra which can be explained as a result of intralocus recombination. Also, a second gliadin-coding locus on chromosome 1A was found in the cross k-46140 x k-46753. This locus recombines with the main Gli-A1 locus with a frequency of about 22% and was clearly analogous to the additional Gli locus found earlier on chromosome 1A of certain polyploid wheats.     

Atrazine-resistant cytoplasmic male-sterile-nigra broccoli obtained by protoplast fusion between cytoplasmic male-sterile Brassica oleracea and atrazine-resistant Brassica campestris

Summary By using restriction endonuclease digestion patterns, the degree of intraspecific polymorphism of mitochondrial DNA in four diploid species of wheat and Aegilops, Ae. speltoides, Ae. longissima, Ae. squarrosa, and Triticum monococcum, was assessed. The outbreeding Ae. speltoides was found to possess the highest degree of variability, the mean number of nucleotide substitutions among conspecific individuals being 0.027 substitutions per nucleotide site. A very low degree of mtDNA variation was detected among Ae. longissima accessions, with most of the enzyme-probe combinations exhibiting uniform hybridization patterns. The mean number of substitutions among Ae. longissima individuals was 0.001 substitutions per nucleotide site. The domesticated diploid wheat T. monococcum var. monococcum and its conspecific variant T. monococcum var. boeoticum seem to lack mitochondrial DNA variability altogether. Thus, the restriction fragment pattern can be used as a characteristic identifier of the T. monococcum cytoplasmic genome. Similarly, Ae. squarrosa accessions were found to be genetically uniform. A higher degree of variation among accessions is observed when noncoding sequences are used as probes then when adjacent coding regions are used. Thus, while noncoding regions may contain regulatory functions, they are subject to less stringent functional constraints than protein-coding regions. Intraspecific variation in mitochondrial DNA correlates perfectly with the nuclear variability detected by using protein electrophoretic characters. This correlation indicates that both types of variation are selectively neutral and are affected only by the effective population size.     

Comparison of the chromosomes of Triticum timopheevi with related wheats using the techniques of C-banding and in situ hybridization

Summary The chromosomes of the tetraploid wheats Triticum timopheevi (Genome AAGG) and T. araraticum (Genome AAGG) were C-banded at mitosis. The identity of the banded and unbanded chromosomes was then established by firstly making comparisons with the hexaploid species T. zhukovskyi which has the genome formula AAAAGG. Secondly, the meiotic pairing in F₁ hybrids between T. timopheevi and diploid wheats was examined by means of C-banding. The results showed that the banded chromosomes belonged to the G genome, while the unbanded chromosomes belonged to the A genome. Only one of the two pairs of satellited chromosomes had strong heterochromatic bands. The relationship between the genomes of T. timopheevi and T. dicoccum (Genome AABB) was then assessed at meiosis in hybrids between these species, using the techniques of C-banding and in situ hybridisation of a cloned ribosomal RNA gene probe. It was concluded that there were differences both in the amount and distribution of heterochromatin and also translocation differences between the species.     

The Phylogeny of Triticum L. and Aegilops L. Inferred from Comparative Analysis of Nucleotide Sequences in rDNA Promoter Regions

The process of accumulation of knowledge on wheat and related wild species during the 20th century is briefly reviewed with special reference to the evidence of the recent years on evolution of polyploid wheats and the role of diploid species. The latter serve as potential donors of the genomes, detection of which is particularly important because of the continuing speciation in the tribe Triticeae and artificial development of synthetic forms. The arguments in favor of the donor role for various diploid wheat and aegilops species from the section Sitopsis are compared. It is stated that in the formation of the both lines of polyploid wheats turgidum–aestivumand timopheevi,diploid Aegilops speltoides acted as a maternal form. In addition to cytoplasmic genomes, this aegilops species introduced into them also the B and G nuclear subgenomes. A comparison of nucleotide sequences in the variable part of the promoter of evolutionary conserved rRNA genes in polyploid wheats with their counterparts in diploid wheats and aegilops species confirmed the accepted wheat phylogenies.     

The molecular basis of genetic diversity among cytoplasms of Triticum and Aegilops

Summary Many related species and strains of common wheat were compared by matching differences among their mitochondrial genomes with their parent nuclear genomes. We examined three species of Aegilops, section Sitopsis (Ae. bicornis, Ae. sharonensis, and Ae. speltoides), emmer wheat (Triticum dicoccoides, T. dicoccum, and T. durum), common wheat (T. spelta, T. aestivum, and T. compaction), and timopheevi wheat (T. araraticum, T. timopheevi, and T. zhukovskyi). A single source of the cytoplasm was used in all the species, except Ae. speltoides (two sources), T. araraticum (two), and T. aestivum (three). Following restriction endonuclease analyses, the mitochondrial genomes were found to comprise seven types, and a dendrogram showing their genetic relatedness was constructed, based upon the percentage of common restriction fragments. MtDNAs from T. dicoccum, T. durum, T. aestivum, and T. compactum yielded identical restriction fragment patterns; these differed from T. dicoccoides and T. spelta mtDNAs in only 2.3% of their fragments. The fragment patterns of T. timopheevi and T. zhukovskyi were identical, and these differed from T. araraticum mtDNA by only one fragment. In both the emmer-dinkel and timopheevi groups, mitochondrial genome differentiation is evident, suggesting a diphyletic origin of each group. MtDNAs from four accessions of the Sitopsis species of Aegilops differ greatly from one another, but those of Ae. bicornis, Ae. sharonensis, and Ae. searsii, belonging to the same subsection Emarginata, are relatively similar. MtDNAs of timopheevi species are identical, or nearly so, to those of Ae. speltoides accession (09), suggesting that the latter was the cytoplasm donor to the former, polyploid group. The origin of this polyploid group seems to be rather recent in that the diploid and polyploid species possess nearly identical mitochondrial genomes. We cannot determine, with precision, the cytoplasm donor to the emmer-dinkel group. However, our results do suggest that mitochondrial DNAs show larger evolutionary divergence than do the ctDNAs from these same strains.Contribution no. 507 from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan     

Comparative study ofTriticum aestivum andT. timopheevi genomes using C-banding technique

The somatic chromosomes ofTriticum timopheevi and those of two varieties ofT. aestivum, Chinese Spring and Bezostaya-1, have been identified by a Giemsa staining technique. The data suggest thatT. timopheevi and tetraploid wheats had a common ancestor from which their genomes differentiated due to chromosomal aberrations and the increase of heterochromatin in the chromosomes of theT. timopheevi G-genome. The differences between the chromosomes of the AB and AG genomes result in substitutions and large translocations between these chromosomes in interspecific hybrids.     

New 18S·26S ribosomal RNA gene loci: chromosomal landmarks for the evolution of polyploid wheats

Three new 18S·26S rRNA gene loci were identified in common wheat by sequential N-banding and in situ hybridization (ISH) analysis. Locus Nor-A7 is located at the terminal area of the long arm of 5A in both diploid and polyploid wheats. Locus Nor-B6 is located in N-band 1BL2.5 of the long arm of chromosome 1B in Triticum turgidum and Triticum aestivum. ISH sites, similar to Nor-B6, were also detected on the long arms of chromosomes 1G in Triticum timopheevii and 1S in Aegilops speltoides, but their locations on the chromosomes were different from that of Nor-B6, indicating possible chromosome rearrangements in 1GL and 1BL during evolution. The third new locus, Nor-D8, was only found on the short arm of chromosome 3D in the common wheat Wichita. The loss of rRNA gene locus Nor-A3 and gain of repetitive DNA sequence pSc119 on the terminal part of 5AS suggest a structural modification of 5AS. Comparative studies of the location of the 18S·26S rRNA gene loci in polyploid wheats and putative A and B (G) genome progenitor species support the idea that: (1) Triticum monococcum subsp. urartu is the donor of both the A and A^t genome of polyploid wheats. (2) Ae. speltoides is closer to the B and G genome of polyploid wheats than Aegilops longissima and is the most probable progenitor of these two genomes.     

Additional evidence implicating Triticum searsii as the B-genome donor to wheat

In vitro DNA:DNA hybridizations and hydroxyapatite thermal-elution chromatography were employed to identify the diploid wheat species ancestral to the B genome of Triticum turgidum. ³H-T. turgidum DNA was hybridized to the unlabeled DNAs of T. urartu, T. speltoides, T. sharonensis, T. bicorne, T. longissimum, and T. searsii. ³H-Labeled DNAs of T. monococcum and a synthetic tetraploid AADD were hybridized with unlabeled DNAs of T. urartu and T. searsii to determine the relationship of the A genome of polyploid wheat and T. urartu. The heteroduplex thermal stabilities indicated that T. searsii was most closely related to the B genome of T. turgidum (AB) and that the genome of T. urartu and the A genome have a great deal of base-sequence homology. Thus, it appears that T. searsii is the B-genome donor to polyploid wheat or a major chromosome donor if the B genome is polyphyletic in origin.Published with the approval of the Director of The West Virginia Agricultural Experiment Station as Scientific Paper No. 1837.     

Molecular phylogeny of the genus Triticum L

The genus Triticum L. includes the major cereal crop, common or bread wheat (hexaploid Triticum aestivum L.), and other important cultivated species. Here, we conducted a phylogenetic analysis of all known wheat species and the closely related Aegilops species. This analysis was based on chloroplast matK gene comparison along with trnL intron sequences of some species. Polyploid wheat species are successfully divided only into two groups – Emmer (sections Dicoccoides and Triticum) and Timopheevii (section Timopheevii). Results reveal strictly maternal plastid inheritance of synthetic wheat amphiploids included in the study. A concordance of chloroplast origin with the definite nuclear genomes of polyploid species that were inherited at the last hybridization events was found. Our analysis suggests that there were two ancestral representatives of Aegilops speltoides Tausch that participated in the speciation of polyploid wheats with B and G genome in their genome composition. However, G genome species are younger in evolution than ones with B genome. B genome-specific PCR primers were developed for amplification of Acc-1 gene.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.