Wheat phylogeny determined by RFLP analysis of nuclear DNA. 2. Wild tetraploid wheats

Intra- and inter-specific variations in the nuclear DNA of Triticum dicoccoides Körn. (2n = 28, genome constitution AABB) and T. araraticum Jakubz. (2n = 28, AAGG), wild species, respectively, of the Emmer and Timopheevi group, were studied by restriction fragment length polymorphism (RFLP) analysis. Total DNAs of 32 T. dicoccoides and 24 T. araraticum accessions, collected from throughout the distribution areas of these species, were treated with two 6-bp cutters and hybridized with 30 nuclear DNA clones as probes to detect RFLPs. A total of 167 hybrid bands were observed per accession. All the enzyme-probe combinations showed RFLPs between accessions. The average genetic distance between the T. dicoccoides accessions was 0.0135 ± 0.0031 and that between the T. araraticum accessions 0.0036 ± 0.0015, indicative of about a four-fold intraspecific variation in T. dicoccoides as compared to T. araraticum in terms of genetic distance. No significant genetic differentiation was found for the geographical populations of these species, the genetic distance between the two species being 0.0482 ± 0.0022. The interspecific divergence corrected for intraspecific divergence was 0.0395, about three times that for T. dicoccoides and 11 times that for T. araraticum. The results show that in the wild state the Emmer and Timopheevi groups are clearly differentiated and that T. dicoccoides has much greater variation than T. araraticum, suggesting a relatively recent origin for the latter and therefore a diphyletic origin for these species.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 535     

Mitochondrial DNA diversity in the genera of Triticum and Aegilops revealed by southern blot hybridization

Summary Southern blot hybridization of total DNA to defined mitochondrial DNA sequences provides a sensitive assay for mtDNA variation in the genera of Triticum and Aegilops. A clear distinction between cytoplasms of tetraploid species sharing the AG haploid genome is reported for the first time. The Sitopsis section of the genus Aegilops showed the most extensive intra- and inter-specific variation, whereas no variation could be detected among the cytoplasms of polyploid Triticum species (wheats) sharing the AB haploid genome. Extensive cytoplasmic intraspecific diversity was revealed in Ae. speltoides.     

Intra- and interspecific variations in vacuolar flavonoids among Ficus species from the Budongo Forest,Uganda

Leaves of 14 species of Ficus growing in the Budongo Forest, Uganda, were analysed for vacuolar flavonoids. Three to six accessions were studied for each species to see whether there was intraspecific chemical variation. Thirty-nine phenolic compounds were identified or characterised, including 14 flavonol O-glycosides, six flavone O-glycosides and 15 flavone C-glycosides. In some species the flavonoid glycosides were acylated. Ficus thonningii contained in addition four stilbenes including glycosides. Most of the species could be distinguished from each other on the basis of their flavonoid profiles, apart from Ficus sansibarica and Ficus saussureana, which showed a very strong intraspecific variation. However, on the whole flavonoid profiles were sufficiently distinct to help in future identifications.     

Interspecific and intraspecific variations in sibling species of sea urchin Echinometra

Interspecific and intraspecific morphological and fertilization variations were studied in three sibling species of Echinometra (known as sp. B, C and D) found off the coast of Okinawa, Japan. Eggs from C and D were readily fertilized by sperm from all three color morphs of B when the sperm concentrations were high, but no fertilization was observed when sperm of the former were mixed with eggs from the latter. Under limiting sperm concentrations, however, both C and D were incapable to fertilize reciprocally with B. In contrast, crossing between C and D produced fertilization membrane at high and limiting sperm concentrations in both directions. Interspecific crosses between B vs. C and B vs. D clearly showed that these combinations were reproductively isolated in contrast to that observed for crossing between C and D. Interestingly, intraspecific fertilization of B showed considerable morphological variation in addition to variability in fertilization success. Intraspecific fertilization and morphological variations may occur due to a number of genetic and/or non-genetic factors. While the underlying cause(s) remain to be elucidated, the results of the present study suggest that B is now speciating very slowly.     

Copy numbers of chloroplast and nuclear genomes are proportional in mature mesophyll cells of Triticum and Aegilops species

The possibility of estimating the proportion of chloroplast DNA (ctDNA) and nuclear DNA (nDNA) in nucleic-acid extracts by selective digestion with the methylation-sensitive restriction enzyme PstI, was tested using leaf extracts from Spinacia oleracea and Triticum aestivum. Values of ctDNA as percentage nDNA were estimated to be 14.58%±0.56 (SE) in S. oleracea leaves and 4.97%±0.36 (SE) in T. aestivum leaves. These estimates agree well with those already reported for the same type of leaf material. Selective digestion and quantitative dot-blot hybridisation were used to determine ctDNA as percentage nDNA in expanded leaf tissue from species of Triticum and Aegilops representing three levels of nuclear ploidy and six types of cytoplasm. No significant differences in leaf ctDNA content were detected: in the diploids the leaf ctDNA percentage ranged between 3.8% and 5.1%, and in the polyploids between 3.5% and 4.9%. Consequently, nuclear ploidy and nDNA amount were proportional to ctDNA amount (r₍₁₉₎=0.935, P>0.01) and hence to ctDNA copy number in the mature mesophyll cells of these species. There was a slight increase in ctDNA copy numbers per chloroplast at higher ploidy levels. The balance between numbers of nuclear and chloroplast genomes is discussed in relation to polyploidisation and to the nuclear control of ctDNA replication.Abbreviations ctDNA chloroplast DNA - nDNA nuclear DNA - RuBPCase ribulose-1,5-bisphosphate carboxylase - DAPI 4,6-diamidine-2-phenylindole     

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.     

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     

RFLP analysis of the wild potato species,Solanum acaule Bitter (Solanum sect. Petota)

Summary Intraspecific variation of a wild potato species, Solanum acaule Bitt., was analyzed by RFLPs of genomic DNA. One hundred and five accessions were selected throughout the distribution area, including all subspecies, i.e., ssp. albicans (hexaploid), ssp. punae (tetraploid), ssp. acaule (tetraploid) and ssp. aemulans (tetraploid). Twenty-seven low-copy DNA clones (probes) were Southern hybridized with EcoRI, EcoRV, HindIII, and XbaI digests of total DNA of all accessions. In total, 238 RFLPs were detected from 94 enzyme x probe combinations. Among them, 49 RFLPs were specific to ssp. albicans, suggesting that the additional third genome is distinct from its two other genomes. RFLPs between and within subspecies were analyzed by principal component analysis. DNA similarities between subspecies coincided with a former taxonomic treatment in the sense that ssp. albicans is the most distantly related to ssp. acaule and ssp. aemulans is distantly related. Subspecies acaule and ssp. punae were indistinguishable. In addition, RFLPs could be used to distinguish groups within subspecies. Subspecies aemulans, confined to Argentina, was divided into two populations, one from the province of La Rioja and the other from the province of Jujuy. In ssp. acaule, some accessions from the southernmost distribution area were clearly distinguishable, while the others varied continuously, showing a geographical cline from Peru to Argentina.Reference to a specific brand or firm name does not constitute endorsement by the US Department of Agriculture over others of similar nature not mentioned     

Analysis of metaphase I chromosome association in species of the genus Aegilops

Summary Metaphase-I chromosome associations in every diploid and polyploid species of the genus Aegilops were studied using C-banding in order to analyse the cytogenetic behaviour of the whole complement as well as of specific genomes in different polyploid species. Differences were observed in the frequency of associations per cell among different species of the same ploidic level and even between species sharing the same genomic constitution. Differences were also found between different genomes within the same polyploid species and between the same genome when present in several diploid and polyploid species. Several factors proposed as having an influence on the frequency of metaphase-I associations, such as chromosome morphology, C-heterochromatin content, genetic control and genome interactions, are discussed. Most of the polyploid Aegilops species showed a diploid-like behaviour at metaphase I although multivalents involving homoeologous associations were occasionally observed in Ae. biuncialis, Ae. juvenalis and Ae. crassa(6x); therefore, the Aegilops diploidising genetic system is not equally effective in all polyploid species.     

Nuclear DNA changes within Helianthus annuus L.: variations in the amount and methylation of repetitive DNA within homozygous progenies

Complex alterations in the redundancy and methylation of repeated DNA sequences were shown to differentiate the nuclear genome of individuals belonging to single progenies of homozygous plants of the sunflower. DNA was extracted from seedlings obtained from seeds collected at the periphery of flowering heads (P DNA) or from seedlings obtained from seeds collected in their middle (M DNA). Three fractions of repeated sequences were isolated from genomic DNA: a highly repetitive fraction (HR), which reassociates within an equivalent Cot of about 2 × 10^-1, and two medium repetitive fractions (MR1 and MR2) having Cot ranges of about 2 × 10^-1-2 and 2-10², respectively. Denaturation kinetics allowed different sequence families to be recognized within each fraction of repetitive DNA, and showed significant differences in sequence redundancy to occur between P and M DNA, particularly as far as the MR2 fraction is concerned. Most DNA sequence families are more represented in P DNA than in M DNA. However, the redundancy of certain sequences is greater in the latter than in the former. Each repetitive DNA fraction was hybridized to Southern blots of genomic P or M DNA which was digested to completion by three pairs of isoschizomeric restriction endonucleases which are either insensitive or sensitive to the methylation of a cytosine in the recognition site. The results obtained showed that the repetitive DNA of H. annuus is highly methylated. Clear-cut differences in the degree of methylation of P and M DNA were found, and these differences were particularly apparent in the MR2 fraction. It is suggested that alterations in the redundancy of given DNA sequences and changes in their methylation patterns are complementary ways to produce continuous genotypic variability within the species which can be exploited in environmental adaptation.Research supported by National Research Council of Italy, Special Project RAISA, Sub-project No. 2     

Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla

The ribosomal DNA repeat units of two closely related species of the genus Fraxinus, F. excelsior and F. oxyphylla, were characterized. The physical maps were constructed from DNA digested with BamHI, EcoRI, EcoRV and SacI, and hybridized with three heterologous probes. The presence or the absence of an EcoRV restriction site in the 18s RNA gene characterizes two ribosomal DNA unit types found in both species and which coexist in all individuals. A third unit type appeared unique to all individuals of F. oxyphylla. It carries an EcoRI site in the intergenic spacer. Each type of unit displayed length variations. The rDNA unit length of F. excelsior and F. oxyphylla was determined with EcoRV restriction. It varied between 11kb and 14.5kb in F. excelsior and between 11.8kb to 13.8kb in F. oxyphylla. Using SacI restriction, at least ten spacer length variants were observed in F. excelsior, for which a detailed analysis was conducted. Each individual carries 2–4 length variants which vary by a 0.3-kb step multiple. This length variation was assigned to the intergenic spacer. By using the entire rDNA unit of flax as probe in combination with EcoRI restriction, each species can be unambiguously discriminated. The species-specific banding pattern was used to compare trees from a zone of sympatry between the two species. In some cases, a conflicting classification was obtained from morphological analysis and the use of the species-specific rDNA polymorphism. Implications for the genetic management of both species are discussed.     

Grain protein variability among species of Triticum and Aegilops: quantitative SDS-PAGE studies

Summary Total proteins were extracted from degermed seeds of various species of Triticum and Aegilops with solutions containing sodium dodecyl sulfate (SDS) and mercaptoethanol. The reduced, dissociated proteins were fractionated according to molecular weight (MW) by high-resolution polyacrylamide gel electrophoresis in buffers containing SDS (SDS-PAGE). Stained SDS-PAGE patterns were measured by densitometric scanning over a suitable range of optical density. The data were normalized to equivalent total areas for each of the densitometric scans by means of a computer program that also permitted the construction of patterns of hypothetical amphiploids by averaging patterns of two or three diploid species. The grain proteins of most species examined had distinctive qualitative and quantitative aspects that were characteristic of the species even though nearly every accession or cultivar of a species exhibited at least minor differences in pattern from other accessions or cultivars. The main protein components (probably prolamins) of Triticum monococcum ssp. monococcum, T. monococcum ssp. boeoticum, T. urartu, and Aegilops squarrosa had MW's in the range 29–36 X 10³ whereas the most important components of Ae. speltoides, Ae. longissima, and Ae. searsii had MW's in the range 37–55 × 10³. Changes in the quantitative expression of particular genes, especially those coding for storage protein components, may have been associated with speciation. The strong predominance of proteins with MW's in the range 29–36 × 10³ in some accessions of AB genome tetraploids, such as T. turgidum ssp. dicoccoides, may indicate contributions to the B genome of these tetraploids by T. monococcum ssp. boeoticum, T. urartu, or Ae. squarrosa.     

Ecological distribution and species diversity of Aegilops L. genus in Bulgaria

The genus Aegilops has an important potential utilization in wheat improvement because of its resistance to different biotic and abiotic stresses and close relation with the cultivated wheat. Therefore, a better knowledge of the eco-geographical distribution of Aegilops species and their collection and conservation are required. A total of 297 Aegilops accessions representing nine (five tetraploid and four diploid) species were collected in different regions of Bulgaria, and the ecological characteristics of the 154 explored sites were recorded. The distribution of the diploid species (Ae. caudata L., Ae. speltoides Tausch, Ae. umbellulata Zhuk. and Ae. comosa Sibth. and Sm.) was limited to specific environments in south-central Bulgaria. Tetraploid species were present in harsher environments than diploid species and showed wider adaptation and distribution. Species–environment relationships were analysed by considering the worldwide distribution of the species and their physiological resistance to abiotic stress. Aegilops cylindrica Host was more frequently found in northern Bulgaria and at high altitudes. Its distribution was closely related to its tolerance to low temperatures. Aegilops geniculata Roth and Ae. neglecta Req. ex Bertol. were absent in the north of Bulgaria, but widely distributed in low rainfall areas. Aegilops neglecta, more frost resistant than Ae. geniculata, was present at higher altitude. Aegilops biuncialis Vis. and Ae. triuncialis L. showed adaptation to a wide range of climatic conditions. The study of Aegilops species ecology and distribution in Bulgaria provided useful information for the future collection and for the genetic resource management in this region.     

Chloroplast DNA variation inDesmodium subgenusPodocarpium (Leguminosae): Infrageneric phylogeny and infraspecific variations

Chloroplast DNA (cpDNA) restriction site variation was examined in five species ofDesmodium subgenusPodocarpium (Leguminosae; Papilionoideae; Desmodieae). Twenty four phylogenetically informative cpDNA mutations were scored. The cladistic analysis of characters based on the 24 mutations resulted in the most parsimonious tree which supports the monophyly of the subgenus.Desmodium elegans of subgenusDollinera was the sister group of subgenusPodocarpium in this tree. The groupings obtained from the cpDNA characters were consistent with the present infrageneric classification system for the subgenus except for the infraspecific taxa ofD. podocarpum. Three groups withinD. podocarpum, which were incongruent with the infraspecific classification of the species, were distinguished by a total of four site mutations. The first group consisted of subsp.podocarpum, subsp.fallax, and subsp.oxyphyllum var.oxyphyllum; the second subsp.oxyphyllum var.oxyphyllum; and the last subsp.oxyphyllum var.oxyphyllum and var.mandshuricum.     

Origin and differentiation of Aegilops triuncialis L. as determined by esterase isozyme analysis

Summary Banding patterns of esterase isozymes in Aegilops triuncialis (2n = 28, genome formula C^uC^uCC) and its putative parental species, Ae. umbellulata (2n = 14, C^uC^u) and Ae. caudata (2n = 14, CC), were studied by the gel isoelectric focusing method using pH 6–8 carrier ampholite. Zymogram phenotypes of both parents were quite uniform. Seven zymogram phenotypes (designated as phenotypes 1 to 7) were found among the 260 strains of Ae. triuncialis examined. Of these phenotypes, phenotype 1 was identical to the zymogram phenotype produced by the ancestral species, Ae. umbellulata, and bands considered to have been derived from Ae. caudata were absent in this phenotype. Phenotype 3 had all bands of both parents. The other phenotypes differed greatly from phenotype 3. Therefore, phenotype 3 was considered to be most primitive of the 7 types, and the Ae. triuncialis strains which showed phenotype 3 to be the most primitive of the strains examined. If Ae. triuncialis originated as a hybrid between Ae. umbellulata and Ae. caudata, the zymogram phenotype must have been phenotype 3, in which the isozymes of both parental species are present. Whether the phenotypes other than type 3 were due to introgressive hybridization could not be verified, but they were considered in this article to be a consequence of a rearrangement of chromosomes.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University No. 432     

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.     

Variability in nuclear DNA content within pigeonpea,Cajanus cajan (Fabaceae)

4C DNA values have been estimated in 16 cultivars ofCajanus cajan by cytophotometry. The values range between 6.19 pg to 7.97 pg, a 23.6% variation. The cultivars form four groups which differ significantly from each other but have insignificant difference within them. The implications of this variability with respect to the heterogeneity and origin of this legume crop are discussed.     

Genetic analysis of stunted growth by nuclear-cytoplasmic interaction in interspecific hybrids of Capsicum by using RAPD markers

When eight cultivars of Capsicum annuum were used as female parents in interspecific crosses with two accessions of C. chinense, dwarfism occurred in hybrids originating from 10 out of 16 combinations, while hybrids of the remaining 6 combinations grew normally. In contrast, when C. chinense was used as female parent, all of the hybrids showed severely stunted growth as if affected by a virus. These results suggested that the stunted growth expressed in the cross of C. chinense x C. annuum is caused by an interaction between nuclear gene(s) from C. annuum and the cytoplasm of C. chinense. To examine the number of nuclear gene(s) which cause(s) the stunted growth, we backcrossed F₁ hybrids of C. annuum x C. chinense to C. chinense. About one-quarter of the progeny in the backcrossed hybrids of C. chinense x (C. annuum x C. chinense) showed the same stunted growth shown by the f₁ hybrids of C. chinense x C. annuum, suggesting that two complementary genes of C. annuum cause the stunted growth. However, the higher abortion rates of ovules and lower germination percentage of seeds in C. chinense x C. annuum than in the selfed C. chinense implied that the genetic ratio of the stunted type would have been higher than that observed in the C. chinense x (C. annuum x C. chinense) progeny. We then attempted a linkage analysis between the stunted growth and randomly amplified polymorphic DNA (RAPD) of C. chinense x (C. annuum x C. chinense) progeny. A RAPD marker that associated with 94% of the stunted plants but not with 94% of the normal one was identified. This confirmed that a single nuclear gene of C. annuum which is linked to the RAPD marker with a recombination value of 6% causes the stunted growth in an interaction with the cytoplasm of C. chinense.     

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.