Alkaloid levels in Duncecap (Delphinium occidentale) and Tall larkspur (D. barbeyi) grown in reciprocal gardens: separating genetic from environmental influences

The objective of this study was to determine whether differences in toxicity between Tall larkspur (Delphinium barbeyi) and Duncecap larkspur (D. occidentale) were genetically inherent within the species, or due to environmental influences unique to the different regions where they grow. There was no difference in the concentration of the toxic alkaloids between the two species when grown in common gardens. However, both species had higher concentration of toxic alkaloids when grown in the southern Rocky Mountain region which is characterized by summer monsoonal thunderstorms, compared to summer drought in the north. In a unique occurrence where Duncecap larkspur grew beside Tall larkspur, toxic alkaloids were very low in Duncecap larkspur, but total alkaloid concentration was often higher than in Tall larkspur.     

Pollination, breeding system, and genetic structure in two sympatric Delphinium (Ranunculaceae) species

Two sympatric Delphinium species, D. barbeyi and D. nuttallianum, are ecologically and morphologically similar. However, D. barbeyi has multiple, large inflorescences while D. nuttallianum has a single, small inflorescence. These differences in floral display should result in greater intraplant pollen transfer in D. barbeyi, leading to higher rates of self-pollination through geitonogamy. Reduced gene flow by pollen should in turn produce greater population differentiation among populations of D. barbeyi relative to D. nuttallianum. We tested these predictions by comparing pollinator behavior, breeding systems, outcrossing rates, and population genetic structure of sympatric populations of the two species in Colorado. Bumble bee and hummingbird pollinators visit more flowers and inflorescences per foraging bout in D. barbeyi than in D. nuttallianum. The species differed in breeding system; D. barbeyi produced more seeds by autogamy (9 vs. 2%) than D. nuttallianum and suffered no reduction in seed set in hand-self vs. outcross pollinations, in contrast to a 41% decline in D. nuttallianum. The outcrossing rate in one D. barbeyi population was 55%, but ranged from 87 to 97% in four D. nuttallianum populations. Genetic differentiation among population subdivisions estimated by hierarchical F statistics was >10 times greater in D. barbeyi ( = 0.055-0.126) than D. nuttallianum ( = 0.004-0.009) at spatial scales ranging from metres to 3.5 km. Spatial autocorrelation analysis also indicated more pronounced local genetic structure in D. barbeyi than D. nuttallianum populations. Fixation indices (F(IS)) of D. barbeyi adults were much lower than expected based on mating system equilibrium and suggest that differences in the degree of self-compatibility and/or the timing of postpollination selection/inbreeding depression between the two species further contribute to the genetic differences between them.     

Taxonomic implications of diterpene alkaloids in three toxic tall larkspur species (Delphinium spp.)

The diterpene alkaloid content was used to assess the chemical taxonomic diversity in three larkspur species. Samples (n=163) were collected from 18 different locations in five western states, extracted and analyzed for diterpene alkaloids using electrospray mass spectrometry. The data were statistically analyzed using canonical discriminant analysis and analysis of variance. Delphinium glaucum samples were easily grouped and were significantly different from all other groups (P<0.005) for two compounds. Delphinium barbeyi and Delphinium occidentale were found to be distinct groups, but more closely related. Samples representing a putative hybrid between D. barbeyi and D. occidentale were found to be closely related to D. occidentale, but were significantly different from all other groups. These data support the classification of D. glaucum, D. barbeyi, and D. occidentale as distinct species and suggest that a possible hybrid is more similar to D. occidentale than D. barbeyi.     

Cyanotypic frequencies in adjacent and mixed populations of Trifolium occidentale Coombe and Trifolium repens L. are regulated by different mechanisms

The cyanogenic polymorphism in Trifolium repens is caused by the variation in two genes, the interaction of which produces four distinct cyanotypes. Along the Atlantic coasts of Bretagne, T. repens is sometimes found in populations mixed with the related species Trifolium occidentale, although the latter species usually occurs only in a narrow fringe along the coast, whereas T. repens is a more inland species. No plants of T. occidentale have ever been reported to have linamarase activity. Indeed, of 763 T. occidentale plants studied, none contained linamarase activity. However, the variation in the proportion of cyanotypes in T. repens was enormous, even between sites less than 2km apart. Our results confirm the presumption that T. repens and T. occidentale are indeed separate species. Both the fact that T. occidentale plants never contain linamarase activity, and the difference in proportion of plants with cyanoglucosides in mixed stands show that gene flow between the species must be rare. These dissimilar distributions strongly indicate that cyanotypic frequencies in adjacent and mixed populations of the very closely related species T. occidentale and T. repens are regulated by different mechanisms     

Relationships between heterozygosity and fitness in the Iberian threatened larkspur Delphinium bolosii (Ranunculaceae)

The relationship between individual heretozygosity and fitness was explored in the perennial larkspur Delphinium bolosii (Ranunculaceae), an endangered species endemic to Catalonia (North-Eastern Spain), as a part of several studies prior to designing a programme for the reintroduction of this species in a locality where it has been extinct for approximately one century. Allozyme electrophoresis was used to quantify the levels of heterozygosity at nearly to one hundred surveyed individuals in two extant populations. At the same time, eight parameters of vegetative and reproductive fitness were measured. A principal component analysis reduced the original fitness variables to three uncorrelated principal components, two associated with the maternal plant and the other one with the offspring. However, none of the components were significantly correlated with heterozygosity. The low number of variable allozyme markers and the likely influence of ecological factors could be responsible for the lack of correlation between individual heterozygosity and fitness.     

烟粉虱Q与B隐种mtCOI基因的遗传变异及其对利用CAPS标记进行隐种鉴别的影响

【目的】为了评估VspI,StyI和StuI为基础的线粒体细胞色素氧化酶I基因（mtCOI）酶切扩增多态性序列（cleaved amplified polymorphic sequence, CAPS)标记方法鉴别烟粉虱Q与B隐种的有效性。【方法】本研究对国内外烟粉虱种群的mtCOI基因进行了测序并鉴定了其隐种;在对464个Q隐种、98个B隐种mtCOI序列分析的基础上,利用VspI,StyI和StuI对Q与B隐种分别进行了CAPS标记验证。检索并比对了GenBank中烟粉虱Q与B隐种mtCOI序列中VspI,StyI和Stu I酶切位点分布情况。【结果】对国内外烟粉虱种群研究发现,以Vsp I为基础的CAPS标记方法能够有效鉴别实验中的Q与B隐种;利用StuI或StyI的CAPS标记方法无法有效鉴别Q与B隐种。对GenBank中烟粉虱Q与B隐种mtCOI序列比对发现,利用VspI,StyI和StuI为基础的CAPS标记方法不能有效鉴别Q与B隐种。【结论】以VspI,StyI和StuI为基础的CAPS标记方法鉴别Q与B隐种方面均有一定的局限性。     

Application of STS markers for leaf rust resistance genes in near-isogenic lines of spring wheat cv. Thatcher

Sequence tagged site (STS) markers for eight resistance genes against Puccinia recondita f. sp. tritici were used to screen a set of near-isogenic lines of wheat cv. Thatcher containing in total 40 different Lr genes and their alleles. Polymerase chain reaction (PCR) analysis was carried out by using STS, SCAR and CAPS primers specific for the leaf rust resistance genes Lr1, Lr9, Lr10, Lr19, Lr24, Lr28, Lr37 and Lr47. The STS, CAPS and SCAR markers linked to resistance genes Lr9, Lr10, Lr19, Lr24, Lr37 and Lr47 were found to be reliable in diverse genetic backgrounds. The amplification product of the Lr1 gene marker was detected in the susceptible cv. Thatcher and in all of the near-isogenic lines examined except Lr2a, Lr2b, Lr2c and Lr19. The sequence analysis of PCR products amplified in lines Lr1, Lr10, Lr28 and in cv. Thatcher indicated that the near-isogenic lines and cv. Thatcher contained in the targeted chromosome region an allele that differed from the original alleles corresponding to Lr1/6*Thatcher (TLR621) and susceptible Thatcher (TH621). The amplification product specific to the STS marker of the Lr1 gene was amplified in almost all Thatcher near-isogenic lines and in cv. Thatcher because their alleles possessed primer sequences identical to the original allele TLR621. The marker for the Lr28 resistance gene was identified in line Lr28, carrying gene Lr28, and in 21 other near-isogenic lines. The sequencing of PCR products specific to Lr28 and generated in lines Lr1, Lr10 and Lr28 indicated that the lines Lr1, Lr10 and Lr28 are heterozygous in this region.     

Genetic diversity and the genetic structure of natural populations of Chamaecyparis obtusa: implications for management and conservation

We investigated 25 natural populations of Chamaecyparis obtusa using 51 cleaved amplified polymorphic sequence (CAPS) markers, which were developed using information on sequence-tagged sites (STS) in Cryptomeria japonica. Most CAPS markers have codominant expression patterns, and are suitable for population studies because of their robustness and convenience. We estimated various genetic diversity parameters, including average heterozygosity (H(e)) and allelic richness and found that the more peripheral populations tended to have lower genetic diversity than central populations, in agreement with a previous theoretical study. The overall genetic differentiation between populations was low, but statistically significant (G(ST)=0.039), and similar to the level reported in a previous allozyme study. We attempted to detect non-neutral loci associated with local adaptation to clarify the relationship between the fixation index (F(ST)) and H(e) values for each locus and found seven candidates non-neutral loci. Phylogenetic tree analysis of the populations and Bayesian clustering analysis revealed a pattern of gradually increasing isolation of populations with increasing geographical distance. Three populations had a high degree of linkage disequilibrium, which we attribute to severe bottlenecks due to human disturbance or competition with other species during their migration from refugia after the most recent glaciation. We concluded that the small populations in western Japan and in Kanto district are more important, from a conservation perspective, than the populations in central Japan, due to their genetic divergence, relatively small sizes and restricted areas.     

Genetic diversity in the endangered dysploid larkspur Delphinium bolosii and its close diploid relatives in the series Fissa of the Western Mediterranean area

Allozyme diversity was evaluated in four closely-related taxa of the Delphinium series Fissa distributed throughout the Western Mediterranean area. All are considered threatened plants. Delphinium bolosii and Delphinium mansanetianum are narrowly endemic to the Eastern Iberian Peninsula, whereas Delphinium fissum ssp. sordidum is found in a few populations across the Peninsula. Delphinium fissum ssp. fissum is more widely distributed but often in small and isolated populations. In this group, Delphinium bolosii is dysploid (2 n  = 18) whereas the other taxa are diploid (2 n  = 16). A total of 12 populations were surveyed, including all known locations for D. bolosii , D. mansanetianum , and D. fissum ssp. sordidum . Eleven enzyme systems were assayed and 15 loci were resolved. Markedly depauperate values for genetic diversity were obtained for D. mansanetianum ( H _e = 0.013) and D. fissum ssp. sordidum ( H _e = 0.044). The estimates for D. fissum ssp. fissum ( H _e = 0.071) were below the values expected for widespread species. Small population size and marginal distribution have probably contributed to the low variability observed in this group. By contrast, D. bolosii exhibited comparatively larger populations and greater genetic diversity ( H _e = 0.138). We suggest that, apart from population size and local adaptation, genetic diversity during speciation may have been promoted by dysploidy through genomic recombination.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 773–784.     

Molecular genotyping of Trapa bispinosa and T. japonica (Trapaceae) based on nuclear AP2 and chloroplast DNA trnL-F region

?Premise of the study: Two marker systems were developed for the molecular identification of three Trapa species based on the length variation of nuclear AP2 and trnL-F chloroplast intergenic spacer region. ?Methods and Results: Our marker systems analyzed 118 individuals among 36 populations from three Trapa species. Trapa incisa was found to have a unique genotype from the other two species. Individuals of T. bispinosa were distinguished from T. japonica because the former had only a single AP2 genotype. Moreover, our results suggest a hybrid status of T. japonica in terms of two bands within an individual. One band was specific to T. japonica and the other one shared with T. bispinosa. ?Conclusions: Our marker system demonstrates that nucleotide sequence variations can serve as a fast, reliable, and reproducible tool for molecular genotyping and examining the natural hybrid of water chestnut species.     

Genetic relationships among Stylosanthes species as revealed by sequence-tagged site markers

 Nineteen sequence-tagged site (STS) primer pairs were designed on coding and non-coding regions in nine published Stylosanthes genes, which were mostly derived from cDNA. Direct sequencing of PCR products derived from genomic DNA allowed us to identify introns and to design specific primers flanking these introns. The use of 24 STS primer pairs for the detection of intra- and inter-specific variation in Stylosanthes based on size differences was tested on a core set of Stylosanthes species. Based on these results, 20 STS markers were selected to determine genetic relationships among 63 genotypes representing 24 Stylosanthes species. A total of 148 alleles were amplified and analyzed, resulting in a genetic similarity value ranging from 0.62 to 0.98 among the species. Based on cluster analysis, three main groups and three subgroups were determined, and most of the species were classified unambiguously. Alloploid species were recognized by the occurrence of more than one allele per STS marker, indicating fixed heterozygosity. Sixteen STS markers were useful for the identification of genotypes within a species. Inter-species relationships, as revealed by STS, were in general agreement with previous morphological and molecular relationship studies. These STS markers are useful as an additional tool for the identification of species, subspecies and genotypes in Stylosanthes, with a view to plant conservation and breeding. Received: 2 June 1998 / Accepted: 28 October 1998     

Molecular Mapping of a Gene for Fertility Restoration of Wild Abortive (WA) Cytoplasmic Male Sterility using a Basmati Rice Restorer Line

The inheritance and molecular mapping of a fertility restorer gene in basmati quality restorer line PRR-78 was carried out using an F₂ mapping population from the cross IR58025A X PRR-78 employing microsatellite markers. Dominant monogenic control of fertility restoration was observed in the F₂, and further confirmed by test cross data. Out of 44 sequence tagged microsatellite (STMS) markers used in the bulked segregant analysis (BSA), four differentiated the fertile bulk from the sterile bulk as well as the two parental lines from each other. One of these markers, RM258 located on chromosome 10, was found linked to the restorer gene at a distance of9.5 cM. Considering the RM258 location, additional STMS (RM171 and RM294A) and sequence tagged site (STS) primers derived from restriction fragment length polymorphic (RFLP) clones (G2155 and C1361) linked to fertility restorer gene(s) in other populations, were also used to find out a marker more tightly linked to the restorer gene. However, of these, RM171, RM294A and G2155 based primers amplified monomorphic fragments between parental lines and no amplification was observed with C1361. Cleaved amplified polymorphic sequence (CAPS) analysis of non-polymorphic STMS and STS markers and random amplified polymorphic DNA (RAPD) analysis using five random primers reportedly linked to restorer gene in other populations, also failed to differentiate the two parents. While, the marker RM258 is being used in the restorer breeding to identify putative restorer lines, search for additional tightly linked markers is underway.     

Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between Brassica napus and Crambe abyssinica.

Sexual progenies of asymmetric somatic hybrids between Brassica napus and Crambe abyssinica were analyzed with respect to chromosomal behavior, fae1 gene introgression, fertility, and fatty-acid composition of the seed. Among 24 progeny plants investigated, 11 plants had 38 chromosomes and were characterized by the occurrence of normal meiosis with 19 bivalents. The other 13 plants had more than 38 chromosomes, constituting a complete chromosomal set from B. napus plus different numbers of additional chromosomes from C. abyssinica. The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridization (GISH) in mitotic and meiotic cells. Furthermore, meiotic GISH enabled identification of intergenomic chromatin bridges and of asynchrony between the B. napus and C. abyssinca meiotic cycles. Lagging, bridging and late disjunction of univalents derived from C. abyssinica were observed. Analysis of cleaved amplified polymorphic sequence (CAPS) markers derived from the fae1 gene showed novel patterns different from the B. napus recipient in some hybrid offspring. Most of the progeny plants had a high pollen fertility and seed set, and some contained significantly greater amounts of seed erucic acid than the B. napus parent. This study demonstrates that a part of the C. abyssinica genome can be transferred into B. napus via asymmetric hybridization and maintained in sexual progenies of the hybrids. Furthermore, it confirms that UV irradiation improves the fertility of the hybrid and of its sexual progeny via chromosomal elimination and facilitates the introgression of exotic genetic material into crop species.     

Effects of local density on pollination and reproduction in Delphinium nuttallianum and Aconitum columbianum (Ranunculaceae)

Plant populations vary in density both naturally and as a consequence of anthropogenic impacts. Density in turn can influence pollination by animals. For example, plants in dense populations might enjoy more frequent visitation if pollinators forage most efficiently in such populations. We explored effects of plant density on pollination and seed set in the larkspur Delphinium nuttallianum and monkshood Aconitum columbianum. At our site in the Colorado Rocky Mountains, flowers of D. nuttallianum are pollinated primarily by queen bumble bees, solitary bees, and hummingbirds, whereas those of A. columbianum are pollinated primarily by queen and worker bumble bees. We found that the quantity of pollination service to both species (pollinator visitation rate and pollen deposition) was at best weakly related to density. In contrast, seed set declined by approximately one-third in sparse populations relative to nearby dense populations. This decline may stem from the receipt of low-quality pollen, for example, inbred pollen. Alternatively, sparsity may indicate poor environmental conditions that lower seed set for reasons unrelated to pollination. Our results demonstrate the value of simultaneously exploring pollinator behavior, pollen receipt, and seed set in attempting to understand how the population context influences plant reproductive success.     

Low genetic diversity and allozymic evidence for autopolyploidy in the tetraploid Pyrenean endemic larkspur Delphinium montanum (Ranunculaceae)

Allozyme electrophoresis was conducted to survey the levels and distribution of genetic diversity in the tetraploid perennial larkspur Delphinium montanum (Ranunculaceae), which is endemic to the eastern Pyrenees of Spain and France and considered to be endangered. Seven populations were sampled, resolving 14 putative enzymatic loci belonging to eight enzymes. Banding patterns stained in gels revealed several enzymatic duplications attributable to autotetraploidy, such as the presence of both balanced and unbalanced heterozygotes and the lack of fixed heterozygosity. However, variability in D. montanum ( P  = 23.8%, A  = 1.48, and H _e = 0.082) was lower than that expected for an autotetraploid species. This, in addition to the scarcity of loci showing three or four different alleles, could indicate that this species has suffered genetic erosion through population bottlenecks, or, alternatively, that it is undergoing diploidization.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 211–222.     

IRIS NELSONII (IRIDACEAE): ORIGIN AND GENETIC COMPOSITION OF A HOMOPLOID HYBRID SPECIES

Nuclear and chloroplast DNA variation was assayed for two populations of Louisiana irises (Bayou Teche and Young's Coulee) that demonstrated extreme morphological variation and for a sample of the putative hybrid species, Iris nelsonii. The genetic markers examined in this analysis were diagnostic for either Iris fulva. Iris hexagona, or Iris brevicaulis. These data demonstrated that the two morphologically variable populations were hybrid associations involving all three of these species and that all three of these species were involved in the origin of I. nelsonii. The distribution of genetic variation in I. nelsonii was significantly different from that present in either of the two hybrid populations. I. nelsonii demonstrated significantly fewer foreign markers than the two hybrid populations. This finding is in accord with the prediction that I. nelsonii is a hybrid species that has undergone stabilization with regard to genetic recombination and segregation. Although the genotypic makeup of I. nelsonii was significantly different from other parental and hybrid populations, individual plants from this species cannot be unequivocally differentiated from either I. fulva or certain hybrid genotypes. This reflects the paradoxical nature of genotypic variation in hybrid species. Thus, a hybrid species may include genotypes that overlap with both parental and contemporary hybrid populations. In the case of I. nelsonii it is necessary to utilize additional information (morphological, chromosomal, ecological) to identify plants belonging to this taxon. One hybrid population (Young's Coulee) is suggested as a paradigm for the progenitor population that gave rise to I. nelsonii.     

Seasonal variation in the secondary chemistry of foliar and reproductive tissues of Delphinium nuttallianum

Plant secondary compounds are critical in affecting interactions between plants and their herbivores. The norditerpene alkaloids are secondary compounds in Delphinium (larkspur) species which are divided into two classes: the N-(methylsuccinimido) anthranoyllycoctonine (MSAL-type) and non MSAL-type, and are known to be toxic to herbivorous insects and livestock. Alkaloid concentrations were measured in a whole plant context in vegetative and reproductive tissues in Delphinium nuttallianum at different stages of plant maturity at two locations to explore how plant maturity affected alkaloid concentrations within a growing season. Alkaloid concentrations differed between vegetative and reproductive tissues, with vegetative tissues having significantly lower alkaloid concentrations than reproductive tissues. However, no systematic differences in alkaloid concentrations were observed at different plant maturity stages across the growing season. Based on the data we suggest that alkaloid allocation in different plant parts of D. nuttallianum is influenced by life history of the plant, consistent with plant defense theory. At one location, as pods mature the qualitative alkaloid composition changed through structural diversification of the alkaloids present. The ecological significance of this structural diversification awaits further exploration.     

Natural hybridization in tropical spikerushes of Eleocharis subgenus Limnochloa (Cyperaceae): Evidence from morphology and DNA markers

? Premise of the study: Natural hybridization represents an important force driving plant evolution and affecting community structure and functioning. Hybridization may be overlooked, however, among morphologically highly uniform congeners. An excellent example of such a group is Eleocharis subgenus Limnochloa, which has no reliably proven hybrids. Does this reflect biological barriers to interspecific crosses or difficulties in detecting the hybrids? We tested the hypothesis that hybridization occurs among sympatric Eleocharis cellulosa, E. interstincta, and E. mutata in northern Belize, Central America. ? Methods: Morphometric study (407 plants) was followed by examination of inter-simple sequence repeat (ISSR) polymorphisms (44 plants) and ITS sequence variation (33 plants). ? Key results: Two putatively hybrid morphotypes were discerned-E. cellulosa-resembling and E. interstincta-resembling. DNA markers of E. cellulosa and E. interstincta displayed additive constitution in plants from one E. cellulosa-resembling population only. The other putatively hybrid populations contained ISSR and ITS markers of the species they resembled morphologically, several unique ISSR markers, and ITS sequences of an undescribed South American Limnochloa entity. DNA markers of E. mutata were absent in the putative hybrids. ? Conclusions: Simultaneous use of various types of molecular markers can overcome many pitfalls of investigations concerning hybridization among closely related and morphologically similar species. Northern Belize represents a hybrid zone of E. cellulosa and E. interstincta. A third participant in the hybridization events occurring in this zone is an unknown Limnochloa lineage but is not E. mutata. Interspecific hybridization may play a significant role in the diversification of Eleocharis.     

Identification and characterization of RAPD–SCAR markers linked to glyphosate-susceptible and -resistant biotypes of Eleusine indica (L.) Gaertn

Eleusine indica is one of the most common weed species found in agricultural land worldwide. Although herbicide-glyphosate provides good control of the weed, its frequent uses has led to abundant reported cases of resistance. Hence, the development of genetic markers for quick detection of glyphosate-resistance in E. indica population is imperative for the control and management of the weed. In this study, a total of 14 specific random amplified polymorphic DNA (RAPD) markers were identified and two of the markers, namely S4R727 and S26R₆976 were further sequence characterized. Sequence alignment revealed that marker S4R727 showing a 12-bp nucleotides deletion in resistant biotypes, while marker S26R₆976 contained a 167-bp nucleotides insertion in the resistant biotypes. Based on these sequence differences, three pairs of new sequence characterized amplified region (SCAR) primers were developed. The specificity of these primer pairs were further validated with genomic DNA extracted from ten individual plants of one glyphosate-susceptible and five glyphosate-resistant (R2, R4, R6, R8 and R11) populations. The resulting RAPD–SCAR markers provided the basis for assessing genetic diversity between glyphosate-susceptible and -resistant E. indica biotypes, as well for the identification of genetic locus link to glyphosate-resistance event in the species.     

Relationships among 3 Kochia species based on PCR-generated molecular sequences and molecular cytogenetics.

Forage kochia (Kochia prostrata ssp. virescens 'Immigrant' is native to the arid and semiarid regions of central Eurasia. It was introduced into the United States in 1966 as PI 314929 and released as a perennial forage shrub in 1984. Kochia americana is a perennial native to the United States, whereas Kochia scorparia is an introduced annual species that became a weed. To assess both the breeding potential and the possibility of genetic contamination, relationships among the 3 Kochia species were analyzed using random amplified polymorphic DNA (RAPD) markers, sequence tagged site (STS) marker sequences of the chloroplast NADH dehydrogenase gene (ndhF), genomic in situ hybridization (GISH), and multicolor fluorescence in situ hybridization (MC-FISH). Seventy decamer random primers yielded 458 polymorphic bands from 9 plants of K. americana, 20 plants of K. prostrata, and 7 plants of K. scoparia. Fifty-four and 55 species-specific RAPD markers were identified for K. americana and K. prostrata, whereas 80 RAPD markers were specific to K. scoparia. Based on the presence or absence of informative RAPD markers, the 3 species always grouped into 3 distinct clusters in a NTSYSpc2.01b-generated dendrogram. The same relationships were found among the 3 Kochia species based on ndhF DNA sequence divergence. Using a set of 7 STS markers that can identify each Kochia species, we did not find a single interspecific hybrid from artificial hybridizations among the 3 Kochia species. In GISH studies, chromosomes of 1 species fluoresced in green only when they were probed by genomic DNA of the same species. Cross-hybridization by genomic DNA of another species was not observed. In FISH studies using pTa71 (for 18S-5.8S-26S rDNAs) and pScT7 (for 5S rDNA) as probes, there were 1, 1 and 3 pTa71 sites and 2, 1, and 1 pScT7 sites in each haplome of K. prostrata, K. americana, and K. scoparia, respectively. It is concluded that these 3 Kochia species are so genomically distinct that gene introgression among them would be extremely rare.