Mitochondrial DNA sequences of greenbug (Homoptera: Aphididae) biotypes

Sequence comparisons were made for 738-bp of mtDNA cloned from seven greenbug, Schizaphis graminum, biotypes (B, C, E, F, G, H and I) obtained from laboratory colonies maintained by USDA-ARS, Stillwater, OK. These sequences include parts of the genes for 16S ribosomal subunit (16S rRNA), tRNAleu, tRNAser, cytochrome b (cytb) and NADH dehydrogenase (ND) subunits one and four. Sequence data revealed considerable variation in 86 (12%) nucleotide sites over the 738-bp sequenced among the seven greenbug biotypes. Nucleotide invariance was observed within the seven greenbug biotypes from both the laboratory colonies and field collected biotype E greenbugs from Kansas, Nebraska, Oklahoma, and Texas.     

Using high-throughput amplified fragment length polymorphism to distinguish sorghum greenbug (Homoptera: Aphididae) biotypes

Abstract 1 The greenbug Schizaphis graminum (Rondani) is a serious pest of Sorghum bicolor L. and small grains in the Southern Plains of the U.S.A. Use of resistant cultivars, the major greenbug management strategy, has been challenged by the rapid development of new greenbug biotypes that overcome plant resistance. 2 We used a high‐throughput amplified fragment length polymorphism (AFLP) fingerprinting method to examine genetic divergence among eight greenbug biotypes (B, C, E, G, I and K, New York and South Carolina). Clustering analysis based on 1775 scored AFLP markers clearly showed that biotypes (C, E, I and K), which are able to infest sorghum fields, share more common polymorphisms among themselves than with other biotypes. 3 This result suggests that common genetic factors exist among these biotypes, enabling them to predominate and thrive in monoculture crops. Our study demonstrated the sensitivity of AFLP in obtaining large quantities of biotype‐associated polymorphic information across the entire greenbug genome.     

Efficacy of pyramiding greenbug (Homoptera: Aphididae) resistance genes in wheat

Durable resistance to greenbug, Schizaphis graminum (Rondani), in wheat is a goal of wheat improvement teams, and one that has been complicated by the regular occurrence of damaging biotypes. Simulation modeling studies suggest that pyramiding resistance genes, i.e., combining more than one resistance gene in a single cultivar or hybrid, may provide more durable resistance than sequential releases of single genes. We examined this theory by pyramiding resistance genes in wheat and testing a series of greenbug biotypes. Resistance genes Gb2, Gb3, and Gb6, and pyramided genes Gb2/Gb3, Gb2/Gb6, and Gb3/Gb6 were tested for effectiveness against biotypes E, F, G, H, and I. By comparing reactions of plants with pyramided genes to those with single resistance genes, we found that pyramiding provided no additional protection over that conferred by the single resistance genes. Based on the results of this test, we concluded that the sequential release of single resistance genes, combined with careful monitoring of greenbug population biotypes, is the most effective gene deployment strategy for greenbug resistance in wheat.     

Molecular analysis of sorghum resistance to the greenbug (Homoptera: Aphididae)

Chromosomal regions of sorghum, Sorghum bicolor (L.) Moench, conferring resistance to greenbug, Schizaphis graminum (Rondani), biotypes C, E, I, and K from four resistance sources were evaluated by restriction fragment-length polymorphism (RFLP) analysis. At least nine loci, dispersed on eight linkage groups, were implicated in affecting sorghum resistance to greenbug. The nine loci were named according to the genus of the host plant (Sorghum) and greenbug (Schizaphis graminum). Most resistance loci were additive or incompletely dominant. Several digenic interactions were identified, and in each case, these nonadditive interactions accounted for a greater portion of the resistance phenotype than did independently acting loci. One locus in three of the four sorghum crosses appeared responsible for a large portion of resistance to greenbug biotypes C and E. None of the loci identified were effective against all biotypes studied. Correspondingly, the RFLP results indicated resistance from disparate sorghums may be a consequence of allelic variation at particular loci. To prove this, it will be necessary to fine map and clone genes for resistance to greenbug from various sorghum sources.     

Characterization of greenbug (Homoptera: Aphididae) resistance in synthetic hexaploid wheats

Twelve greenbug (Schizaphis graminum (Rondani)) biotype E-resistant synthetic hexaploid wheats synthesized by crossing Triticum dicoccum Schrank. and Aegilops tauschii (Coss.) Schmal. were evaluated for the three known insect resistance categories, including antibiosis, anti-xenosis, and tolerance. Different methods were evaluated for calculating antibiosis and tolerance. Calculating intrinsic rate of population increase and measuring leaf chlorophyll content with a SPAD chlorophyll meter proved to be time- and labor-efficient for antibiosis and tolerance determination, respectively. The resistance in all synthetic hexaploids proved to be the result of a combination of antibiosis, antixenosis, and tolerance, which makes them valuable sources of greenbug resistance. To assist plant breeders in selecting the best germplasm for greenbug resistance, a plant resistance index was created that revealed differences among the synthetic hexaploid wheats.     

Plant resistance components of two greenbug (Homoptera: Aphididae) resistant wheats

Several biotypes of the greenbug, Schizaphis graminum (Rondani), attack winter wheat, Triticum aestivum L., on the Southern Plains every year. Two wheat germplasm sources of resistance ('Largo' and 'GRS 1201') have been developed that provide protection against the three predominant greenbug biotypes (E, I, and K). Each source has agronomic and end-use quality advantages and disadvantages for the breeder to consider in choosing a greenbug-resistant breeding line. We compared these two germplasms to determine their levels of resistance against biotype E. Components of resistance (i.e., antibiosis, antixenosis, and tolerance) were measured on seedlings of GRS 1201, Largo, and 'TAM W-101' (a susceptible control). Several aphid and plant measurements (e.g., total number of aphids produced per plant, aphid selection preferences, and plant damage ratings) were recorded for each plant entry. Select data recorded for each resistance component were normalized and combined to derive a plant resistance index for each wheat entry. Results indicated that GRS 1201 had a higher level of combined resistance components than did Largo, followed by TAM W-101, the susceptible control. These data provide additional information for the breeder to consider in selecting a greenbug-resistant breeding line.     

Categories of resistance to greenbug (Homoptera: Aphididae) biotype I in Aegilops tauschii germplasm

Categories of resistance to greenbug, Schizaphisgraminum (Rondani), biotype I, were determined in goatgrass, Aegilops tauschii (Coss.) Schmal., accession 1675 (resistant donor parent), 'Wichita' wheat, Triticum aestivum L., (susceptible parent), and an Ae. tauschii-derived resistant line, '97-85-3'. Antibiosis was assessed using the intrinsic rate of increase (rm) of greenbugs confined to each of the three genotypes. Neither parent nor the resistant progeny expressed antibiosis. Mean rm values for greenbug I on Wichita (0.0956), and Ae. tauschii (0.10543) were not significantly different. Mean rm values for Wichita and 97-85-3 were also not significantly different. Antixenosis was determined by allowing aphids a choice to feed on plants of each of the three genotypes. Ae. tauschii 1675 exhibited antixenosis, but this resistance was not inherited and expressed in '97-85-3'. In experiments comparing Wichita and Ae. tauschii 1675, greenbug I population distributions were not significantly different on Wichita at 24 h, but were shifted toward Wichita at 48 h. In the second antixenosis experiment, there were no significant differences in greenbug I population distributions on 97-85-3 or Wichita at 24 or 48 h. When all three lines were compared, there were no significant differences in greenbug biotype I populations at 24 or 48 h after infestation. Comparisons of proportional dry plant weight loss (DWT) and SPAD meter readings were used to determine tolerance to greenbug I feeding. Ae. tauschii 1675 and 97-85-3 were highly tolerant compared with Wichita. Infested and uninfested Ae. tauschii 1675 DWT was nonsignificant, and infested Wichita plants weighed significantly less than uninfested plants. When Wichita and 97-85-3 were contrasted, DWT of infested and uninfested Wichita plants were significantly different, but those of 97-85-3 were not. Mean percent leaf chlorophyll losses for the three genotypes, as measured by the SPAD chlorophyll meter, were as follows: Wichita = 65%; Ae. tauschii 1675 = 25%; and 97-85-3 = 39%. Percent leaf chlorophyll losses caused by greenbug feeding was significantly different in comparisons between Wichita and Ae. tauschii 1675, and comparisons between Wichita and 97-85-3, although feeding damage was not significantly different in comparisons between Ae. tauschii 1675 and 97-85-3. These data provided further evidence of the expression of tolerance to greenbug feeding in Ae. tauschii 1675 and 97-85-3.     

Molecular mapping of sorghum genes expressing tolerance to damage by greenbug (Homoptera: Aphididae)

Genetic linkage maps are fundamental for the localization of genes conferring tolerance to greenbug, Schizaphis graminum (Rondani), feeding damage in sorghum, Sorghum bicolor (L.) Moench. Thirteen linkage groups (LGs) containing 60 simple sequence repeat (SSR) loci were mapped by using a set of sorghum recombinant inbred lines (RILs) obtained from the cross '96-4121' (greenbug-tolerant parent) x Redlan (greenbug-susceptible parent). The LG spanned a distance of 603.5 cM, with the number of loci per LG varying from 2 to 14. Seventeen additional SSR loci were unlinked at a log of odds value of 3.0. Based on chlorophyll loss occurring after greenbug feeding, visual damage ratings, and soil plant analysis development (SPAD), chlorophyll-loss indices were recorded for each RIL and for the parents used in the cross. Composite-interval mapping identified three quantitative trait loci (QTLs) associated with biotype I and five QTLs associated with biotype K. The amount of phenotypic variation explained by these QTLs ranged from 9 to 19.6%. The identification of QTLs that influence greenbug tolerance will not only facilitate the use of marker-assisted selection in sorghum breeding programs but also will provide a solid foundation for detailed characterization of individual loci implicated in greenbug tolerance in sorghum.     

Differential colonization of wheat cultivars by two biotypes of Russian wheat aphid （Homoptera： Aphididae）

Susceptible and resistance wheat cultivars, Triticum aestivum L, were presented to two biotypes of Russian wheat aphid, Diuraphis noxia （Mordvilko）, in multiple choice tests to assay their relative acceptability as host plants. Both apterae （third and fourth instars） and alate adults were offered plants at the two-leaf stage in different cultivar combinations at 22±1℃ and 16：8 （L： D） hour photoperiod. Apterae were released from Petri dishes in the center of a circle of test plants, whereas alatae dispersed from a mature aphid colony to settle on plants arranged in rows. Both alatae and apterous nymphs of both biotypes readily colonized all cultivars tested：‘2137＇, ‘Akron＇,‘Ankor’,‘ Halt’ ,‘ Jagger’ ,‘ Prairie Red’ , ‘Stanton＇,‘TAM 107＇,‘TAM 110＇,‘Trego＇, ‘ Yuma＇, and ‘Yumar＇. Fewer biotype I apterae responded （settled and fed） in the combination containing more resistant （Dn4- and Dny-expressing） cultivars, compared to the combinations that had fewer. The reverse was true for biotype 2 apterae; more aphids responded in the combination containing the largest number of Dn4 expressing cultivars. Differential colonization of cultivars was observed in only one combination, in which biotype 2 apterae colonized Akron and Yumar in larger numbers than they did Stanton and Yuma. A separate experiment confirmed that, 48 hours after infestation, more biotype 2 apterae abandoned plants of Yuma than plants of Yumar. This differential response was likely due to genetic differences between the two ＇ near isogenic＇ lines that include the lack of Dn4 expression in Yuma. Choice tests with alatae did not result in differential rates of cultivar colonization by either biotype in any combination tested. These results suggest that young wheat plants appear to lack any meaningful antixenosis toward D. noxia, even though the aphids appear to perceive, and sometimes respond to, certain differences in cultivar suitability.     

Phylogeny of the genus Aphis Linnaeus, 1758 (Homoptera: Aphididae) inferred from mitochondrial DNA sequences

Aphis is the largest aphid genus in the world and contains several of the most injurious aphid pests. It is also the most reluctant aphid genus to any comprehensive taxonomic treatment: while most species are easily classified into "species groups" that form well defined entities, numerous species within these groups are difficult to tell apart morphologically and identification keys remain ambiguous and mostly rely on host plant affiliation. In this paper, we used partial sequences of COI/COII and CytB genes to reconstruct the first phylogeny of Aphis and discuss the present systematics. The monophyly of the subgenus Bursaphis and of the tree major species groups, Black aphid, Black backed aphid and frangulae-like species was recovered by all phylogenetic analyses. However our data suggested that the nominal subgenus was not monophyletic. Relationships between major species groups were often ambiguous but "Black" and "Black backed" species groups appeared as sister clades. The most striking result of this study was that our molecular data met the same limits as the morphological characters used in classifications: mitochondrial DNA did not allow the differentiation of species that are difficult to identify. Further, interspecies relationships within groups of species for which taxonomic treatment is difficult stayed unresolved. This suggests that species delineation in the genus Aphis is often ambiguous and that diversification might have been a rapid process.     

Phenotypic mechanisms of host resistance against greenbug (Homoptera: Aphididae) revealed by near isogenic lines of wheat

Interactions between biotype E greenbug, Schizaphis graminum (Rondani), and wheat, Triticum aestivum L., were investigated using resistant and susceptible near isogenic lines of the greenbug resistance gene Gb3. In an antixenosis test, the greenbugs preferred susceptible plants to resistant ones when free choice of hosts was allowed. Aphid feeding resulted in quick and severe damage to susceptible plants, which seemed to follow a general pattern spatially and was affected by the position where the greenbugs were initially placed. Symptom of damage in resistant plants resembled senescence. Within-plant distribution of aphids after infestation was clearly different between the two genotypes. Significantly more greenbugs fed on the first (oldest) leaf than on the stem in resistant plants, but this preference was reversed in the susceptible one. After reaching its peak, aphid population on the susceptible plants dropped quickly. All susceptible plants were dead in 10-14 d after infestation due to greenbug feeding. Aphid population dynamics on resistant plants exhibited a multipeak curve. After the first peak, the greenbug population declined slowly. More than 70% of resistant plants were killed 47 d after infestation. Performance of both biotype E and I greenbugs on several Gb3-related wheat germplasm lines were also examined. It seems that the preference-on-stem that was characteristic of biotype E greenbugs on the susceptible plants was aphid biotype- and host genotype-dependent. Results from this study suggested that antixenosis, antibiosis, and tolerance in the resistant plants of wheat might all contribute to resistance against greenbug feeding.     

Categories of resistance to greenbug (Homoptera: Aphididae) biotype K in wheat lines containing Aegilops tauschii genes

The wheat lines (cultivars) 'Largo', 'TAM110', 'KS89WGRC4', and 'KSU97-85-3' conferring resistance to greenbug, Schizaphis graminum (Rondani), biotypes E, I, and K were evaluated to determine the categories of resistance in each line to greenbug biotype K. Our results indicated that Largo, TAM110, KS89WGRC4, and KSU97-85-3 expressed both antibiosis and tolerance to biotype K. Largo, KS89WGRC4, and KSU97-85-3, which express antixenosis to biotype I, did not demonstrate antixenosis to biotype K. The results indicate that the same wheat lines may possess different categories of resistance to different greenbug biotypes. A new cage procedure for measuring greenbug intrinsic rate of increase (r(m)) was developed, by using both drinking straw and petri dish cages, to improve the efficiency and accuracy of r(m)-based antibiosis measurements.     

Life history study of multiple clones of insecticide resistant and susceptible greenbug Schizaphis graminum (Homoptera: Aphididae)

Comparative differences and similarities in prereproductive time (d), progeny production in a time equal to d (Md), and intrinsic rate of increase (rm) were established for one susceptible (S) and three resistant (R) strains of the greenbug, Schizaphis graminum (Rondani), reared on sorghum hybrids Dekalb G550E and Cargill 607E. The R strains showed three patterns of elevated esterase activity. Four R1 clones, four R2 clones, one R3 clone, and four S clones were evaluated. The interaction of sorghum hybrid and greenbug strain did not significantly influence any of the parameters measured. However, R1 greenbugs exhibited a significantly longer prereproductive period than the other strains. In addition, the R1 strain had a significantly slower intrinsic rate of increase than the R2 or S greenbug strains, but did not differ significantly from the R3 strain. These results suggest that R1 greenbugs may be less fit than the other strains studied.     

Response of resistant and susceptible barley to infestations of five Diuraphis noxia (Homoptera: Aphididae) biotypes

Since 2003, four new biotypes of the Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), RWA2-RWA5, have been discovered that have the ability to damage most of the wheat germplasm resistant to the original Russian wheat aphid population (RWA1). Barley germplasm lines with resistance to RWA1 have not yet been evaluated against the newest biotypes. Our study compared how biotypes RWA1-RWA5 affected the growth and leaf damage of RWA1-resistant germplasm (STARS 9301B, STARS 9577B), moderately resistant germplasm (MR-015), and susceptible varieties (Schuyler, Harrington, and Morex) under greenhouse conditions. Russian wheat aphid population levels also were determined 14 d after plant infestation. STARS 9301B exhibited strong resistance by showing only small differences in leaf damage and growth parameters from the feeding by the biotypes. STARS 9577B showed greater differences in damage by the Russian wheat aphid biotypes than STARS 9301B, yet, the ratings were still within the resistant category (e.g., chlorosis rating 2.3-4.9). Leaf chlorosis ratings for MR-015 ranged from 5.0 to 6.9 and fell within the moderately resistant to susceptible categories for all the biotypes. The greatest difference in leaf chlorosis occurred in Morex where RWA2 showed less virulence than the other biotypes. Feeding by the Russian wheat aphid biotypes produced only small differences in leaf rolling and plant growth within plant entries. Population levels of the Russian wheat aphid biotypes did not differ within barley entries (n = 610-971) at the termination of the study (14 d). From our research, we conclude that the new Russian wheat aphid biotypes pose no serious threat to the key sources of resistance in barley (STARS 9301B and 9577B).     

Fixed precision sequential sampling plans for the greenbug and bird cherry-oat aphid (Homoptera: Aphididae) in winter wheat

The numbers of greenbugs, Schizaphis graminum (Rondani), and bird cherry-oat aphids, Rhopalosiphum padi L., per wheat tiller (stem) were estimated in 189 production winter wheat (Triticum aestivum L.) fields located throughout Oklahoma. Taylor's power law regressions were calculated from these data and used to construct fixed precision sequential sampling schemes for each species. An evaluation data set was constructed from 240 samples taken during three growing seasons from winter wheat fields at four locations in Oklahoma. Wheat cultivar and growth stage were recorded for each field on the day of sampling. Taylor's power law parameters for evaluation fields differed significantly for both species among growing seasons, locations, and plant growth stages. Median precision achieved using the fixed precision sequential sampling schemes for each species departed <20% from expected precision over the range population intensity in the evaluation data. For the 10% of samples with greatest deviation between observed and expected precision, observed precision was 13.8-81.8% greater than that expected precision depending on aphid species and population intensity. For the greenbug, the distribution of the percentage deviation between observed and expected precision was positively skewed, so that the sampling scheme tended to over-predict precision. For the bird cherry-oat aphid, the distribution was more symmetric. Even though precision observed using the sampling schemes frequently varied from expected precision, because of the inevitable consequence of sampling error and environmental variation, the sampling schemes yielded median observed precision levels close to expected precision levels over a broad range of population intensity.     

Positive correlation of fitness with group size in two biotypes of Russian wheat aphid (Homoptera: Aphididae)

Changes in fitness parameters as a function of colony size (one versus 10 aphids) were measured in two biotypes (RWA1 and RWA2) of the Russian wheat aphid, Diuraphis noxia (Mordvilko) (Homoptera: Aphididae), feeding on three cultivars of wheat, Triticum aestivum L., at two temperatures. 'Trego' is a cultivar with no specific resistance to D. noxia, whereas, 'Stanton' and 'Halt' express Dny and Dn4 resistance sources, respectively. Feeding in a group accelerated the development of RWA1 on Trego and Stanton at 20 degrees C, but not at 24 degrees C, whereas grouped RWA2 developed faster than solitary RWA2 on all three cultivars at 24 degrees C, but not at 20 degrees C. Survival (first instar-adult) of RWA2 also was improved by grouping on Stanton and Halt at 24 degrees C, but solitary RWA2 survived better at 20 degrees C on all three cultivars. The reproductive rate of RWA1 was improved by grouping on Trego and Stanton at both temperatures, but only on Halt at 24 degrees C. Lifetime fecundity of RWA1 also was increased by grouping in all cases except for Trego at 20 degrees C. Grouped development increased the reproductive rate of RWA2 on all three cultivars at 24 degrees C, but had no effect at 20 degrees C. Grouped RWA2 developed and reproduced faster than grouped RWA1 on all three cultivars at 24 degrees C. Thus, the fitness of D. noxia was positively correlated with group size during colony establishment, but the effects were sensitive to temperature, being more pronounced at 20 degrees C for RWA1 and at 24 degrees C for RWA2.     

Purification and characterization of a carboxylesterase associated with organophosphate resistance in the greenbug,Schizaphis graminum (Homoptera: Aphididae)

The Type II esterase associated with organophosphate resistance in the greenbug, Schizaphis graminum, was purified by column chromatography and preparative electrophoresis resulting in over 100-fold purification and approximately 11% recovery. The native enzyme appears to exist as a heterodimer with the subunits equal to 52 and 56 kDa. The mass of the native enzyme was estimated at 102 kDa by gel filtration chromatography and the isoelectric focusing point was 4.8. The enzyme was inhibited by both paraoxon and mercuric chloride, suggesting that it is a serine hydrolase, although it was not inhibited by carbamate insecticides or eserine. The enzyme was active toward both β- and α-naphthol esters, although the length of the side chain (C-2 or C-4) also affected activity. The enzyme displayed no activity toward acetylthiocholine. N-Terminal amino acid sequence analysis of the enzyme subunits indicates that residues Val-4 and Gly-10 of the larger fragment were highly conserved among 11 other carboxylesterase sequences. Sequence data from the smaller fragment did not reveal any similarity with other esterase sequences. Arch. Insect Biochem. Physiol. 36:229–240, 1997. © 1997 Wiley-Liss, Inc.     

Host-plant resistance of sorghum: Differential hydrolysis of sorghum pectic substances by polysaccharases of greenbug biotypes (Schizaphis graminum,homoptera: Aphididae)

Pectic substances extracted from different varieties of sorghum are hydrolyzed at differing rates by unfractionated polysaccharases isolated from two biotypes (C, GBC; and E, GBE) of the sorghum pest, Schizaphis graminum (the greenbug). A higher degree of susceptibility of a sorghum variety is associated with a greater rate of hydrolysis of sorghum pectic substances by a greenbug biotype. Increases in the specific activity of polysaccharases on the pectic substances from a resistant sorghum variety are dependent on the duration that a biotype is maintained as a colony on that variety. Polysaccharase activity of GBE on arabinogalactan was significantly greater than GBC. However, there were no differences between the biotypes on the depolymerization of a variety of other plant matrix polysaccharides and a synthetic polysaccharide. The sequence of substrates of increasing refractoriness to hydrolysis are: arabinogalactan < microcrystalline cellulose < xylan < pectin < 2,3-diacetyl pectin < α-1,4-galacturonan. Pectic substances from sorghum varieties resistant to GBC but susceptible to GBE are relatively lower in arabinogalactan with elevated levels of uronic acid (UA) compared to varieties susceptible to both biotypes. A sorghum variety resistant to both GBC and GBE was lowest in levels of arabinogalactan, highest in UA, and highest in fructan content, which in the other varieties occurred only in trace amounts. Pectic composition of rhamnose, xylose, and glucose showed no relationship to resistance. Bound phenolics (potential inhibitors of enzyme activity) were not detected in any of the sorghum pectic substances. The relationship of plant matrix polysaccharides to host-plant aphid biotype compatibility is discussed.     

Bemisia tabaci (Homoptera: Aleyrodidae) biotypes in India

Host plant performance, esterase, and virus transmission tests revealed cassava-strain and sweetpotato-strain populations of whitefly Bemisia tabaci (Gennadius) biotypes in India. Individuals from the sweetpotato-reared population did not breed on cassava, Manihot esculenta Crantz, and the cassava-strain-reared individuals failed to develop on sweetpotato, Ipomoea batatus (L.) Lam. Eggplant, Solanum melongena L., and tobacco, Nicotiana tabacum L., were common hosts for both biotypes. The cassava-strain whiteflies but not the sweetpotato-reared whiteflies successfully transmitted cassava mosaic virus from disease-infected cassava seedlings to healthy cassava seedlings. Presence of biotypes in B. tabaci is reported for the first time from India.     

A ribosomal DNA probe to distinguish populations of Rhopalosiphum maidis (Homoptera: Aphididae)

Total DNA from clones of Rhopalosiphum maidis was digested with restriction endonucleases and hybridised with a heterospecific ribosomal DNA probe. DNA banding patterns exhibited sufficient differences to distinguish five different clones, and provided greater resolving power than gel electrophoresis of allozymes. The mechanisms and applications of this marking technique are discussed.