Tertiary Gene Pool Research Articles

Status of the Global Cotton Germplasm Resources

The cultivated Gossypium spp. (cotton) represents the single most important, natural fiber crop in the world. In addition to its fiber, the oil and protein portion of the cottonseed also represents significant economic value. To protect the worldwide economic value of cotton fiber and cotton byproducts, coordinated efforts to collect and maintain cotton genetic resources have increased over the last 100 yr. The classified genetic resources of cotton are extensive and include five tetraploid species in the primary gene pool, 20 diploid species in the secondary gene pool, and 25 diploid species in the tertiary gene pool. This report provides information on the status and contents of eight major cotton germplasm collections present across the world. Based on the findings of this report, a number of classified Gossypium species are not maintained in these collections, and several are underrepresented and vulnerable to extinction. This report presents several critical challenges and opportunities facing international efforts to enhance and preserve the world's Gossypium genetic resources. Multinational communication and collaboration are essential to protect, secure, and evaluate the global cotton germplasm resources. Without global, collaborative efforts, the rarest and most unique cotton germplasm resources are vulnerable to extinction.

The species of the genus Oryza and transfer of useful genes from wild species into cultivated rice, O. sativa

The genus Oryza has 24 species out of which two are cultivated (O. sativa and O. glaberrima) and 22 are wild species. Of the 22 wild species, six are in the primary gene pool of O. sativa complex and these wild species are easily crossable with the major cultivated species. These have the same AA genome as O. sativa. However, there are 10 wild species under O. officinalis complex having BB, CC, BBCC, CCDD, EE and FF genomes. The wild species of this complex are in the secondary gene pool and are cross incompatible with O. sativa. There are six most distantly related wild species with either diploids or tetraploids of GG, HHJJ and HHKK genomes and are highly cross incompatible with O. sativa. All the 22 wild species of Oryza are a vast reservoir of genes for biotic and abiotic stresses resistance. Some of the yield enhancing traits/genes from AA genome wild species have been identified and mapped with molecular markers for their integration into O. sativa genome. A broad-spectrum resistance gene for bacterial blight resistance (Xa21) has been identified in O. longistaminata and introduced into many rice cultivars. Advances in biotechnology have facilitated the development of interspecific hybrids between O. sativa and wild species of secondary and tertiary gene pools. Some important genes Pi40 and Bph18 for resistance to blast and brown planthopper, respectively, have been successfully transferred into elite cultivars from O. australiensis and the function of one blast resistance gene (Pi9) derived from O. minuta is elucidated. Many important genes from the most distantly related wild species such as O. alta, O. granulata, O. longiglumis and O. coarctata are expected to be transferred into cultivated rice in the future using the latest tools of molecular genetics and biotechnology.

A novel genome of C and the first autotetraploid species in the Setaria genus identified by genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to investigate the genomic relationships among some newly collected species of genus Setaria. Previous work identified that S. viridis and S. adhaerens carry genomes A and B, respectively. GISH patterns obtained in this report clearly distinguished the genome of S. grisebachii from the known genomes A and B, and indicated its new genomic constitution which we suggest to name genome C of the Setaria genus. The two sets of chromosomes of tetraploid S. queenslandica hybridized well with the A genome of S. viridis indicating its autotetraploid nature. This is the first autotetraploid identified in the Setaria genus, which should be classified into the primary A genome gene pool rather than the tertiary gene pool as previously classified. GISH patterns did not distinguish the genome of S. leucopila from the A genome of S. viridis and S. italica, suggesting its close relation with foxtail millet. Strong hybridization signals were observed when S. adhaerens genomic DNA was used as probe to hybridize the chromosomes of diploid S. verticillata, inferring its B genome nature. Combined with morphological observation and previous work, we deduce that diploid S. verticillata and S. adhaerens are probably taxonomically the same species with different names.

Crossability Barriers in the Interspecific Hybridization between Oryza sativa and O. meyeriana

Oryza meyeriana Baill (GG genome) is a precious germplasm in the tertiary gene pool of cultivated rice (AA genome), and possesses important traits such as resistance and tolerance to biotic and abiotic stress. However, interspecific crossability barrier, a critical bottleneck restricting genes transfer from O. meyeriana to cultivars has led to no hybrids through conventional reproduction. Therefore, the reasons underlying incrossability were investigated in the present report. The results showed that: (i) at 3-7 d after pollination (DAP), many hybrid embryos degenerated at the earlier globular-shaped stage, and could not develop into the later pear-shaped stage. Meanwhile, free endosperm nuclei started to degenerate at 1 DAP, and cellular endosperm could not form at 3 DAP, leading to nutrition starvation for young embryo development; (ii) at 11-13 DAP, almost all hybrid ovaries aborted. Even though 72.22% of hybrid young embryos were produced in the interspecific hybridization between O. sativa and O. meyeriana, young embryos were not able to further develop into hybrid plantlets via culturing in vitro. The main reason for the incrossability was hybrid embryo inviability, presenting as embryo development stagnation and degeneration since 3 DAP. Some possible approaches to overcome the crossability barriers in the interspecific hybridization between O. sativa and O. meyeriana are discussed.

Novel Germplasm Resources for Improving Environmental Stress Tolerance of Hexaploid Wheat

Wheat (Triticum spp. L.) breeders have significantly improved wheat adaptation to stress‐prone environments around the world. This progress has largely been achieved using empirical selection and genetic variability within the primary wheat gene pool. As most stress tolerance traits are quantitatively inherited, expansion of the available genetic diversity for stress tolerance is necessary if rates of genetic progress are to be maintained. This review explores three sources of novel genetic variability, namely synthetic wheat, landrace cultivars, and alien introgressions and their applicability to applied wheat breeding. Synthetic hexaploid wheat, derived by crossing tetraploid wheat with Aegilops tauschii, provides new genetic variability for adaptation to drought, high temperature, salinity, waterlogging, and soil micronutrient imbalances from the secondary wheat gene pool. Synthetic‐derived materials have performed well in many stressed environments globally. There is significant unexploited variation among landraces and modern wheat cultivars to improve the stress adaptation of cultivated wheat. The tertiary gene pool, with a few significant exceptions, has been more difficult to exploit due to complex inheritance, meiotic instability, and linked deleterious effects. Nevertheless, there is sufficient genetic variation in the wheat gene pool to ensure the continued improvement of wheat adaptation to abiotic stress.

Chickpea Improvement: Role of Wild Species and Genetic Markers

Chickpea is an important grain legume of the semi arid tropics and warm temperate zones, and forms one of the major components of human diet. However, a narrow genetic base of cultivated chickpea (Cicer arietinum L.) has hindered the progress in realizing high yield gains in breeding programs. Furthermore, various abiotic and biotic stresses are the major bottlenecks for increasing chickpea productivity. Systematic collection and evaluation of wild species for useful traits has revealed presence of a diverse gene pool for tolerance to the biotic and abiotic stresses. Relationships among the species of genus Cicer are presented based on crossability, karyotype and molecular markers. The reproductive barriers encountered during interspecific hybridization are also examined. Recent information on genetic linkage maps, comparison of isozymes The functional markers generated from genic sequence (e.g. ESTs) have definite advantage over the markers generated from anonymous random regions of the genome, because they are completely linked to desired trait alleles (Anderson and Lubberstedt, 2003; Varshney et al., 2005; Buhariwala et al., 2005). At ICRISAT, Buhariwala et al. (2005) have developed an EST library from two very closely related chickpea genotypes (Cicer arietinum). A total of 106 EST-based markers were designed from 477 sequences with functional annotations and tested on C. arietinum. Forty-four EST markers were polymorphic when screened across nine Cicer species (including the cultivated species) and were used to study the species relationship. Most of the species clustered as reported in the previous studies, and hence it further supported the classification of primary (C. arietinum, C. echinospermum and C. reticulatum), secondary (C. pinnatifidum, C bijugum and C. judaicum), and tertiary (C. yamashitae, C. chrossanicum and C. cuneatum) gene-pools. A large proportion of EST alleles (45%) were only present in one or two of the accessions tested whilst the others were represented in up to twelve of the accessions tested.

Landmark research in legumes

Legumes are members of the family Fabaceae or Leguminosae and include economically important grain legumes, oilseed crops, forage crops, shrubs, and tropical or subtropical trees. Legumes are a rich source of quality protein for humans and animals. They also enrich the soil by producing their own nitrogen in symbiosis with nitrogen-fixing bacteria. International centers and national institutes collect, maintain, distribute, and produce high-yielding legumes (grain-pulses, oilseeds, forages, nutraceuticals, medicinal shrubs, and trees). Legume breeders are confined within the primary gene pools (GP-1) in their varietal improvement programs and have not exploited secondary gene pools (GP-2), tertiary gene pools (GP-3), or quaternary gene pools (GP-4). Legumes are also an excellent source of timber, medicine, nutraceuticals, tannins, gums, insecticides, resins, varnish, paints, dyes, and eco-friendly by-products such as soy diesel. Three forage crops, Medicago truncatula, Lotus japonicus, and Trifolium pratense, are model legumes for phylogenetic studies and genome sequencing. This paper concludes that a "protein revolution" is needed to meet the protein demands of the world.

Crossability and Characterization of Interspecific Hybrids between Sexual Pennisetum glaucum (Pearl Millet) and a New Cytotype (2n=56) of Apomictic P. squamulatum

Pennisetum squamulatum is an important species belonging to tertiary gene pool of cultivated pearl millet as potential donor of several genes. Recently a new cytotype with 2n=56 chromosmes has been identified. Crossability of this cytotype has been studied with diploid and induced tetraploid pearl millet. The hybrids produced with this novel cytotype were compared for their characteristics with the hybrids previously reported with widely reported 2n=54 P. squamulatum cytotypes. Strong incompatability barriers were observed between 2n=56 cytotype with diploid pearl millet, and hybrids could not be obtained even after embryo rescue. Limited crossability was obtained with tetraploid pearl millet and 2 successful hybrids were produced. Their hybridity was confirmed by morphology, flowcytometry and cytology. The hybrids were highly fertile, perennial and segregated for mode of reproduction. Segregation for other traits confirmed heterozygous nature of P. squamulatum 2n=56 cytotype. Cytological investigations suggested 2n=8x nature of this cytotype. Successful hybridization between pearl millet and this new cytotype shall be important for introgression of useful traits as well as to study inheritance of traits such as perenniality, apomixis and tolerance to stresses.

Genotype Dependent Interspecific Hybridization of Sorghum bicolor

Wild Australian Sorghum species are a tertiary gene pool to grain sorghum [Sorghum bicolor (L.) Moench], and they are of interest to breeders because of their resistance to important insects and pathogens. However, strong reproductive barriers have prevented hybridization between S. bicolor and these wild species. The purpose of this study was to determine if the recessive iap allele (dominant allele Iap = inhibition of alien pollen) would reduce or eliminate the pollen–pistil incompatibilities that prevent hybridization between S. bicolor and divergent Sorghum species. Cytoplasmic male‐sterile S. bicolor plants, homozygous for the iap allele, were pollinated with three divergent Sorghum species, S. angustum Blake, S. nitidum (Vahl) Pers., and S. macrospermum Garber. The pollen of these three wild species readily germinated and the pollen tubes grew to the base of the S. bicolor ovary within 2 h after pollination. Hybrid embryos were detected in the S. bicolor florets 13 to 20 d post‐pollination. Sorghum bicolor × S. angustum and S. bicolor × S. nitidum hybrids were obtained using embryo rescue followed by in vitro culture techniques and hybrids between S. bicolor and S. macrospermum were obtained by simply germinating the hybrid seed. These hybrids were confirmed by their morphological and cytological traits. These findings clearly demonstrate that the recessive iap allele circumvents pollen–pistil incompatibilities in the genus Sorghum and permits hybrids to be made between S. bicolor and species of the tertiary gene pool.

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F1 hybrids. The F1 hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F2, F3 and F4 progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F0 parental cross.

Characterization of derivatives from wheat-Thinopyrum wide crosses

Partial amphiploids are lines that contain 42 (38–42) wheat and 14 (14–18) alien chromosomes. They are derived by backcrossing wheat onto hybrids between wheat and either Thinopyrum intermedium (6x) or Th. ponticum (10x). GISH analysis has shown that, with possibly one exception, the alien genomes (chromosome sets) in partial amphiploids are found to be hybrids i.e. composed of chromosomes from more than one alien genome. The individual partial amphiploids are meiotically stable and nearly perfectly fertile, but hybrids between different lines were characterized by varying numbers of unpaired chromosomes and consequently variable degrees of sterility. Translocated chromosomes involving different Thinopyrum genomes or Thinopyrum and wheat genomes were found in partial amphiploids and consequently in the addition lines derived from them. Partial amphiploids have proven to be an excellent tertiary gene pool for wheat improvement, containing resistance to biotic stresses not present in wheat itself. Resistance to Barley Yellow Dwarf Virus (BYDV) and Wheat Streak Mosaic Virus (WSMV) have been found in partial amphiploids and addition lines derived from both Th. intermedium and Th. ponticum. Excellent resistance to Fusarium head blight has been found on a Th. intermedium chromosome that had substituted for chromosome 2D in wheat. Genes for resistance to leaf rust and stem rust have already been incorporated into wheat and tagged with molecular markers.

Development of ESTs from chickpea roots and their use in diversity analysis of the Cicer genus

BackgroundChickpea is a major crop in many drier regions of the world where it is an important protein-rich food and an increasingly valuable traded commodity. The wild annual Cicer species are known to possess unique sources of resistance to pests and diseases, and tolerance to environmental stresses. However, there has been limited utilization of these wild species by chickpea breeding programs due to interspecific crossing barriers and deleterious linkage drag. Molecular genetic diversity analysis may help predict which accessions are most likely to produce fertile progeny when crossed with chickpea cultivars. While, trait-markers may provide an effective tool for breaking linkage drag. Although SSR markers are the assay of choice for marker-assisted selection of specific traits in conventional breeding populations, they may not provide reliable estimates of interspecific diversity, and may lose selective power in backcross programs based on interspecific introgressions. Thus, we have pursued the development of gene-based markers to resolve these problems and to provide candidate gene markers for QTL mapping of important agronomic traits.ResultsAn EST library was constructed after subtractive suppressive hybridization (SSH) of root tissue from two very closely related chickpea genotypes (Cicer arietinum). A total of 106 EST-based markers were designed from 477 sequences with functional annotations and these were tested on C. arietinum. Forty-four EST markers were polymorphic when screened across nine Cicer species (including the cultigen). Parsimony and PCoA analysis of the resultant EST-marker dataset indicated that most accessions cluster in accordance with the previously defined classification of primary (C. arietinum, C. echinospermum and C. reticulatum), secondary (C. pinnatifidum, C. bijugum and C. judaicum), and tertiary (C. yamashitae, C. chrossanicum and C. cuneatum) gene-pools. A large proportion of EST alleles (45%) were only present in one or two of the accessions tested whilst the others were represented in up to twelve of the accessions tested.ConclusionGene-based markers have proven to be effective tools for diversity analysis in Cicer and EST diversity analysis may be useful in identifying promising candidates for interspecific hybridization programs. The EST markers generated in this study have detected high levels of polymorphism amongst both common and rare alleles. This suggests that they would be useful for allele-mining of germplasm collections for identification of candidate accessions in the search for new sources of resistance to pests / diseases, and tolerance to abiotic stresses.

Geographical patterns of genetic variation in the world collections of wild annual Cicer characterized by amplified fragment length polymorphisms

Cicer reticulatum, C. echinospermum, C. bijugum, C. judaicum, C. pinnatifidum, C. cuneatum and C. yamashitae are wild annual Cicer species and potential donors of valuable traits to improve chickpea (C. arietinum). As part of a large project to characterize and evaluate wild annual Cicer collections held in the world gene banks, AFLP markers were used to study genetic variation in these species. The main aim of this study was to characterize geographical patterns of genetic variation in wild annual Cicer germplasm. Phylogenetic analysis of 146 wild annual Cicer accessions (including two accessions in the perennial C. anatolicum and six cultivars of chickpea) revealed four distinct groups corresponding well to primary, secondary and tertiary gene pools of chickpea. Some possible misidentified or mislabelled accessions were identified, and ILWC 242 is proposed as a hybrid between C. reticulatum and C. echinospermum. The extent of genetic diversity varied considerably and was unbalanced between species with greatest genetic diversity found in C. judaicum. For the first time geographic patterns of genetic variation in C. reticulatum, C. echinospermum, C. bijugum, C. judaicum and C. pinnatifidum were established using AFLP markers. Based on the current collections the maximum genetic diversity of C. reticulatum, C. echinospermum, C. bijugum and C. pinnatifidum was found in southeastern Turkey, while Palestine was the centre of maximum genetic variation for C. judaicum. This information provides a solid basis for the design of future collections and in situ conservation programs for wild annual Cicer.

The extent of embryo and endosperm growth following interspecific hybridization between Cicer arietinum L. and related annual wild species

In vivo interspecific pollinations were performed and immature seed development investigated by histological methods in order to study crossability barrier(s) in Cicer L. species wide hybridization. Seven of the eight wild annual Cicer species, belonging to the secondary and tertiary gene pools, were used in reciprocal crosses with the cultivated chickpea. It was confirmed that the zygote was formed in all interspecific crosses. The embryos showed continued and retarded growth at different rate in various crosses, but eventually aborted at an early pro-embryo stage in all crosses except C. arietinum L. ×C. echinospermum Dav. Reciprocal cross differences were observed in early embryo growth rate and could have implications in obtaining hybrids. This study further emphasizes the necessity for developing appropriate and efficient in vitro procedures for rescuing immature globular hybrid pro-embryos, which will make the wild Cicer gene resources amenable to chickpea improvement.

Importance of the Secondary Genepool in Barley Genetics and Breeding. I. Cytogenetics and Molecular Analysis

There have been no plant breeding developments using species from the tertiary genepool of cultivated barley for breeding or genetics since the VIII<sup>th</sup> International Barley Genetics Symposium in 2000. Hence, the first part of this review describes progress since 2000 in developing and characterising recombinant lines derived from hybridisations between the sole species in the secondary genepool, Hordeum bulbosum L., and cultivated barley, Hordeum vulgare L. The topics discussed in part I are cytogenetics and molecular analysis of recombinant lines.  

Geographical Distribution of Wild Lactuca Species (Asteraceae, Lactuceae)

I. Abstract A broad survey of available world literature showed that at least 98 wild Lactuca spp. (Asteraceae) have been described taxonomically. The distribution of the genus Lactuca worldwide includes 17 species in Europe, 51 in Asia, 43 in Africa, and 12 in the Americas (mostly the North American subcontinent). Species originating in Asia, Africa, and the Americas form ca. 83% of known Lactuca spp. richness; however, they are very poorly documented from the viewpoint of taxonomic relationships, ecogeography, and variability. The phytogeography of Lactuca spp. regarding their distribution on different continents and in relation to the structure of the lettuce gene pool is discussed. A more detailed analysis of geographical distribution and habitats is given for some species (L. serriola, L. saligna, L. virosa, L. perennis, L. quercina, L. tatarica), which represent the primary, secondary, and tertiary gene pools of cultivated lettuce (L. sativa). Original and synanthropic distributions of Lactuca spp. ...

Resistance of Phaseolus species to ice crystallization at subzero temperature.

Dry bean (Phaseolus vulgaris L.) cultivars possess little or no freezing tolerance and are killed at the temperature of ice formation in their tissues. An increase in frost tolerance by 2-3 degrees C would expand dry bean production in the short growing seasons of the Canadian prairies and possibly to higher altitudes in the tropics where episodic frosts occur during the growing season. The objective of this study was to determine the differences in frost resistance of Phaseolus species in both controlled and field environments. Leaflets of dry bean cv. CDC Nighthawk, and wild relatives from the primary gene pool (P. vulgaris var. mexicanus Freytag and P. vulgaris var. aborigineus (Burkart) Baudet) and the tertiary gene pool (P. acutifolius var. tenuifolius A. Gray, P. filiformis Bentham, P. angustissimus A. Gray and P. ritensis M.E. Jones) were subjected to subzero temperatures with and without ice nucleation to determine the levels of tolerance and avoidance, respectively. The lethal temperature at which 50% of the leaflets were killed (LT(50)) was 0.5-1 degrees C lower for species of the tertiary gene pool compared to those from the primary gene pool. Leaflets of species from the tertiary gene pool were also characterized by extensive supercooling compared to leaflets of species from the primary gene pool. Resistance of Phaseolus species to spring and autumn frosts were determined on seedlings transplanted to the field. Phaseolus angustissimus, a species of the tertiary gene pool had the highest seedling survival in response to both autumn and spring frosts, when the minimum air temperatures were -5 and -7 degrees C, respectively. Frost resistance of Phaseolus angustissimus, if successfully introgressed into bean germplasm, may enable the development of frost resistant dry bean cultivars.

Genetic differentiation of autochthonous populations of Pinus sylvestris (Pinaceae) from the Iberian peninsula

Genetic structure and genetic diversity in fourteen autochthonous populations of Pinus sylvestris L. from the Iberian peninsula and two populations from the Massif Central in France were studied at eleven isoenzymatic polymorphic loci. The studied populations formed two genetically similar but heterogeneous groups. The first group consist of populations localized in North-Eastern Spain with two genetically distinct populations. The second includes populations of the West-Central part of the Iberian peninsula. Populations from the Massif Central in France are diverse from those of Spain, but are similar to the group from North- part of the Iberian peninsula. The existence of significant numbers of private alleles in Spanish populations which were not observed in populations from the Massif Central, suggests that the Scots pine from the Iberian peninsula probably did not take part in the colonization of Europe after the last glaciations and represents original ancient tertiary gene pools.

Evaluation of Spring Cereal Grains and Wild Triticum Germplasm for Resistance to Rhizoctonia solani AG‐8

Rhizoctonia root rot, caused by Rhizoctonia solani Kuhn AG-8 (Anastomosis Group 8), is a yield-limiting disease of direct-seeded cereals in the Pacific Northwest (PNW) region of the USA, and to date, no resistant Triticum germplasm has been identified. The objective of this research was to identify potential sources of genetic resistance among selected members of the primary, secondary, and tertiary gene pools of wheat (Triticum aestivum L.) for use in cultivar improvement. Members of the primary gene pool {spring wheat germplasm, synthetic hexaploids, triticale (x Triticosecale spp.), Triticum turgidum var. durum (Desf.) Bowden [= T. turgidum subsp. durum (Desf.) Husn.]}, secondary gene pool [Aegilops cylindrica Host, Dasypyrum villosum (L.) P. Candargyl, and tertiary gene pool (Hordeum vulgare L.) of wheat were screened for disease response to two isolates (C1 and D2) of R. solani AG-8 in controlled environment assays. Variation for magnitude of susceptibility to both isolates was detected, but no sources of resistance were identified among primary or tertiary gene pool members. Isolate D2 generally produced more severe disease than isolate C1, with the exception of the most susceptible accessions for which both isolates caused equally severe disease. All accessions of D. villosum exhibited some level of seedling resistance to isolate Cl, and 73% also displayed a resistance response to isolate D2. All D. villosum/durum amphiploids, as well as Chinese Spring/D. villosum addition lines, were susceptible to both isolates. Differences in disease response between the two isolates suggest that varying levels of virulence, or aggressiveness, exist among R. solani AG-8 isolates.

Evaluation of Spring Cereal Grains and Wild Germplasm for Resistance to AG-8

Rhizoctonia root rot, caused by Rhizoctonia solani Kühn AG-8 (Anastomosis Group 8), is a yield-limiting disease of direct-seeded cereals in the Pacific Northwest (PNW) region of the USA, and to date, no resistant Triticum germplasm has been identified. The objective of this research was to identify potential sources of genetic resistance among selected members of the primary, secondary, and tertiary gene pools of wheat (Triticum aestivum L.) for use in cultivar improvement. Members of the primary gene pool {spring wheat germplasm, synthetic hexaploids, triticale (× Triticosecale spp.), Triticum turgidum var. durum (Desf.) Bowden [= T turgidum subsp. durum (Desf.) Husn.]}, secondary gene pool [Aegilops cylindrica Host, Dasypyrum villosum (L.) P. Candargy], and tertiary gene pool (Hordeum vulgare L.) of wheat were screened for disease response to two isolates (C1 and D2) of R solani AG-8 in controlled environment assays. Variation for magnitude of susceptibility to both isolates was detected, but no sources of resistance were identified among primary or tertiary gene pool members. Isolate D2 generally produced more severe disease than isolate C1, with the exception of the most susceptible accessions for which both isolates caused equally severe disease. All accessions of D villosum exhibited some level of seedling resistance to isolate C1, and 73% also displayed a resistance response to isolate D2. All D villosum/durum amphiploids, as well as Chinese Spring/D villosum addition lines, were susceptible to both isolates. Differences in disease response between the two isolates suggest that varying levels of virulence, or aggressiveness, exist among R solani AG-8 isolates.