Genetic analysis and QTL mapping of the seed hardness trait in a black common bean (Phaseolus vulgaris) recombinant inbred line (RIL) population.

Seed hardness trait has a profound impact on cooking time and canning quality in dry beans. This study aims to identify the unknown genetic factors and associated molecular markers to better understand and tag this trait. An F2:7 recombinant inbred line (RIL) population was derived from a cross between the hard and soft seeded black bean parents (H68-4 and BK04-001). Eighty-five RILs and the parental lines were grown at two locations in southern Manitoba during years 2014-2016. Seed samples were harvested manually at maturity to test for seed hardness traits. The hydration capacity and stone seed count were estimated by soaking the seeds overnight at room temperature following AACC method 56-35.01. Seed samples from 2016 tests were also cooked to determine effect of seed hardness on cooking quality. For mapping of genomic regions contributing to the traits, the RIL population was genotyped using the genotype by sequencing (GBS) approach. The QTL mapping revealed that in addition to the major QTL on chromosome 7 at a genomic location previously reported to affect seed-hydration, two novel QTL with significant effects were also detected on chromosomes 1 and 2. In addition, a major QTL affecting the visual appeal of cooked bean was mapped on chromosome 4. This multi-year-site study shows that despite large environmental effects, seed hardness is an oligo-genic and highly heritable trait, which is inherited independently of the cooking quality scored as visual appeal of cooked beans. The identification of the QTLs and development of SNP markers associated with seed hardness can be applied for common bean variety improvement and genetic exploitation of these traits.

Variation in Growth, Colonization of Maize, and Metabolic Parameters of GFP- and DsRed-Labeled Fusarium verticillioides Strains.

Autofluorescent proteins are frequently applied as visual markers in the labeling of filamentous fungi. Genes gfp and DsRed were transformed into the genome of Fusarium verticillioides via the Agrobacterium tumefaciens-mediated transformation method. The selected transformants displayed a bright green or red fluorescence in all the organelles of the growing fungal mycelia and spores (except for the vacuoles) both in cultures and in the maize (Zea mays) roots they colonized. The results of gene-specific polymerase chain reaction (PCR) analysis and the thermal asymmetrical interlaced (TAIL)-PCR analysis demonstrated that gfp and DsRed were integrated on different chromosomes of the fungus. Reductions in the colony growth on the plates at pH 4.0 and 5.5 was observed for the green fluorescent protein (GFP)-transformant G3 and the DsRed-transformant R4, but transformants G4 and R1 grew as well as the wild-type strain at pH 4.0. The speed of growth of all the transformants was similar to the wild-type strain at pH ≥ 7. The insertion of gfp and DsRed did not alter the production of extracellular enzymes and fumonisin B by F. verticillioides. The transformants expressing GFP and DsRed proteins were able to colonize maize roots. However, the four transformants examined produced fewer CFU in the root samples than the wild-type strain during a sampling period of 7 to 28 days after inoculation.

First Report of Fusarium cuneirostrum Causing Root Rot Disease in Dry Bean (Phaseolus vulgaris) in Canada.

Root rot is a major disease of dry bean and can cause significant yield reductions due to weakened root systems and poor plant stands. An in-depth study on root rot pathogen identification was conducted in 2011 in three commercial dry bean fields from the major production areas in Manitoba. Ten plants, sampled at each of four random sites within each field, were rated for disease severity. Twenty roots were processed for pathogen isolation and identification in the laboratory. Roots were cut into eight sections (~1 cm) and surface-sterilized in a laminar flow bench. Four root sections were placed on potato dextrose agar plates amended with 0.02% streptomycin sulfate (PDA-Strep) and four root sections were placed on peptone-pentachloronitrobenzene agar amended with 0.1% streptomycin sulfate and 0.012% neomycin sulfate. Afterward, 960 monosporic cultures were obtained representing 320 single spore isolates of potential root rot pathogens per commercial field. Common monosporic cultures from each field were subcultured on PDA-Strep and Spezieller Nährstoffarmer Agar (SNA) media. Based on morphological characteristics, 74 isolates were identified as Fusarium cuneirostrum (1). Colonies grew slowly on PDA-Strep with undulated margins, radial cream-grey mycelia, and conidia pustules with a cream-greyish pigmentation. Sporodochial conidia were falcate, mostly 5-septate, with a wedge shape and slightly protruding basal foot cell (56.3 to 71.8 × 4.6 to 6.2 µm on average). Species identity was confirmed for two isolates by sequencing the translation elongation factor 1 alpha (EF1-α) gene (2), the internal transcribed spacer (ITS) region (4), and the ribosomal intergenic spacer (IGS) (3) (GenBank Accession Nos. KF530848, KF530849, and KF025648 to 51). Sequence homology was compared using BLAST analysis and the FUSARIUM-ID database. The F. cuneirostrum isolates were deposited at the Canadian Collection of Fungal Cultures (DAOM 242540 and 242541). Pathogenicity screenings of two isolates was performed using sterilized seed of navy bean cv. Envoy. Seeds were germinated on moist filter paper for 3 days at 25°C and then inoculated by immersion in a prepared conidial suspension (2.5 × 105 conidia/ml) for 5 min. Seeds of the controls were immersed in sterile water. After inoculation, the germinated seeds were planted in 10-cm diameter pots, filled with sterile soilless mix (Sunshine #3). In the greenhouse, the experiment was arranged as a completely randomized design with three replicates with four germinated seeds per isolate, and was repeated twice. Disease assessment was performed 14 days after inoculation. Infected plants displayed dark brown lesions on the hypocotyl and primary root with a disease severity of 4 scored on a 0 to 5 scale. Fusarium cuneirostrum was re-isolated from roots of symptomatic plants. To our knowledge, this is the first report of F. cuneirostrum causing root rot of dry bean in Canada. It has been previously isolated from mung bean (Vigna radiata) in Ontario (1). References: (1) T. Aoki et al. Mycoscience. 46:162, 2005. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (4) H. Wang et al. J. Clin. Microbiol. 49:1890, 2011. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.

First Report of Rhizome Blight of Ginger Caused by Binucleate Rhizoctonia AG-R in China.

In 2004, rhizome blight of ginger (Zingiber officinale (Willd.) Roscoe) (cv. Yunnanxiaojiang) occurred in the Kunming District of China. The surface of ginger rhizome after harvest was crimpled and covered with white hyphae. Initial symptoms on ginger were wilting on the stem and the color of the rhizome turned from white to light brown with no lesion formation. After 2 weeks, the surface of ginger rhizome was covered with white hyphae and a dry rot set in under humid conditions. The yield loss in ginger almost reached 50% because of the disease. An AG-R tester isolate paired with the unknown 37 isolates of Rhizoctonia spp. from the diseased ginger rhizomes caused a C2 reaction that confirmed their identity. Isolates of AG-R (GenBank Accession Nos. DQ885780 and DQ885781) had 100% sequence similarity with 5.8S rDNA-ITS with the AG-R tester isolate (GenBank Accession No. AF354082). To produce infected soil inoculum, 10 isolates were cultured on potato dextrose agar in a 9-cm petri dish for 3 to 4 days and then covered with approximately 20 g of autoclaved soil and kept at 25°C for 3 to 4 days. Seedlings of ginger (cv, Yunanxiaojiang) were planted in natural potting soil at a density of one plant per vinyl pot (8 cm in diameter, 9 cm high) and grown in the greenhouse for 7 days. Each seedling was inoculated with 7 g of infested soil by placing it around the rhizome. Control plants were inoculated with autoclaved soil. The experiments were carried out three times, each time with three replicates in a growth chamber kept at 25 and 16°C with a 16-h light and 8-h dark photoperiod. After 14 days, the disease severity was recorded based on a scale in which - = no symptoms; + = small lesions on seedlings, no blight; ++ = seedling blight; and +++ = plant dead. All of the 10 tested AG-R isolates caused ginger seedling blight. Rhizoctonia spp. was reisolated from these plants, confirming its pathogenicity. To our knowledge, this is the first report of rhizome blight of ginger caused by Rhizoctonia spp. and binucleate Rhizoctonia AG-R in China. References: (1) B. Sneh et al. Page 135 in: Identification of Rhizoctonia Species. The American Phytopathological Society, St. Paul, MN, 1998.

A Specific and Sensitive Method for the Detection of Colletotrichum lindemuthianum in Dry Bean Tissue.

To facilitate early diagnosis and improve control of bean anthracnose, a rapid, specific, and sensitive polymerase chain reaction (PCR)-based method was developed to detect the causal agent, Colletotrichum lindemuthianum, in bean (Phaseolus vulgaris) seed. Based on sequence data of the rDNA region consisting of the 5.8S gene and internal transcribed spacers (ITS) 1 and 2 of four C. lindemuthianum races and 17 Colletotrichum species downloaded from GenBank, five forward primers were designed and evaluated for their specificity. Among them, one forward primer was selected for use in combination with ITS4 to specifically detect C. lindemuthianum. A 461-bp specific band was amplified from the genomic DNA template of 16 representative isolates of C. lindemuthianum, but not from 58 representative isolates of 17 other Colletotrichum species or 10 bean pathogens. Moreover, to enhance the sensitivity of detection, nested PCR was applied, which allowed the detection of as little as 10 fg of C. lindemuthianum genomic DNA and 1% infected seed powder, which was mixed with 99% healthy seed powder. The diagnostic analysis can be completed within 24 h, compared with about 2 weeks required for culturing. Furthermore, this method can be performed and interpreted by personnel with no specialized taxonomic expertise.

First Report of Damping-Off of Swiss Chard Caused by Rhizoctonia solani AG-4 HG I and Binucleate Rhizoctonia AG-A in China.

During July, 2003, damping-off of Swiss chard (Beta vulgaris subsp. cicla L.) was observed in a seedling (approximately 1 month after germination) field (approximately 2 ha) in Yuanmou County in the Cuxiong District of Yunnan, China. More than 80% of the seedlings showed symptoms of the disease. Symptoms on newly emerged plants consisted of wilting, a brown necrosis of the lower taproot, and eventual death of seedlings. Among the 15 isolates of Rhizoctonia spp. isolated from Swiss chard with damping-off symptoms, 12 isolates of Rhizoctonia solani with dark brown sclerotia on potato dextrose agar (PDA) anastomosed with tester isolates of each subgroup AG-4 HG I, AG-4 HG II, and AG-4 HG III, giving a C2 hyphal fusion (1) reaction at a high frequency. The other three binucleate Rhizoctonia spp. (BNR) isolates whose mycelia were white with floccose aerial hyphae on PDA anastomosed freely with two BNR AG-A tester isolates producing a C2 hyphal reaction. The 5.8S rDNA-ITS of a single isolate of R. solani and a single isolate of BNR was sequenced. The sequence of the AG-4 isolate (GenBank Accession No. EF679777) exhibited 99 to 100% homology with isolates of R. solani AG-4, subgroup 4HG I (GenBank Accession No. AY154307). The sequence from the AG-A isolate (GenBank Accession No. EF679778) exhibited 98% homology with BNR AG-A (GenBank Accession Nos. AB000040 and AF354092). Swiss chard (cv. Baijin) seedlings (approximately 5 cm high) were planted in potting soil at a density of one seedling per vinyl pot (8 cm diameter, 9 cm high). Two isolates each of R. solani and BNR were used in pathogenicity testing. Each seedling was inoculated in the root zone with approximately 7 g of artificially infested soil. Control plants were inoculated with autoclaved soil. The experiments were conducted three times, each time with three replicates, in a greenhouse with a photoperiod of 16 h of light and 8 of h dark at 30 and 16°C, respectively. After 7 days, disease severity was measured based on a scale in which 0 = no symptom; 1 = small lesions on seedlings, no blight; 2 = leaves blight, no stem blight; 3 = stem blight; and 4 = plant dead. The two AG-4 and two of AG-A isolates were pathogenic on the Swiss chard seedlings and caused damping-off symptoms with a disease index 1.7 to 4.0, and there were no significant differences (P = 0.05) among them. We reisolated and confirmed the presence of R. solani and BNR AG-A from diseased plants. AG-3 isolates were reported to cause the damping-off of Swiss chard in the United States (2). To our knowledge, this is the first report of damping-off of Swiss chard caused by Rhizoctonia solani AG-4 HG I and BNR AG-A. References: (1) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reaction. Kluwer Academic Publishers, Dordecht, the Netherlands, 1996. (2) S. T. Koike and K. V. Subbarao. Plant Dis. 83:695, 1999.

Characterization of Wheat-Triticale Lines Resistant to Powdery Mildew, Stem Rust, Stripe Rust, Wheat Curl Mite, and Limitation on Spread of WSMV.

High yield potential and the wide adaptability of wheat-rye T1BL·1RS translocation lines are attractive to breeders. The wheat-rye lines Lankao 1, 3, 4, and 5 were resistant to a wide spectrum of wheat powdery mildew (Blumeria graminis f. sp. tritici) isolates from both China and Canada. They also were resistant to a mixture of wheat stem rust (Puccinia graminis f. sp. tritici) pathotypes (98WSR) and wheat stripe rust (P. striiformis f. sp. tritici) races from western Canada and China. Colonization of wheat curl mite (WCM) (Aceria tosichella) resulted in slower development of rolling and trapping leaves in the Lankao lines than in the WCM-susceptible check cultivars. The delayed development of Wheat streak mosaic (WSM) symptoms on Lankao lines was observed when transmitted by viruliferous WCM, even though they were susceptible to Wheat streak mosaic virus (WSMV). This effect of Lankao lines on limiting the spread of WSM was comparable with other known sources of WCM resistance. Sequential C-banding and genomic in situ hybridization analyses revealed the presence of a pair of T1BL·1RS translocated chromosomes in the Lankao lines. Segregation analysis of the F2 progeny plants derived from crosses between Lankao 4 and the susceptible wheat cvs. Mingxian 169 and Lovrin 13 indicated that a single dominant gene was responsible for the isolate-specific resistance against wheat powdery mildew in Lankao 4. Polymerase chain reaction analysis using an STS marker amplified rye chromatin in powdery mildew-resistant and -susceptible F2 plants of the Mingxian 169 × Lankao 4 cross demonstrated that the resistance of Lankao 4 was not controlled by a gene or genes located on the rye chromosome arm of T1BL·1RS. The resistance of the Lankao lines to diseases and limitation of the spread of WSMV, in combination with good quality and high yield potential, makes them useful for wheat improvement and production.

Molecular cytogenetic discrimination and reaction to wheat streak mosaic virus and the wheat curl mite in Zhong series of wheat--Thinopyrum intermedium partial amphiploids.

Thinopyrum intermedium (2n = 6x = 42, JJJsJsSS) is potentially a useful source of resistance to wheat streak mosaic virus (WSMV) and its vector, the wheat curl mite (WCM). Five partial amphiploids, namely Zhong 1, Zhong 2, Zhong 3, Zhong 4, and Zhong 5, derived from Triticum aestivum x Thinopyrum intermedium crosses produced in China, were screened for WSMV and WCM resistance. Zhong 1 and Zhong 2 had high levels of resistance to WSMV and WCM. The other three partial amphiploids, Zhong 3, 4, and 5, were resistant to WSMV, but were susceptible to WCM. Genomic in situ hybridization (GISH) using a genomic DNA probe from Pseudoroegneria strigosa (SS, 2n = 14) demonstrated that two partial amphiploids, Zhong 1 and Zhong 2, have almost the identical 10 Th. intermedium chromosomes, including four Js, four J, and two S genome chromosomes. Both of them carry two pairs of J and a pair of Js genome chromosomes and two different translocations that were not observed in the other three Zhong lines. The partial amphiploids Zhong 3, 4, and 5 have another type of basic genomic composition, which is similar to a reconstituted alien genome consisting of four S and four Js genome chromosomes of Th. intermedium (Zhong 5 has two Js chromosomes plus two Js-W translocations) with six translocated chromosomes between S and Js or J genomes. All three lines carry a specific S-S-Js translocated chromosome, which might confer resistance to barley yellow dwarf virus (BYDV-PAV). The present study identified a specific Js2 chromosome present in all five of the Zhong lines, confirming that a Js chromosome carries WSMV resistance. Resistance to WCM may be linked with J or Js chromosomes. The discovery of high levels of resistance to both WSMV and WCM in Zhong 1 and Zhong 2 offers a useful source of resistance to both the virus and its vector for wheat breeding programs.

Characterisation of Triticum vavilovii-derived stripe rust resistance using genetic, cytogenetic and molecular analyses and its marker-assisted selection.

Stripe rust resistance was identified in Triticum vavilovii( T. vaviloviiAus22498)-derived Russian wheat aphid (RWA)-resistant germplasm. Inheritance studies indicated monogenic control of resistance. The resistance gene was tentatively designated as Yrvav and was located on chromosome 1B by monosomic analysis. A close association (1.5+/-0.9% recombination) of Yrvav with a T. vavilovii-derived gliadin allele ( Gli-B1vav) placed it in chromosome arm 1BS. Yrvavwas allelic with Yr10. Tests with Yr10 avirulent and virulent pathotypes showed that Yrvav and Yr10 possess identical pathogenic specificity. Yrvav and Yr10 showed close genetic associations with alternate alleles at the Xpsp3000(microsatellite marker), Gli-B1 and Rg1 loci. Based on these observations Yrvav was named as Yr10vav. The close association between Xpsp3000 and Gli-B1 was also confirmed. The Yr10vav-linked Xpsp3000 allele (285 bp) was not present in 65 Australian cultivars, whereas seven Australian wheats lacking Yr10 carried the same Xpsp3000 allele (260 bp) as Yr10carrying wheat cultivar Moro. Xpsp3000 and/or Gli-B1 could be used in marker-assisted selection for pyramiding Yr10vavor Yr10 with other stripe rust resistance genes. Yr10vav was inherited independently of the T. vavilovii-derived RWA resistance.

Different Reactions to the Wheat Curl Mite and Wheat streak mosaic virus in Various Wheat-Haynaldia villosa 6V and 6VS Lines.

Wheat curl mite (WCM), Aceria tosichella, is the vector of Wheat streak mosaic virus (WSMV), a destructive viral pathogen in wheat (Triticum aestivum). Genetic resistance to WCM colonization can reduce the incidence of wheat streak mosaic. Chromosome 6V in Hay-naldia villosa is a new source of WCM resistance. We compared variation in resistance among different sources of H. villosa chromosome 6V and 6VS lines to WCM and WSMV and their effectiveness in controlling the incidence of WSMV following exposure to viruliferous WCM. WCM resistance varied among the 6V and 6VS lines depending on the H. villosa parent. The 6V substitution lines Yi80928, GN21, and GN22 derived from an accession of H. villosa from China, and the 6VS translocation lines 92R137, 92R178, and Sub6V from an H. villosa accession collected from the United Kingdom were uniformly resistant to WCM colonization. In contrast, the 6V substitution line RW15 and a 6VS translocation line Pm33 developed from an H. villosa collection from the former Union of Soviet Socialist Republics were susceptible to WCM. All 6V and 6VS lines were susceptible to WSMV when manually inoculated. However, symptom expression was delayed in the WCM-resistant 6V and 6VS lines after exposure to viruliferous WCM. The 6V and 6VS lines differed in their ability to control WSMV infection. WCM-susceptible lines RW15 and Pm33 had no effect on controlling the infection by WSMV. Lines GN21 and GN22 were the most effective of the three H. villosa sources in limiting the spread of WSMV. Their high yield potential and protein content, in combination with resistance to stripe rust (Puccinia striiformis f. sp. tritici) and powdery mildew (Erysiphe graminis f. sp. tritici), make GN21 and GN22 promising sources of WCM resistance.

Characterization of wheat-Thinopyrum partial amphiploids by meiotic analysis and genomic in situ hybridization.

A combination of genomic in situ hybridization (GISH) and meiotic pairing analysis of wheat-Thinopyrum partial amphiploids was employed to identify the genomic constitution and relationships between partial amphiploids derived from wheat and wheatgrass crosses. On the basis of similarities in the meiotic behavior and GISH patterns, the alien chromosomes of two of eight partial amphiploids, TAF46 and 'Otrastayuskaya 38', were judged to originate from Th. intermedium, whereas Th. ponticum was one of the parents of the other six partial amphiploids; PWM706, PWM206, PWM209, PWMIII, OK7211542, and Ag-wheat hybrid. Each of these partial amphiploids was found to contain a synthetic alien genome composed of different combinations of St-, J-, or Js-genome chromosomes. For relatedness of partial amphiploid lines, meiotic analysis of F1 hybrids and GISH results were generally complementary, but the latter offered greater precision in identifying constituent genomes.

Genome analysis of Thinopyrum intermedium and Thinopyrum ponticum using genomic in situ hybridization.

Genomic in situ hybridization (GISH) using genomic DNA probes from Thinopyrum elongatum (Host) D.R. Dewey (genome E, 2n = 14), Thinopyrum bessarabicum (Savul. & Rayss) A. Löve (genome J, 2n = 14), and Pseudoroegneria strigosa (M. Bieb.) A. Löve (genome S, 2n = 14), was used to examine the genomic constitution of Thinopyrum intermedium (Host) Barkworth & D.R. Dewey (2n = 6x = 42) and Thinopyrum ponticum (Podp.) Barkworth & D.R. Dewey (2n = 10x = 70). Evidence from GISH indicated that hexaploid Th, intermedium contained the J, Js, and S genomes, in which the J genome was related to the E genome of Th. elongatum and the J genome of Th. bessarabicum. The S genome was homologous to the S genome of Ps. strigosa, while the Js genome referred to modified J- or E-type chromosomes distinguished by the presence of S genome specific sequences close to the centromere. Decaploid Th. ponticum had only the two basic genomes J and Js. The Js genome present in Th. intermedium and Th. ponticum was homologous with E or J genomes, but was quite distinct at centromeric regions, which can strongly hybridize with the S genome DNA probe. Based on GISH results, the genomic formula of Th. intermedium was redesignated JJsS and that of Th. ponticum was redesignated JJJJsJs. The finding of a close relationship among S, J, and Js genomes provides valuable markers for molecular cytogenetic analyses using S genome DNA probes to monitor the transfer of useful traits from Th. intermedium and Th. ponticum to wheat.

Molecular characterization of Haynaldia villosa chromatin in wheat lines carrying resistance to wheat curl mite colonization.

Wheat-Haynaldia villosa (L.) Schur, hybrid lines were tested as potential sources of resistance to colonization by the wheat curl mite, the vector of wheat streak mosaic virus. Two lines, Add 6V-1 and Sub 6V-1, were found to be mite-resistant. Fluorescence in situ hybridization using total genomic DNA, from H. villosa in the presence of unlabelled wheat DNA, confirmed that Add 6V-1 is a disomic wheat-H. villosa chromosome addition line. Sub 6V-1 turned out to be a homoeologous wheat-H. villosa chromosome translocation line rather than a substitution. The translocation in Sub 6V-1 occurred between a wheat chromosome and a chromosome from H. villosa through Robertsonian fusion of misdivided centromeres. Only the short arm of the group 6 chromosome of H. villosa was involved in the genetic control of mite resistance, a conclusion based on the genomic in situ hybridization signal and specific DNA fragments obtained by polymerase chain reaction.

Identification of the parental chromosomes of the wheat-alien amphiploid Agrotana by genomic in situ hybridization.

Labelled total genomic DNA from four alien species, Thinopyrum ponticum (Host) Beauv. (2n = 70, genomes J1J1J1J2J2), Th. bessarabicum (Savul. &Rayss) Love (2n = 14, genome J), Th. elongatum (Host) Beauv. (2n = 14, genome E), and Haynaldia villosa (L.) Schur. (2n = 14, genome V), were used as probes in combination with blocking wheat DNA for in situ hybridization of the chromosomes of Agrotana, a wheat-alien hybrid (2n = 56) of unknown origin. The results showed that genomic DNA probes from Th. ponticum and Th. bessarabicum both clearly revealed 16 alien and 40 wheat chromosomes in Agrotana, indicating that the J genome present in these two species has a high degree of homology with the alien chromosomes in Agrotana. Biotinylated genomic DNA probe from Th. elongatum identified 10 chromosomes from Agrotana, while some regions of six other chromosomes yielded a weak or no signal. The probe from H. villosa produced no differential labelling of the chromosomes of Agrotana. The genomic formula of Agrotana was designated as AABBDDJJ. We suggest that the alien parent donor species of Agrotana is Th. ponticum rather than Th. bessarabicum. Genomic relationships of the three Thinopyrum species are discussed in relation to the distribution of GISH signals in the chromosomes of Agrotana.

C-banding and fluorescence in situ hybridization studies of the wheat-alien hybrid 'Agrotana'.

'Agrotana', a wheat-alien hybrid (2n = 56), is a potential source of resistance to common root rot, stem rust, wheat streak mosaic virus, and the wheat curl mite. However, the origin of 'Agrotana', reported to be durum wheat x Agropyron trichophorum (pubescent wheatgrass), is uncertain. The objective of this investigation was to determine the chromosome constitution of 'Agrotana' using C-banding and fluorescence in situ hybridization techniques. The F1 hybrid of 'Agrotana' x 'Chinese Spring' wheat showed 7 I + 21 II in 14.9% of the pollen mother cells, evidence of the presence of the A, B, and D genomes in 'Agrotana'. The hybrid had 16 heavily C-banded chromosomes, namely 4A, and 1-7B of wheat, and a translocation that probably involved wheat chromosomes 2A and 2D. In situ hybridization using biotinylated genomic DNA of Ag. trichophorum cv. Greenleaf blocked with CS DNA failed to identify the alien chromosomes in 'Agrotana', indicating that the alien chromosomes were not likely derived from pubescent wheatgrass. In situ hybridization using labelled wheat genomic DNA blocked with 'Agrotana' DNA revealed that 'Agrotana' had 40 wheat, 14 alien, and 2 (a pair) wheat-alien translocated chromosomes. There was no homology between wheat and the alien chromosomes or chromosome segments involved in the wheat-alien recombinant. Two of the seven pairs of alien chromosomes were homoeologous to each other. The ability to identify alien chromatin in wheat using labelled wheat DNA instead of labelled alien DNA will be particularly useful in chromosome engineering of wheat germplasms having alien chromatin of unknown origin.

Intravarietal variation in satellites and C-banded chromosomes of Agropyron intermedium ssp. trichophorum cv. Greenleaf.

A high amount of intravarietal variation in satellites and C-banded chromosomes was observed in the hexaploid wheatgrass synthetic cultivar 'Greenleaf' (Agropyron intermedium ssp. trichophorum (Link) A. &Gr., 2n = 6x = 42, genome E1E1E2E2SS). The cultivar is an open-pollinated perennial that shows extensive interplant polymorphism for many biological characters. Maximum number of satellites detected varied among plants from zero to six. In 61% of the plants, we observed two large satellites in association with zero, one, or two small ones. Chromosome constitution differed significantly among plants as revealed by analysis of variance based on the total number of banded chromosomes and the number of banded chromosomes with telomeric bands at either one or both ends. Heteromorphism in C-banding patterns between homologues was found in most of the chromosomes and was classified into four types: (i) difference in band size, (ii) difference in presence/absence of one or two bands, (iii) completely different banding patterns, and (iv) banded versus unbanded. Homologous chromosomes having types iii and iv heteromorphism could only be matched by their relative length and arm ratio instead of C-banding patterns. Deletions were detected in two chromosomes. Overall, C-banded chromosomes of this cultivar were characterized by the presence of large telomeric bands and were quite different from the previously reported karyotypes of the supposed diploid ancestor Agropyron elongatum (Host) P. Beauv. (genome EE) and an Ag. intermedium (Host) P. Beauv. accession (E1E1E2E2SS) The results suggest that dramatic chromosome modifications have occurred in this species during the course of evolution. The study sheds light on the extent of intrapopulation polymorphism present in the karyotypes of outcrossing polyploids and synthetic cultivars and has implications regarding strategies for chromosomal manipulation involving open-pollinated species.

Reaction of alien chromosome substitution and addition lines of hard red spring wheat to common root rot and black point.

Resistance to common root rot and black point, caused by Cochliobolus sativus, was evaluated in alien chromosome substitution and addition lines of the cultivars 'Cadet' and 'Rescue'. Substitution of chromosome 5B in 'Rescue' with 5Ag from Agropyron elongatum decreased root rot susceptibility to a level intermediate between that in the susceptible 'Rescue' and the resistant 'Cadet'. The substitution of 'Rescue' chromosome 5A or 5D with 5Ag, or the addition of 5Ag to 'Rescue' complement had no consistent effect on root rot susceptibility. The root rot resistance of 'Cadet' was unaffected by substitution of chromosomes 5A, 5B, or 5D with 5Ag, or the addition of 5Ag. This indicates that the susceptible allele of the gene Crr is the primary determinant in the reaction of wheat to common root rot. Black point resistance in the susceptible cultivar 'Rescue' was significantly increased by substitution of chromosome 5B with 5Ag, or the addition of 5Ag. No corresponding effect was demonstrated for black point incidence in the moderately resistant cultivar 'Cadet' with substitution of chromosome 5Ag for 5B, or the addition of 5Ag. Chromosome 5Ag apparently carries one or more genes conferring resistance to black point. The identity of these lines was confirmed by restriction fragment length polymorphism analysis using group 5 chromosome arm specific probes. This extends the use of these molecular probes to the Agropyron genome.

Nimodipine alters acquisition of a visual discrimination task in chicks.

Chicks 5 days old received intraperitoneal injections of nimodipine 30 min before training on either a visual discrimination task (0, 0.5, 1.0, or 5.0 mg/kg) or a test of separation-induced distress vocalizations (0, 0.5, or 2.5 mg/kg). Chicks receiving 1.0 mg/kg nimodipine made significantly fewer visual discrimination errors than vehicle controls by trials 41-60, but did not differ from controls 24 h later. Chicks in the 5 mg/kg group made significantly more errors when compared to controls both during acquisition of the task and during retention. Nimodipine did not alter separation-induced distress vocalizations at any of the doses tested, suggesting that nimodipine's effects on learning cannot be attributed to a reduction in separation distress. These data indicate that nimodipine's facilitation of learning in young subjects is dose dependent, but nimodipine failed to enhance retention.

Microinjections of leupeptin in the frontal cortex or dorsal hippocampus block spatial learning in the rat.

Adult male Long-Evans hooded rats were given bilateral microinjections of 18 mM leupeptin or saline through cannulae implanted with tips aimed at the frontal cortex or CA1 or CA3 hippocampal cell fields. Five minutes following injections animals were allowed to complete an eight-arm radial maze. The acquisition criterion required that the animal make 7 correct choices of the first 8, and 8 correct choices of the first 10, for five consecutive sessions. Leupeptin slowed acquisition of the eight-arm radial maze task when injected into the CA1 and CA3 hippocampal fields or the frontal cortex, but did not influence spontaneous activity. These results suggest that earlier reports of the amnestic effect of leupeptin when administered into the lateral cerebral ventricle may have been due to effects within the cortex and hippocampus. The present experiment represents the first attempt to identify behaviorally those brain areas in which leupeptin acts to alter learning.

Opiate effects on social behavior of juvenile dogs as a function of social deprivation.

The relationship between opioids and social behavior was examined by administering morphine (an opioid agonist) and naloxone (an opioid antagonist) to juvenile dogs and measuring various social behaviors (e.g., tail wagging) in a large room. Drugs were administered following social deprivation and nondeprivation. It was hypothesized that morphine would ease effects of social deprivation while naloxone would result in behavior typical of untreated socially-deprived dogs. Social deprivation (24 hr) resulted in more contact with the experimenter and increased tail wagging relative to nondeprivation. Morphine (0.25 mg/kg) resulted in more contacts with the experimenter and entrances into the "experimenter's area" relative to vehicle injections. Further, morphine decreased and naloxone increased tail wagging in the dog's area and there was a significant social condition X drug interaction for that measure. Naloxone (0.25 mg/kg) increased wagging following nondeprivation while morphine decreased wagging following deprivation. These data support the hypotheses that social deprivation can increase social behaviors, and that social behavior is regulated by activity in brain opioid systems.