Molecular cytogenetic characterization of alien introgressions with gene Fhb3 for resistance to Fusarium head blight disease of wheat.

Fusarium head blight (FHB) resistance was identified in the alien species Leymus racemosus, and wheat-Leymus introgression lines with FHB resistance were reported previously. Detailed molecular cytogenetic analysis of alien introgressions T01, T09, and T14 and the mapping of Fhb3, a new gene for FHB resistance, are reported here. The introgression line T09 had an unknown wheat-Leymus translocation chromosome. A total of 36 RFLP markers selected from the seven homoeologous groups of wheat were used to characterize T09 and determine the homoeologous relationship of the introgressed Leymus chromosome with wheat. Only short arm markers for group 7 detected Leymus-specific fragments in T09, whereas 7AS-specific RFLP fragments were missing. C-banding and genomic in situ hybridization results indicated that T09 has a compensating Robertsonian translocation T7AL.7Lr#1S involving the long arm of wheat chromosome 7A and the short arm of Leymus chromosome 7Lr#1 substituting for chromosome arm 7AS of wheat. Introgression lines T01 (2n = 44) and T14 (2n = 44) each had two pairs of independent translocation chromosomes. T01 had T4BS.4BL-7Lr#1S + T4BL-7Lr#1S.5Lr#1S. T14 had T6BS.6BL-7Lr#1S + T6BL.5Lr#1S. These translocations were recovered in the progeny of the irradiated line Lr#1 (T5Lr#1S.7Lr#1S). The three translocation lines, T01, T09, and T14, and the disomic addition 7Lr#1 were consistently resistant to FHB in greenhouse point-inoculation experiments, whereas the disomic addition 5Lr#1 was susceptible. The data indicated that at least one novel FHB resistance gene from Leymus, designated Fhb3, resides in the distal region of the short arm of chromosome 7Lr#1, because the resistant translocation lines share a common distal segment of 7Lr#1S. Three PCR-based markers, BE586744-STS, BE404728-STS, and BE586111-STS, specific for 7Lr#1S were developed to expedite marker-assisted selection in breeding programs.

[Molecular markers linked to dwarf gene Rht3 in wheat].

The dwarf gene Rht3 derived from Tom Thumb variety, a Tibetan wheat of China, is a dominant gene with the insensitivity to gibberellic acid. Rht3 shows to depress alpha-amylase activity and hence enchance the resistance to preharvest sprouting mainly through its effect on the expression of alpha-Amy1. Near isogenic lines with Rht3 and their segregating population were analyzed by PCR and RFLP techonology. In RAPD analysis, out of 310 random primers (10 bp) screened, only three primers of UBC389, OPV-06 and S1060 revealed polymorphisms in NIL from 310 random primers. Fragments S1060(1900) and S1060(2000) amplified by primer S1060 were shown to be linked to Rht3 with a genetic distance 7.1 cM and 9.2 cM. Fifty-three probes specific for short arms of homoeologous group 4 were screened in RFLP analysis. Xpsr584, XksuF8 and Xcdo38 showed polymorphisms between the NIL. The linkage analysis indicateded that Xpsr584 was linked to Rht3 with a genetic distance 8.0 cM.

[Cloning and characterization of pathogenesis-related protein 1 from wheat-H. villosa translocation lines].

A Triticum aestivum-Haynaldia villosa 6VS/6AL translocation line contained powdery mildew resistance gene Pm21 which is effective against all the current biotypes of Erygsiphe gramins. Cloning of the related genes for powdery mildew resistance is significance for understanding its resistance mechanism and disease resistance breeding. Using RT-PCR and RACE technology, a wheat pathogenesis related protein 1 cDNA clone (TaPr-1) corresponding to a mRNA differentially induced in resistant 6VS/6AL translocation line compared to susceptible wheat cultivar "Yangmai 5" by powdery mildew infection was isolated and characterized. This sequence contained 823 bp and had an open reading frame (ORF) containing 164 amino acids with 24 amino acids in the putative signal peptide and 140 amino acids comprising the mature peptide (15.1 kD). The deduced amino acid sequence showed close homology to PR-1 like proteins, which have been isolated from many plants. Northern blot analysis revealed the most abundantly accumulation of the corresponding mRNA 12 h after infection in translocation line (6VS/6AL). The obviously difference in the expression of the PRw-1 was also observed between resistant translocation line (6VS/6AL) and susceptible parent "Yangmai 5", it showed the TaPr-1 gene is related to powdery mildew resistance. Southern blot indicated that the wheat genome contains more than one copies of TaPr-1 genes, and there are polymorphism between translocation lines and "Yanmai 5", the result shows that 6VS may contain TaPr-1 genes.

[Pyramiding and marker-assisted selection for powdery mildew resistance genes in common wheat].

This project used the strategy of discarding susceptible individual plants and keeping resistance ones by resistance identification in breeding populations and planted resistant plants next in earlier generations (F1-F3), and performing marker-assisted selection combining resistance identification in later generation (F4 generation) for pyramiding powdery mildew resistance. F4 populations from crosses of elite parents possessing different Pm genes were screened by using molecular markers tightly linked to Pm2, Pm4a, Pm8 and Pm21 genes, respectively. Fourteen individual plants with Pm4a and Pm21 out of 40 F4 plants were identified. Sixteen individual plants containing Pm2 and Pm4a out of 80 F4 plants and six plants possessing Pm8 + Pm21 out of 58 F2 plants were screened. Notably, the plants with Pm2 and Pm4a showed high level resistance or immunity to powdery mildew, while plants with single Pm2 or Pm4a had lower resistance. The results suggest that the strategy of pyramiding different Pm genes provide an alternative way of utilizing resistance gene resources for breeding new types of resistance lines and cultivars, which will have significance not only in breeding practice but also in theoretical research.

[Development and identification of T. aestivum-S. cereale-H. villosa double translocation line 1RS.1BL, 6VS.6AL via chromosome C-banding and dual color FISH].

In order to enhance the disease resistance of wheat-rye 1BL.1RS translocation line and broaden its genetic bases, a 1RS.1BL line was crossed to 6VS.6AL and from its F2 progenies, a double translocation line designated as 1RS.1BL, 6VS.6AL (2n = 42) was identified by (root tip cell) RTC chromosome C-banding. Its mean chromosome configuration at PMC MI was 19.14II + 1.86[symbol: see text] II indicating genetic stability. Rye and H. villosa genomic DNA labelled by biotin- and digoxingenin-11-dUTP respectively were used as probes simultaneously for dual color FISH identification. The results confirmed the C-banding results. Rye and H. villosa chromatin after FISH expressed green or red signals respectively in both RTC and PMC of the double translocation line. This line showed normal fertility, desirable agronomic traits and resistance to powdery mildew, and might be of interest for wheat improvement.

[Development of Triticum aestivum-Haynaldia villosa 6VS ditelosomic substitution line via phlb mutant].

Chinese Spring phlb mutant (C S phlbphlb) was crossed to Triticum aestivum-Haynaldia villosa 6V (6A) alien substitution line and F1 back was crossed with C. S phlbphlb. One LV 02 with varied H. villosa 6V chromosome and one LV 02-01 with 40 T. aestivum chromosome, one H. villosa 6V and 6VS chromosome were screened in BC1F1 and BC1F2 respectively by C-banding and the fluorescence in situ hybridization (FISH). In segregated generation of LV 02-01, eight T. aestivum-H. villosa 6VS ditelosomic substitution lines were screened by FISH and C-banding.

[RFLP analysis of wheat-L. racemosus translocation lines].

A number of wheat-L. racemosus translocation lines were developed by irradiation, pollen culture and gametocidal chromosome methods. In order to identify homozygous translocation lines and determine the exact location of the breakpoints involved in the translocations, 67 probes genetically or physically mapped previously on wheat chromosomes belonging to seven homoeologous groups were used for RFLP analysis. Three homozygous translocation lines were identified: T1BL.7Lr # 1S, T4BS.4BL-7Lr # 1 and T6AL.7Lr # 1S. In lines T1BL.7Lr # 1S and T6AL.7Lr # 1S, the breakpoint of chromosome 7Lr # 1 was located in the short arm between the area marked by clone MWG808 and that of ABG476.1, and the breakpoints of chromosomes 1B and 6A were both located near the centromere. In line T4BS.4BL-7Lr # 1S, the breakpoint of chromosome 7Lr # 1 was located in the short arm between the area marked by clone BCD349 and that of CDO595, the breakpoint of chromosome 4B was located in the long arm between the area marked by clone CDO541 and that of PSR164.

[Studies of development of disomic addition lines of Triticum aestivum-Haynaldia villosa via AABBDDDD octaploid].

Hexaploid hybrid between Triticum aestivum-Aegilops squarrosa (2n = 8x = 56, AABBDDDD) octaploid and Triticum durum-Haynaldia villosa (2n = 6x = 42, AABBVV) was self pollinated. The putitative T. aestivum-H. villosa 6V and 2V addition lines were creened by C-banding in F4 and chromosome configurations of PMC at MI were 0.14I + 20.42II + 1.50II and 0.10 I + 20.07II + 1.82II, respectively. Genomic DNA of 95-7 and 26-7 were digested by EcoRI, and Southern bloting was employed using group 6 probe Psr113 for 95-7 and group 2 probe BCD240 fro 26-7. The results showed that 95-7 and 26-7 had the same special bands as H. villosa did. Therefore, 95-7 and 26-7 further identified were T. aestivum-H. villosa 6V and 2V disomic addition lines.

[Construction of a transformation-competent artificial chromosome (TAC) library of a wheat-Haynaldia villosa translocation line].

Transformation-competent artificial chromosome (TAC) vector is able to clone and transfer large DNA fragments in plants and is a powerful tool for plant gene isolation and transformation. To clone important genes from wheat, a TAC genomic library was constructed from nuclear DNA of a 6VS/6AL wheat-Haynaldia villosa translocation line that harbor the gene Pm21 for resistance to powdery mildew. The library consists of 2.1 x 10(6) clones with an average DNA insert size of 35 kb, and represents in total 4.9 genome equivalents. The library was stored as clone pools in 96-well plates, and each pool contained about 1000 clones. TAC clones containing gene(s) of interest can be screened by a pooled-PCR/colony-hybridization strategy.

[Studies of the development of Triticum aestivum-Leymus racemosus translocation lines by pollen irradiation].

Pollens of 94G15 and 94G45, two wheat lines added to chromosome Lr.14 and Lr.2 of Leymus racemosus respectively were irradiated via 60Co-gamma rays and then used to hybridize with two common wheat varieties--Yangmai 5 and Mianyang 11. The five plants showing chromosome pairing between T. aestivum and L. racemosus at PMC MI stage were selected from seventeen examined plants of M1 progeny as the result of meiosis configuration analysis of chromosomes treated with Giemsa C-banding and fluorescent in situ hybridization. Among which, two T. aestivum-L. racemosus alien translocation lines--LW8(3)1 and LW11(3)1 were developed on the basis of further identification of M2 RTC chromosomes by C-banding and in situ hybridization. In addition, feasibility and effectiveness of the pollen irradiation in the development of T. aestivum-relatives alien translocations and the availability of the translocation lines of T. aestivum-L. racemosus are also discussed.

[Screening and study of RAPD markers tightly linked to wheat powdery mildew resistance gene Pm2].

The random amplified polymorphic DNA (RAPD) analysis was made in Pm2 near-isogenic lines (NILs) with 265 random primers. Seventeen out of the 256 tested primers amplified the polymorphic DNA in the NILs. Five of them showed the same discriminating results in more than four replications. The polymorphic bands were assigned to be OPM08(1600), OPI04(1700), OPH19(1100) and OPE09(900), respectively. Using these five polymorphic primers to detect 14 resistant materials with Pm2 and 9 susceptible materials without Pm2, only OPI04(1700) was amplified in 12 of the 14 resistant materials and absent in all of the 9 susceptible materials. Further RAPD analysis of (Chancellor x Ulka/8*Cc) F2 plants by primer OPI04 indicated that the genetic distance of OPI04(1700) to Pm2 was 12.2 +/- 3.3 cM.

Identification of RAPD markers tightly linked to wheat powdery mildew resistance gene Pm6.l.

Pm6 transferred from Triticum timopheevii L. to common wheat, is an effective resistance gene to powdery mildew disease caused by Erysiphe graminis f. sp. tritici. The RAPD technique, employing a total of 700 decamer primers, was used to identify polymorphic markers between resistant (IGVI463) and susceptible (Prins) near-isogenic lines. Primer OPV20 produced a 2,000 base pair (bp) reproducible fragment only in the resistant near-isogenic line. The 2,000-bp DNA fragment was present in all other introgression lines containing Pm6. Using the F2 mapping population from a cross of IGVI-463 (PI170914/7*Prins) x Prins, Pm6 was shown to be closely linked to the marker OPV20-2000 at a genetic distance of 3.0 +/- 2.2 cM. The marker was successfully used in detecting the presence of Pm6 in different genetic backgrounds.

Postexposure immunoprophylaxis of primary isolates by an antibody to HIV receptor complex.

mAb B4 is a monoclonal antibody directed against HIV receptor complex. The antibody had broad neutralizing activity against HIV and provided postexposure prophylaxis to hu-peripheral blood leukocyte (PBL)-severe combined immunodeficient mice and chimpanzees. B4 recognized a complex receptor site for HIV on the T cell surface that includes CD4 and also may be influenced by interaction with HIV coreceptors. mAb B4 preferentially neutralized primary HIV-1 isolates compared with T cell line-adapted strains, including syncytium-inducing and non-syncytium-inducing phenotypes, representatives from HIV-1 subtypes A-G, as well as HIV-2, simian immunodeficiency virus, and chimeric simian/human immunodeficiency virus (SHIV). Neutralization was demonstrated in both pre- and postinfection models. The administration of mAb B4 after infectious challenge totally interrupted the infection of hu-PBL-severe combined immunodeficient mice by PBL-grown HIV-1 and the infection of chimpanzees by chimp-adapted HIV-1. This mode of protection suggested that the anti-HIV receptor antibody is efficacious for prophylaxis after exposure to HIV and for prevention of maternal transmission and may be an effective antiretroviral agent for treatment.

Differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide ELISA.

We have identified continuous antigenic determinants within the amino acid sequences of the conserved nonstructural region containing proteins 2C and 3ABC of foot-and-mouth disease virus which can distinguish between the sera from vaccinated and infected animals. An ELISA based on a 3B peptide gave a positive reaction with sera from cattle, pigs, sheep and guinea pigs infected with all seven serotypes of the virus, but not with sera from vaccinated animals. In experiments with cattle and pigs to determine the duration of the antibody response, positive reactions were obtained as late as one year after infection. The advantages of using peptides from the nonstructural viral proteins instead of recombinant proteins for differentiating vaccinees from infected animals include their exquisite specificity, nonreactivity with antibodies against host cell-derived proteins (e.g. E. coli and insect cell proteins), and their ease of preparation.

Synthetic peptides entrapped in microparticles can elicit cytotoxic T cell activity.

Peptides from Plasmodium berghei circumsporozoite protein (CS) and influenza A virus nucleoprotein (NP) were entrapped in microparticles prepared from poly (lactide-co-glycolide) polymers, and the microparticles were administered parenterally to mice. After immunization with single or multiple doses, splenocytes were tested for a cytotoxic T cell (CTL) response and high levels of CTL activity were detected. The CTL induced were CD8+, MHC class I restricted, and could recognize virus infected cells. Peptide entrapped in microparticles of mean size < 500 nm were better inducers of CTL than larger microparticles (mean > 2 microns and above). Microparticles could also be used to deliver lipid modified peptides (lipopeptides) and elicited higher levels of cytolytic activity than either free peptide in microparticles or lipopeptide alone. Microparticles provide a novel way of inducing a CTL response using synthetic peptides.

Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew.

Several Triticum aestivum L.-Haynaldia villosa disomic 6VS/6AL translocation lines with powdery mildew resistance were developed from the hybridization between common wheat cultivar Yangmai 5 and alien substitution line 6V(6A). Mitotic and meiotic C-banding analysis, aneuploid analysis with double ditelosomic stocks, in situ hybridization, as well as the phenotypic assessment of powdery mildew resistance, were used to characterize these lines. The same translocated chromosome, with breakpoints near the centromere, appears to be present in all the lines, despite variation among the lines in their morphology and agronomic characteristics. The resistance gene, conferred by H. villosa and designated as Pm21, is a new and promising source of powdery mildew resistance in wheat breeding.

Isolation, characterization and application of a species-specific repeated sequence from Haynaldia villosa.

A species-specific repeated sequence, pHvNAU62, was cloned from Haynaldia villosa, a wheat relative of great importance. It strongly hybridized to H. villosa, but not to wheat. In situ hybridization localized this sequence to six of seven H. villosa chromosome pairs in telomeric or sub-telomeric regions. Southern hybridization to whea-H. villosa addition lines showed that chromosomes 1V through 6V gave strong signals in ladders while chromosome 7V escaped detection. In addition to H. villosa, several Triticeae species were identified for a high abundance of the pHvNAU62 repeated sequence, among which Thinopyrum bassarabicum and Leymus racemosus produced the strongest signals. Sequence analysis indicated that the cloned fragment was 292 bp long, being AT rich (61%), and showed 67% homology of pSc7235, a rye repeated sequence. Isochizomer analysis suggested that the present repeated sequence was heavily methylated at the cytosine of the CpG dimer in the genome of H. villosa.It was also demonstrated that pHvNAU62 is useful in tagging the introduced 6VS chromosome arm, which confers a resistance gene to wheat powdery mildew, in the segregating generations.

Transfer of Ph (I) genes promoting homoeologous pairing from Triticum speltoides to common wheat.

Diploid-like chromosome pairing in polyploid wheat is controlled by several Ph (pairing homoeologous) genes with major and minor effects. Homoeologous pairing occurs in either the absence of these genes or their inhibition by genes from other species (Ph (I) genes). We transferred Ph (I) genes from Triticum speltoides (syn Aegilops speltoides) to T. aestivum, and on the basis of further analysis it appears that two duplicate and independent Ph (I) genes were transferred. Since Ph (I) genes are epistatic to the Ph genes of wheat, homoeologous pairing between the wheat and alien chromosomes occurs in the F1 hybrids. Using the Ph (I) gene stock, we could demonstrate homoeologous pairing between the wheat and Haynaldia villosa chromosomes. Since homoeologous pairing occurs in F1 hybrids and no cytogenetic manipulation is needed, the Ph (I) gene stock may be a versatile tool for effecting rapid and efficient alien genetic transfers to wheat.

Serological analysis and biochemical characterization of monoclonal antibodies defining antigens of human hepatocellular carcinoma.

A panel of seven murine monoclonal antibodies reactive with human hepatocellular carcinoma (HCC) cell line, SK- HEP-1, resulted in the definition of four distinct antigen systems, designated HB4, HB5, HB1 and HJ2. HB4 antigen was found to be expressed specifically on HCC cell lines and fresh HCC specimens but not on normal liver. Immunoprecipitation tests suggest that the HB4 epitope may be a heat-stable carbohydrate determinant on a high molecular mass molecule. HB5 antigen was found to have less-restricted expression on a panel of normal adult tissues and on melanoma, astrocytoma, sarcoma, neuroblastoma and epithelial cancer cell lines. In fetal and adult liver, HB5 antigen localized to bile canaliculi and ducts. Under reducing conditions, three mAbs detected a Mr 140,000 glycoprotein using lysates of [125-I], [3-H]-glucosamine and [35-S]-methionine labeled SK-HEP-1 cells. Under non-reducing conditions an additional component of greater than Mr 200,000 was also detected. HB1 antigen was found on almost all monolayer cell lines and not on most cultured suspension cells. This antigen was also detected on cultured HCC cells inoculated into nu/nu mice. Immunoprecipitation experiments revealed that the HB1 antigen is a bimolecular complex with an Mr 170,000 alpha chain and Mr 130,000 beta chain under non-reducing conditions, and three subunits of Mr 140,000, Mr 30,000 and Mr 130,000 under reducing conditions. Two antibodies reacted with epitopes on the alpha chain. HJ2 antigenic determinant is a heat-stable component which could not be immunoprecipitated. This most widely expressed antigen was found in secreted form in many of the cells and tissues examined. These antibodies introduce new antigens which may serve as useful markers for the diagnosis, classification and investigation of HCC and other liver diseases.

Role of cytoplasm-specific introgression in the evolution of the polyploid wheats.

Studies of N-banded mitotic and meiotic karyotypes of Triticum turgidum L. (2n = 28; AABB) and Triticum timopheevii Zhuk. (2n = 28; AAGG) and hybrids between them, along with observations of meiotic pairing between telocentrics of the AB-genome chromosomes and their respective homologues and homeologues in T. timopheevii, showed that chromosome 4 (m4) of Triticum monococcum L. is present (as 4A(t)) in T. timopheevii but is lacking in T. turgidum. Neither 4A nor 4B pairs with 4A(t), but 4A pairs with 4G and, for this reason and because of its banding pattern, must be considered a B-genome chromosome. T. timopheevii chromosomes 4A(t) and 3A(t) are involved in a reciprocal translocation, and 2A(t), 1G, 2G, and 5G are also involved in translocations. Chromosome arm 4BL occasionally pairs with 7G. The satellites are on the short arms of chromosomes 6A(t) and 6G of T. timopheevii and 1B and 6B of T. turgidum. It is suggested that (i) T. timopheevii orginated as an allotetraploid of Aegilops speltoides Tausch/T. monococcum and (ii) T. turgidum was derived from T. timopheevii by introgressive hybridization with an unknown diploid species, which contributed its distinctive cytoplasm, chromosome 4B or a substantial portion of it, and additional chromosome segments. Rapid fixation of 4B in T. turgidum was ensured by cytoplasm-specific transmission.