Specific Hybrids Research Articles

Genetic research in a public–private research consortium: prospects for indirect use of Elite breeding germplasm in academic research

The creation of a public–private research partnership between plant breeding industry and academia can be beneficial for all parties involved. Academic partners benefit from the material contributions by industry and a practically relevant research focus, while industry benefits from increased insights and methodology tailored to a relevant set of data. However, plant breeding industry is highly competitive and there are obvious limits to the data and material partners are willing and able to share. This will usually include current and historic released cultivated materials, but will very often not include the elite germplasm used in-house to create new cultivars. Especially for crops where hybrid cultivars dominate the market, parental lines of hybrid cultivars are considered core assets that are never provided to outside parties. However, this limitation often does not apply to DNA or genetic fingerprints of these parental lines. We developed a procedure to take advantage of elite breeding materials for the creation of new promising research populations, through indirect selection of parents. The procedure starts with the identification of a number of traits for further study based on the presence of marker-trait associations and a priori knowledge within the participating companies about promising traits for quality improvement. Next, regression-based multi-QTL models are fitted to hybrid cultivar data to identify QTLs. Fingerprint data of parental lines of a limited number of specific hybrids are then used to predict parental phenotypes using the multi-QTL model fitted on hybrid data. The specific hybrids spanned the whole of the sensory space adequately. Finally, a choice of parental lines is made based on the QTL model predictions and new promising line combinations are identified. Breeding industry is then asked to create and provide progeny of these line combinations for further research. This approach will be illustrated with a case study in tomato.

Comprehensive Mapping of Receptor-Functioning Domains in Feline Leukemia Virus Subgroup C Receptor FLVCR1

Infection of cells by the highly anemogenic feline leukemia virus subgroup C (FeLV-C) is mediated by the heme exporter FLVCR1, a cell surface protein containing 12 potential transmembrane segments with six presumptive extracellular loops (ECLs). To identify FLVCR1 residues critical for mediating FeLV-C infection, we first independently isolated a human cDNA encoding the FLVCR2 protein that shares 52% identity to human FLVCR1, and we show that FLVCR2 does not function as a receptor for FeLV-C. Then, by generating specific hybrids between FLVCR1 and FLVCR2 and testing susceptibility of mouse cells expressing these hybrids to beta-galactosidase encoding FeLV-C, we identify FLVCR1 ECLs 1 and 6 as critical for mediating FeLV-C infection. Mouse cells expressing a hybrid protein containing FLVCR2 backbone with the ECL6 sequence from FLVCR1 were highly susceptible to FeLV-C infection. Using site-directed mutagenesis, we show that a single mutation of Asn463 in FLVCR2 ECL6 to an acidic Asp residue (a residue present in the corresponding position 487 in FLVCR1 ECL6) is sufficient to render FLVCR2 functional as an FeLV-C receptor. However, an Asp487Asn mutation in FLVCR1 ECL6 or substitution of the entire FLVCR1 ECL6 sequence for FLVCR2 ECL6 sequence does not disrupt receptor function. Subsequent substitutions show that residues within FLVCR1 ECL1 also contribute to mediating FeLV-C infection. Furthermore, our results suggest that FLVCR1 regions that mediate FeLV-C surface unit binding are distinct from ECL1 and ECL6. Our results are consistent with previous conclusions that infection of cells by gammaretroviruses involves interaction of virus with multiple receptor regions.

An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases

Bacterial type I polyketide synthases (PKSs) generate a structurally diverse group of natural products with a wide range of biological activities. Hybrid type I PKSs in which domains of one multifunctional polypeptide are replaced with components from heterologous systems have generated significant interest over the past decade. Almost invariably only one or several specific hybrids are made at a time and tested for functionality. This approach is slow, dependent upon a fortuitous choice of specific fusions points, and often leads to inactive or minimally active hybrid systems. We describe herein a method for generating and screening a library of hybrid pikAI complementation plasmids (encoding the loading domain and the first two extension domains of pikromycin PKS) able to restore pikromycin in a BB138 Streptomyces venezuelae pikAI-deletion mutant. In the first step the plasmid sequence encoding the loading domain AT(0)-ACP(0) was replaced by a counter selectable marker, sacB. DNA family shuffling was then used to generate a diverse library of chimeric AT(0)-ACP(0) fragments, which were used to replace sacB by lambda-Red-mediated in vivo recombination in an Escherichia coli host. This method resulted in the rapid and efficient generation of a large number of hybrid pikAI complementation plasmids, which were used to transform S.venezuelae BB138. A bioassay of over 4000 of these transformants successfully revealed three different PikAI hybrids which were able to lead to pikromycin production. The study suggests that most of the hybrids are not detectably functional, and underscores the need to generate and screen large and diverse libraries in which different fusion points are tried. The methodologies applied in this study address this need and can be used for directed evolution of any component of the PikPKS, and potentially other type I PKS systems.

About wild vine plants

<p style="text-align: justify;">Former frequent wild vines, in France, until the end of the 19th century are rare today, because of phylloxera and parasitic fungi appearances in Europe. Yet, ultimate plants of this keep genetic and phylogenic interest.</p><p style="text-align: justify;">A modest author’s experience on this subject is recorded.</p><p style="text-align: justify;">A more precise climatic natural conditions of their old expansion in France is proposed.</p><p style="text-align: justify;">Some botanic stamples discovered in Nantes Museum dated from 1846 demonstrate the presence of wild vines above the PLANCHON (1887) limit and show natural vegetation possibilities more to the north than the maximum line given by this author.</p><p style="text-align: justify;">Some wild plants still present in some islands of the Loire river are probably to day inter specific hybrids descendants.</p>

Variações genéticas em populações de Eucalyptus spp. detectadas por meio de marcadores moleculares

A tecnologia de marcadores moleculares, aliada às técnicas clássicas do melhoramento, pode contribuir significativamente para o conhecimento básico da cultura e do caráter estudado e para a geração e o desenvolvimento de produtos melhorados. O objetivo deste trabalho foi utilizar marcadores RAPD para detectar e maximizar a variabilidade genética em genótipos Eucalyptus, identificando cruzamentos favoráveis para um programa de melhoramento florestal, visando o uso múltiplo. Foram analisados 44 genótipos de híbridos naturais do gênero Eucalyptus, plantados na região noroeste de Minas Gerais. Os marcadores moleculares RAPD apresentaram poder de discriminação eficiente entre os 44 genótipos avaliados, constatando-se uma distância genética média entre os genótipos de Eucalyptus de 54% e divergência genética variando de 24 a 73%. Este fato indica que entre os indivíduos analisados existe uma ampla base genética, o que possibilita a manipulação desse material em programas de melhoramento. A distância genética entre os genótipos 5 e 9; 9 e 10; 9 e 19; 9 e 25; 9 e 33; 9 e 35; 9 e 36; 9 e 44; 10 e 33; 12 e 19; 12 e 33; e 12 e 39 apresentou-se maior ou igual a 70%. A análise de agrupamento estabelecida, utilizando UPGMA e o critério de corte de 80% da distância genética total, permitiu a formação de nove grupos distintos. Esses grupos apresentaram divergência genética média superior a 60%. A maior média de distância ocorreu entre o grupo I e os demais, com 67%. A avaliação por marcadores moleculares RAPD forneceu uma identificação direta da variação genética dos genótipos e, neste sentido, novos cruzamentos para produção de híbridos específicos poderão ser gerados, aumentando, assim, a divergência genética e a produtividade de derivados de madeira de qualidade superior para usos múltiplos em programas de melhoramento florestal.

Identification of Mytilus galloprovincialis larvae from the Galician rı́as by mouse monoclonal antibodies

The larvae of different species of bivalves are very similar in appearance during the early stages of development (D-shaped larvae). The method available today for an accurate identification of these larvae, based on the study of several ultrastructural morphological traits of the larval hinge, is impractical when the rapid identification and sorting of the larvae of bivalves are required for the numerous and complex plankton samples that field investigations generate. Therefore, in recent years, new methods such as molecular biology, biochemical, or immunological techniques are being used for efficient and accurate identification of those larvae. Here, we follow this trend and have attempted to produce specific monoclonal antibodies (mAbs) that could be used in immunological techniques for the identification of the larvae of the mussel Mytilus galloprovincialis in plankton samples from the Galician rı́as (NW of Spain). Mice were immunized with different amounts of D-shaped larvae of mussels and with larvae treated in different ways, then mouse spleen cells were fused with myeloma tumoral cells to obtain specific hybrids. We have obtained 12 mAbs against the immunizing larvae, and two of them, M2 and M3, showed a high specific recognition to M. galloprovincialis. These two antibodies do not show any cross-reaction when tested with the larvae of some other abundant species of bivalves coexisting in the area, such as Cerastoderma edule, Ostrea edulis, Aequipecten opercularis, Tapes decussatus, and Tapes philippinarum. Furthermore, each antibody displays a different pattern of staining in the larvae of mussels visualized by confocal microscope, indicating that they are directed against different antigen epitopes. The mAbs obtained, either used alone or in combination, could be very useful for the specific identification of the larvae of M. galloprovincialis, which is the only mussel species present in the area.

Yield and quality of three corn hybrids as affected by broiler litter fertilization and crop maturity

Specific hybrids, fertilization and maturity impact yields and nutrient quality of corn silage. To address these effects, three corn hybrids ( Zea mays L.) (Pioneer 3136, 3140, 3154) with five N fertilization (F) treatments were planted for 2 years and harvested at approximately 1/3, 2/3 or 100% milk line in a split–split plot design. Main treatments were No-F, 4.5, 9.0, 13.5 (Year 1) or 18.0 (Year 2) Mg ha −1 of broiler litter (BrL) or 202 kg ha −1 of NH 4NO 3. At harvest, milk lines and whole plant, stover and grain yields were determined. Whole plant and stover portions were chopped and subsampled for subsequent nutrient component and in vitro dry matter disappearance (IVDMD). Milk line advanced rapidly, especially during Year 1 that was a dry year. Whole plant yields were greatest for the 13.5/18.0 BrL treatments with no difference between 9.0 BrL and NH 4NO 3 treatments. Nutrient content varied with fertilizer treatment and year; during Year 1, NH 4NO 3 treatments had greater CP and lower ADF values, but the 13.5/18.0 BrL had the greatest CP content in Year 2, a wetter year. Whole plant IVDMD values were greater for NH 4NO 3 than the 13.5/18.0 BrL treatments during both years, but this occurred only during Year 1 for stover. Corn hybrid did not consistently affect yield, but during both years stover ADF was lower and IVDMD was higher for Hybrid 3136. These advantages existed in the whole plant only during Year 1. Increased maturity enhanced grain and whole plant yield, but effects on nutrient quality were variable. Appropriate application rates of BrL provide comparable yields to commercial fertilizer without an adverse effect on nutrient composition, although digestibility may be reduced.

T-cell antigen discovery (T-CAD) assay: a novel technique for identifying T cell epitopes

The identification of T cell epitopes is a critical step in evaluating and monitoring T cell mediated immune responses. Here, we describe a novel technique for simultaneously identifying class I and class II MHC restricted epitopes using a one-step protein purification system. This method uses Ni/chelate coated magnetic beads and magnetic separation to isolate poly-histidine tagged recombinant antigen from bacterial lysates. These beads, once coated with antigen, are also used to deliver antigen to APC where it is processed and presented to T cells. A colorimetric assay and ovalbumin specific, lac Z inducible, T cell hybridomas were used to validate the system. Further, using PSA specific hybrids, generated from T cells isolated from PSA secreting tumors, both class I and class II MHC restricted epitopes of PSA were identified. Additional characterization has shown that these peptides contribute significantly to the overall PSA specific response in vivo, and may represent the dominant epitopes of PSA.

Cytological and Karyotypic Studies in Four Beta Species

The karyotype and its components have been used to decipher karyo-evolutionary trends within taxa. With this in mind, chromosomal characteristics of four species of the genus Beta were studied to find karyotype relation­ ships, vis-a-vis meiotic features, and to ascertain the feasibility of using these species for development of inter­ specific hybrids and polyploids in sugarbeet (B. vulgaris L.) breeding programs in subtropical India. Four species of Beta were studied; B. vulgaris L., B. vulgaris ssp. maritima, B. vulgaris ssp. oriel/talis, and B. lomantogona; all with chromosome numbers of2n=18. Their karyotypes were generally asymmetric. Total haploid chromatin length ranged from 17.92 to 24.17 ~m, whereas individual chromosome size ranged from 1.47 to 3.15 ~m. According to Stebbin's classification of asymmetry, these species ranged from 2A to 4A, thereby confirming an evolution­ ary trend among the karyotypes. The karyotype of B. vul­ garis L. var LS-6 was most advanced and was classified as 4A. The karyotypes of B. vulgaris ssp. maritima and ssp. orientalis were classified as 3A. To further quantify gradations within a class of asymmetry, a chromosome Dispersion Index (DI) was calculated. A DI value of 0.479 for B. vulgaris L. var LS-6 confirmed its high degree of karyotypic specialization. Meiotically, all the species formed predominantly open bivalents with distal chiasma localization. Chiasma formation per bivalent decreased as the length of pairing blocks increased. This suggested a species specific chromosome condensation gradient because sugarbeet karyotypes are relatively constant during somatic and meiotic phases.

Introdução e avaliação de clones de capim-elefante (Pennisetum purpureum Schum.) em Campos dos Goytacazes, RJ

Quinze novos clones de capim-elefante e duas cultivares-testemunhas foram avaliados em um delineamento em blocos ao acaso, com quatro repetições. O experimento foi realizado no Setor de Forragicultura do LZNA/CCTA da Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, no período de fevereiro de 1995 a dezembro de 1997, totalizando 12 cortes. Avaliaram-se produção de matéria seca (MS), em kg/ha/corte, média de 12 cortes; produção de MS da época da seca, em kg/ha; participação da produção da época da seca em relação ao total anual, proporção de folhas na planta, altura das plantas (m), diâmetro do colmo na base (mm) e número de perfilhos por metro linear. Com exceção dos clones CNPGL 91: F28-1, F01-2, F13-2 e F10-2 e da cultivar Mineiro, todos os demais destacaram-se quanto à produção de MS por corte, enquanto as variáveis produção de MS da época da seca, participação da produção da época da seca em relação ao total anual e proporção de folhas na planta (com base em MS) não apresentaram diferenças significativas entre os clones avaliados. A análise multivariada demonstrou a superioridade dos clones dos grupos 2 (Pioneiro e CNPGL 91 F27-5) e 5 (CNPGL 91 F25-3 e CNPGL 91 F06-3) para produção de matéria seca, constituindo-se em grupos contrastantes e altamente adaptados às condições edafoclimáticas de Campos dos Goytacazes. As variáveis diâmetro do colmo, altura das plantas no corte na época das águas e altura das plantas no corte na época da seca foram consideradas as mais importantes para explicar a dispersão dos clones no plano bidimensional.

Highly sensitive detection of hybridization of oligonucleotides to specific sequences of nucleic acids by application of fluorescence resonance energy transfer.

We show a new application of fluorescence resonance energy transfer (FRET) in two stages to detect specific sequences of nucleic acids. In the first stage, two fluorescently tagged oligonucleotides hybridize with a complementary target molecule to produce FRET. The sequences of the oligonucleotides and spectral properties of fluorophores are chosen to provide a basis for an efficient energy transfer. In the next step, the specificity of hybridization is tested by competition of labeled probes with an excess of unlabeled oligonucleotides of the same sequence. The resulting emission spectra, one obtained in the excess of unlabeled donor probe and the other produced in the excess of unlabeled acceptor probe, are compared with the spectrum from the first stage to look for differences in the emission pattern of the fluorescent labels. We show that it is possible to detect the existence of specific hybrids composed of the two probes and complementary target molecule even in very unfavorable conditions, such as the presence of unhybridized probes in the final reaction mixture, secondary nonacceptor quenching of donor probe fluorescence, and strong background emission of acceptor produced by its direct excitation with a donor excitation light.

Mid-Season Stalk Breakage in Corn: Hybrid and Environmental Factors

Mid-season corn (Zea mays L.) stalk breakage in south central Nebraska resulted from 100 mph winds on 8 July 1993 and 80 mph winds on 1 July 1994. Corn growth stages at the time of damage ranged from V10 to V14. Observations of field studies were taken to determine: (i) the impact of stalk breakage on grain yields of different hybrids; (ii) yield compensation by remaining plants; and (iii) the influence of soil and cultural factors on susceptibility to wind damage. We recorded stalk breakage on over 100 corn hybrids in evaluation trials at one site in 1993 and at two sites in 1994 to address objective i. Twelve hybrids were chosen at each site and yield components from broken and standing plants were recorded separately to determine whether remaining standing plants compensated for broken plants (objective ii). We also evaluated stalk breakage on a N management study and a site-specific management/ variable rate study to address objective iii. In 1993 stalk breakage ranged from 7 to 88%, and grain yield was reduced 1.5 bu/acre for every 1% increase in stalk breakage. Breakage in 1994 ranged from 1 to 37% at one site and from 5 to 51% at the other site. Grain yield was reduced by 1.5 and 1.8 bu/acre for every 1% increase in stalk breakage, respectively. Remaining plants did not compensate for grain loss from broken plants at any site. Percentage yield loss is directly related to percentage stalk breakage. Stalk breakage increased with increasing N rate but was reduced with sidedress relative to preplant N application and no-till relative to conventional tillage. Stalk breakage in the site-specific management study correlated (positively) with soil organic matter content. Factors that accelerated plant growth early in the growing season increased susceptibility to stalk breakage. Unfortunately, management practices that result in slow early plant growth also limit yield potential. Planting tolerant hybrids is the best way to avoid losses. More information is needed on why individual plants break and why specific hybrids are more prone than others to mid-season stalk breakage.

INFLUÊNCIA DA POLINIZAÇÃO E DA MORFOLOGIA FLORAL NA FRUTIFICAÇÃO DE VARIEDADES DE LARANJA-DOCE

A espécie de laranjas doces (Citrus sinensis [L.] osbeck) é citada como sendo autógama. No entanto a alogamia tem sido demonstrada pelo aumento da produção de frutos com o uso de polinizadores eficientes, assim como pela existência de bons híbridos inter-específicos e ausência de bons híbridos intra-específicos. Como os programas de melhoramento via hibridação sexual em laranja doce dependem de maiores estudos, buscou-se avaliar o prendimento de frutos em 34 diferentes variedades representativas da espécie, no banco de germoplasma de Citrus do Centro de Citricultura Sylvio Moreira do Instituto Agronômico de Campinas, sob diferentes tratamentos. Foi observado que 68% das variedades prenderam frutos sob polinização aberta, 15% sob auto-polinização, 35% após cruzamento com o tangeleiro Minneola e 15% delas apresentaram prendimento de fruto após o cruzamento com o citrangeiro Troyer. Apenas uma variedade prendeu fruto quando as flores foram emasculadas e protegidas. Foi ainda observado que sob polinização aberta ocorreu maior prendimento nas variedades que apresentaram os estigmas acima das anteras e em pistilos maiores. Estes dados podem ser indicativos da predominância de alogamia nestas variedades, nas quais a polinização mostrou ser importante para o prendimento de frutos.

Fruit quality evaluation of plantains, plantain hybrids, and cooking bananas

Abstract Triploid plantains (Musa AAB) are among the most important horticultural crops of West Africa. However, all landraces are susceptible to black Sigatoka, a fungal disease caused by Mycosphaerella fijiensis, Morelet. To ameliorate the spread of this epidemic, exotic triploid cooking bananas (ABB), with resistance to black Sigatoka, were introduced into West Africa and new disease resistant polyploid Musa (AAAB) germplasm developed through conventional cross-breeding of French plantains (AAB). To ensure that released materials would compete with landraces on a fruit quality basis, an evaluation methodology was developed to compare exotic and hybrid Musa genotypes with West African triploid landraces. Tests showed that plantain hybrids and cooking bananas had heavier bunches than plantain landraces. The plantain hybrids generally produced more but smaller fruit compared with False Horn plantain (AAB). The fruit ripening time of plantain hybrids and the cooking banana cultivar Pelipita was longer than that of plantain landraces, and selected plantain hybrids had fruit with a dry matter content similar to that of the female parent. The fruit of ripe plantain landraces had a firmer texture than plantain hybrids and the landraces Agbagba and Bobby Tannap had lower levels of soluble solids at specific stages of ripening compared with the other Musa germplasm. These results showed that simple physical tests can be used to select for higher quality cooking bananas and plantain hybrids and that specific plantain hybrids, within the test group, showed a higher potential for market acceptance.

Triple Testcross Analysis to Detect Epistasis in Maize

Maize (Zea mays L.) breeders have successfully exploited heterosis by crossing inbred lines to develop hybrids. Epistatic effects can contribute to the expression of heterosis for specific hybrids. The hybrid B73 × Mo17 was a widely grown hybrid with exceptional performance in the central U.S. Corn Belt during the late 1970s and early 1980s. The objective of this study was to determine if epistatic effects contribute significantly to the performance of B73 × Mo17. With use of the triple testcross design, 100 random F2 plants from B73 × Mo17 were testcrossed to B73, Mo17, and their F1. The 300 testcrosses were evaluated at three locations in 1992 and two locations in 1993. Triple testcross analysis suggested that epistatic effects were important for ear length, number of kernel rows, ear height, and flowering traits. In the analysis of variance, additive by additive effects were not significant for grain yield, whereas additive by dominance and dominance by dominance effects were significant. The additive by additive by environment interaction was more important than additive by additive effects per se for grain yield. Epistatic deviations from the comparison of testcross means suggest that B73 had favorable additive by additive effects for grain yield, barren plants, kernel‐row number, ear height, and silk delay. Inbred Mo17 had favorable additive by additive effects for ear length. The presence of significant positive epistatic effects may have contributed to the expression of heterosis and could explain why B73 × Mo17 was an exceptional and widely grown hybrid.

Leaf area, light interception, and crop development in maize

Few models used to estimate the fraction of photosynthetically active radiation intercepted by maize crops (fIPAR) as a function of leaf area index (LAI) account for genotype differences and ontogenetic stage. In this study, the development of the fIPAR/GLAI relationship of three maize hybrids having contrasting plant type and grown in different environmental conditions was characterized. Three field experiments were conducted at Rojas (34°08′S), Argentina, on a silty clay loam soil, without nutrient restriction. The effects of sowing date and water regime were tested. Plant density was always 7 plants m −2. The fIPAR was calculated from measurements above and below the canopy, before and after maximum GLAI was attained. Hybrids differed significantly ( P < 0.05) in individual leaf area, leaf angle, and GLAI evolution. Exponential models adequately described ( r 2 > 0.77; n ≥ 15) the fIPAR/GLAI relationship for pre-maximum GLAI data of all hybrids. Nevertheless, maximum fIPAR was always below values quoted in the literature (< 0.90), and differences were detected among hybrids in the attenuation coefficient ( k) that were probably related to leaf angle and leaf area. For hybrids with a similar leaf angle at all leaf positions no difference existed in the fIPAR/GLAI relationship along the cycle (pre- and post-maximum GLAI). The relationship changed after tasselling for the hybrid with erect upper leaves. Ignoring such differences could be misleading when fIPAR/GLAI models are used to estimate canopy photosynthesis and hence biomass production, radiation-use efficiency, and kernel set of specific hybrids and growth stages.

G/C-modified oligodeoxynucleotides with selective complementarity: synthesis and hybridization properties.

Modified oligodeoxyribonucleotides (ODNs) that have unique hybridization properties were designed and synthesized for the first time. These ODNs, called selective binding complementary ODNs (SBC ODNs), are unable to form stable hybrids with each other, yet are able to form stable, sequence specific hybrids with complementary unmodified strands of nucleic acid. To make SBC ODNs, deoxyguanosine (dG) and deoxycytidine (dC) were substituted with deoxyinosine (dI) and 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one (dP), respectively. The hybridization properties of several otherwise identical complementary ODNs containing one or both of these nucleoside analogs were studied by both UV monitored thermal denaturation and non-denaturing PAGE. The data showed that while dI and dP did form base pairs with dC and dG, respectively, dI did not form a stable base pair with dP. A self-complementary ODN uniformly substituted with dI and dP acquired single-stranded character and was able to strand invade the end of a duplex DNA better than an unsubstituted ODN. This observation implies that SBC ODNs should effectively hybridize to hairpins present in single-stranded DNA or RNA.

Anti-DNA.RNA antibodies: an efficient tool for non-isotopic detection of Listeria species through a liquid-phase hybridization assay.

This study was undertaken to evaluate the potential of a new approach using anti-DNA.RNA monoclonal antibodies to detect Listeria in both pure culture and inoculated meat and meat products. A sensitive liquid-phase assay was first developed, based on the formation in solution of a hybrid between a 784-bp DNA probe, specific for the genus Listeria, and target rRNA. Monoclonal antibody and antisera raised against hybrid nucleic acids were then used in various immunoenzymatic assays to detect specific hybrids formed in solution. System 2, using a double sandwich enzyme-linked immunosorbent assay, and system 1, using a biotinylated probe, proved to be very effective. The method using biotin-streptavidin complex, however, resulted in a higher background signal. System 2 described here, using unlabeled probe, was more effective. This strategy allowed the detection of as little as 2.5 pg target RNA from pure culture and 500 cells from inoculated meat homogenate, even in the presence of other contaminating bacteria. The assay was more sensitive and could be completed within 3 h, as opposed to several days when conventional culture methods were used.

Xenia and Maternal Effects on Maize Agronomic Traits at Three Plant Densities

Kernel mass is a primary yield component in maize (Zea mays L.) governed by both xenia and maternal effects. Because kernel growth depends on assimilate supply, we wanted to learn if kernel‐weight genes also affect plant size and agronomic characters. We reciprocally crossed three strains known to differ for rates of grain filling [high (HC), random (RC), and low (LC) R‐nj color] with two kernel‐weight strains [large (LG), and small (SM)] to produce 12 hybrid strains. For 2 yr at two locations, we grew plants of each hybrid at 24 000, 48 000, and 72 000 plants ha−1 in plots arranged so that wind pollinated silks with either LG or SM pollen. Leaf number, leaf width, leaf length, and time to silk were measured to compute leaf area and average leaf expansion rate (ALER). We also measured grain yield and grain moisture concentration at harvest. In one of four environments, hybrids pollinated by the LG strain had 34% higher yields and 24 g kg−1 lower harvest moisture than hybrids pollinated by the SM strain. Increasing plant density delayed silking, increased leaves per plant, and decreased leaf width. Hybrid plants from the LG strain yielded 10% more than hybrid plants from the SM strain. They also had 12% higher grain moisture at harvest, 11% greater leaf surface area (mainly from more and wider leaves), and silked 0.6 d later than SM plants. Hybrid plants from the HC strain produced higher grain yields and greater leaf surface areas than plants from the RC or the LC strains. Leaf surface area was correlated (r = 0.80, P &lt; 0.01) with grain yield. Kernel growth genes influenced yield via xenia effects but mainly by genes in the plant. Although R‐nj color genes only increased yields of specific reciprocal hybrids, in properly planned hybrids these genes may complement genes that increase yield via larger kernels.

Liver regeneration: a comparison of in situ hybridization for histone mRNA with bromodeoxyuridine labeling for the detection of S-phase cells.

We developed an in situ hybridization technique for measurement of proliferative cell numbers through detection of histone mRNA in routinely fixed, paraffin-embedded tissue sections. Histone gene expression is coordinated with the cell cycle, and the increase in expression during S-phase permits unambiguous identification of cells undergoing DNA replication. Histone mRNAs were identified in routinely processed rat liver tissue by non-isotopic in situ hybridization with digoxigenin-labeled oligonucleotide probes. Specific hybrids were detected with alkaline phosphatase-labeled anti-digoxigenin antibody and visualized by BCIP-nitroblue tetrazolium indicator substrate. Unequivocal cytoplasmic labeling was observed in various cell types in the liver remnant during the first 72 hr after a two-thirds partial hepatectomy. The spatial and temporal patterns of histone labeling were almost identical to those obtained by staining with an antibody to bromodeoxyuridine. The identification of histone mRNA appears to be a reliable marker of the S-phase fraction, a technique with the further advantage that the tissue does not have to be first exposed to a nucleotide analogue. Hence, retrospective studies are possible. The probes can be applied to human and animal cells and tissues because the nucleotide sequences of histone genes are conserved.