Development of a marker assisted selection program for cacao.

ABSTRACT Production of cacao in tropical America has been severely affected by fungal pathogens causing diseases known as witches' broom (WB, caused by Moniliophthora perniciosa), frosty pod (FP, caused by M. roreri) and black pod (BP, caused by Phytophthora spp.). BP is pan-tropical and causes losses in all producing areas. WB is found in South America and parts of the Caribbean, while FP is found in Central America and parts of South America. Together, these diseases were responsible for over 700 million US dollars in losses in 2001 (4). Commercial cacao production in West Africa and South Asia are not yet affected by WB and FP, but cacao grown in these regions is susceptible to both. With the goal of providing new disease resistant cultivars the USDA-ARS and Mars, Inc. have developed a marker assisted selection (MAS) program. Quantitative trait loci have been identified for resistance to WB, FP, and BP. The potential usefulness of these markers in identifying resistant individuals has been confirmed in an experimental F(1) family in Ecuador.

Resistance gene homologues in Theobroma cacao as useful genetic markers.

Resistance gene homologue (RGH) sequences have been developed into useful genetic markers for marker-assisted selection (MAS) of disease resistant Theobroma cacao. A plasmid library of amplified fragments was created from seven different cultivars of cacao. Over 600 cloned recombinant amplicons were evaluated. From these, 74 unique RGHs were identified that could be placed into 11 categories based on sequence analysis. Primers specific to each category were designed. The primers specific for a single RGH category amplified fragments of equal length from the seven different cultivars used to create the library. However, these fragments exhibited single-strand conformational polymorphism (SSCP), which allowed us to map six of the RGH categories in an F(2) population of T. cacao. RGHs 1, 4 and 5 were in the same linkage group, with RGH 4 and 5 separated by less than 4 cM. As SSCP can be efficiently performed on our automated sequencer, we have developed a convenient and rapid high throughput assay for RGH alleles.

In situ reverse transcription to detect the cbbL gene and visualize RuBisCO in chemoautotrophic nitrifying bacteria.

AIMS: In situ methodologies targeting the cbbL gene were used to visualize cells of nitrifying bacteria. Both procaryotic in situ PCR (IS-PCR) and in situ reverse transcription (ISRT) protocols were employed to determine gene presence and expression, respectively. METHODS AND RESULTS: Aged-oligotropic seawater samples were inoculated with microbial assemblages containing a mixture of actively growing nitrifying bacteria, starved nitrifying bacteria, and heterotrophic bacteria without cbbL. After the molecular manipulations, we found that while all the nitrifiers (healthy or starved) with the cbbL gene were detected by IS-PCR, only the actively growing autotrophic nitrifiers with detectable levels of carbon fixation and nitrification activity were detected by ISRT analysis. CONCLUSION: These results show how IS-PCR and ISRT supplement each other, and their potential for the analysis of heterogeneous populations where an assortment of healthy and starved/dormant cells are expected.

Detection of avocado sunblotch viroid variants using fluorescent single-strand conformation polymorphism analysis.

A specific reverse transcription-polymerase chain reaction (RT-PCR) protocol has been developed for routine detection of avocado sunblotch viroid (ASBVd). Modifications in this diagnostic technique were made to enable fluorescent detection and variant identification using automated capillary electrophoresis (CE) and fluorescent single-strand conformation polymorphism (SSCP) analysis. Sixteen sequence variants characterized in a previous study were analyzed using CE-SSCP on two ABI 310 Genetic Analyzers. Significant differences were detected between data obtained from the two ABI 310 Genetic Analyzers indicating that an internal control must be run concurrently with the samples. The 16 variants could be classified into 11 groups based on the SSCP patterns. The statistical analysis of the migration rate data provided support for the visual differences in SSCP patterns. The use of SSCP in the ASBVd assay is easily accomplished and gives an estimate of the number of variants in crude samples extracted from infected avocado plants.

Application of RT-PCR for Indexing Avocado Sunblotch Viroid.

A method for the routine detection of avocado sunblotch viroid (ASBVd) in nucleic acid extracts of infected avocado tissues by reverse transcription-polymerase chain reaction (RT-PCR) was developed using ASBVd-specific primers. Amplified cDNA products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide slab gels. The size of the major RT-PCR product from ASBVd-infected tissue was estimated to be 250 bp. This product was absent from amplified extracts of uninfected tissue. The amplification product from ASBVd was sequenced by the dideoxynucleotide chain termination method, and the sequence was over 97% identical to the published sequence. The RT-PCR assay is sensitive enough to allow viroid detection without requiring large amounts of tissue, highly purified ASBVd, or molecular hybridization.

Amplification of the amoA Gene from Diverse Species of Ammonium-Oxidizing Bacteria and from an Indigenous Bacterial Population from Seawater.

Vol. 61, no. 7, p. 2705, column 2, lines 9 to 16: these sentences should read as follows. "These two sequences show 75% identity at the DNA level to the published N. europaea sequence and show 85% identity to each other. Interestingly, the regions that our primers were designed to target show 5 and 3 mismatches with the Nitrosospira sequence and 2 and 2 mismatches with the Nitrosolobus sequence for the forward and reverse primers, respectively." [This corrects the article on p. 2702 in vol. 61.].

Amplification of the amoA gene from diverse species of ammonium-oxidizing bacteria and from an indigenous bacterial population from seawater.

Because the chemolithotrophic ammonium-oxidizing bacteria are an integral component of nitrogen biogeochemistry, a sensitive and accurate method to detect this ecologically important group of microorganisms is needed. The amoA gene of these organisms encodes the active site of ammonia monooxygenase, an enzyme unique to this group of nitrifying bacteria. We report here the use of the PCR technique to detect the amoA gene from pure cultures of chemolithotrophic ammonium-oxidizing bacteria, ammonium oxidizers introduced into filtered seawater, and the natural bacterial population of an unfiltered seawater sample. Oligonucleotide primers, based on the published amoA sequence from Nitrosomonas europaea, were used to amplify DNA from pure cultures of Nitrosomonas europaea, Nitrosomonas cryotolerans, and Nitrosococcus oceanus and from bacteria in seawater collected offshore near the Florida Keys. Partial sequencing of the amplification products verified that they were amoA. These primers, used in conjunction with a radiolabeled amoA gene probe from Nitrosomonas europaea, could detect Nitrosococcus oceanus inoculated into filter-sterilized seawater at 10(4) cells liter-1. Native marine bacteria containing amoA could also be detected at their naturally occurring titer in oligotrophic seawater. Amplification of the gene for ammonia monooxygenase may provide a method to estimate the distribution and relative abundance of chemolithotrophic ammonium-oxidizing bacteria in the environment.

A cDNA encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Solanum tuberosum L.

A cDNA encoding potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, was cloned into phage lambda gt11. The clone represents the first cDNA for this enzyme from any eukaryotic source. The nucleotide sequence of the cDNA was determined, and its identity was confirmed through partial amino acid sequence analysis of the encoded enzyme. The cDNA contains a 1527-base pair open reading frame that encodes a polypeptide with a calculated molecular weight of 56,153. The amino terminus of the deduced polypeptide resembles a chloroplast transit sequence. Amino acid sequence identities between the mature potato enzyme and the homologous isoenzymes from Escherichia coli are only about 22%. The potato cDNA hybridized to various plant mRNAs that are all about 2 kilobases in size.

Increase of chalcone synthase mRNA in pathogen-inoculated soybeans with race-specific resistance is different in leaves and roots.

Soybeans (Glycine max [L.] Merr.) respond to pathogens by producing isoflavonoid-derived phytoalexins. Chalcone synthase (CHS) is the first enzyme of the flavonoid/isoflavonoid biosynthetic pathway. We investigated changes in the steady state levels of CHS mRNA and other specific mRNAs at increasing times after inoculation in two different race-specific interactions, one between leaves and the bacterium Pseudomonas syringae pv glycinea (Psg), and one between roots and the fungus, Phytophthora megasperma f. sp. glycinea (Pmg). The amount of CHS mRNA increases significantly in soybean leaves inoculated with an avirulent race of Psg but not with a virulent race or water. In contrast, the increase in CHS mRNA is similar in roots inoculated with zoospores of either an avirulent or virulent race of Pmg. CHS mRNA increases significantly in pathogen inoculated roots but not in uninoculated controls. Hydroxyproline-rich glycoprotein (HRGP) has been observed by others to increase in wounded or pathogen-inoculated plants. We report here that HRGP mRNA levels are greater in roots inoculated with an avirulent Pmg race than with a virulent race, but inoculation with either race causes a significant increase in HRGP mRNA with respect to controls. Calmodulin or ubiquitin mRNA do not increase in either uninoculated or inoculated roots and leaves. The possibility that race-specific resistance in soybeans is expressed differently in different organs of the plant is discussed.

Preparation of functional acetyl-CoA carboxylase mRNA from rat mammary gland.

Poly(A)+ RNA from lactating rat mammary glands was fractionated according to size by isokinetic sucrose gradient centrifugation to obtain a fraction enriched for acetyl-CoA carboxylase. In vitro translation of this RNA preparation yielded apparent full-length acetyl-CoA carboxylase with a molecular weight of 260,000. The synthesized protein was identified as acetyl-CoA carboxylase by specific immunoprecipitation. Tests with antiserum to fatty acid synthetase, revealed that the fractions containing acetyl-CoA carboxylase mRNA also contained mRNA for fatty acid synthetase; both of these mRNAs were approximately 10 kb. Fatty acid synthetase with a molecular weight of 250,000 was synthesized. Using an in vitro rabbit reticulocyte lysate translation system, we have shown that the amount of translatable acetyl-CoA carboxylase mRNA increases during lactation. On the fifth day postpartum the level of translatable acetyl-CoA carboxylase mRNA increased to a peak level seven times that on the day of parturition.

Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene.

A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.

Induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNAs in cultured plant cells by UV light or fungal elicitor.

The mRNAs encoding two enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and 4-coumarate:CoA ligase (4CL; EC 6.2.1.12), were induced in cultured parsley cells (Petroselinum hortense) either by irradiation with UV light or by treatment with elicitor, a cell-wall fraction of the fungus Phytophthora megasperma f. sp. glycinea. Two-dimensional gel electrophoresis of the encoded PAL and 4CL proteins revealed that the mRNAs induced by either treatment were very similar if not identical. RNA blot hybridization with cDNAs complementary to these mRNAs was used to measure changes in the mRNA amounts at various times after either treatment. Total cellular PAL and 4CL mRNA amounts increased coordinately after UV irradiation to a maximum at 7 hr and then decreased to uninduced levels by 30 hr with the same kinetics as observed previously for the changes in the translational activities. Treatment with the fungal elicitor also caused coordinated, but more rapid, changes in PAL and 4CL mRNA translational activities, with a sharp peak occurring 3 hr after the addition of elicitor. Corresponding changes in mRNA amounts were observed only for 4CL, whereas the amount of PAL mRNA continued to increase at least up to 20 hr after elicitor addition. Our results suggest that parsley cells respond to UV irradiation or addition of fungal elicitor by increased rates of transcription of genes involved in the synthesis of compounds related to UV or disease resistance, respectively.

UV-induction of chalcone synthase mRNA in cell suspension cultures of Petroselinum hortense.

DNAs complementary to poly(A)(+) mRNAs from UV-irradiated cell suspension cultures of parsley (Petroselinum hortense) were inserted into pBR322 and used to transform Escherichia coli strain RR1. A clone containing a DNA complementary to chalcone synthase mRNA was identified by hybrid-selected and hybrid-arrested translation. Large and rapid changes in the amount of chalcone synthase mRNA in response to irradiation of the cells was detected by RNA blot hybridization experiments. The pattern of changes coincided with that previously determined for the rate of chalcone synthase synthesis as measured either in vivo or with polyribosomal mRNA in vitro. The results are consistent with the hypothesis that induction of chalcone synthase by UV light is due to a transient increase in the rate of synthesis of chalcone synthase mRNA.

Coordinated regulation of 4-coumarate:CoA ligase and phenylalanine ammonia-lyase mRNAs in cultured plant cells.

4-Coumarate:CoA ligase (EC 6.2.1.12) from cell suspension cultures of parsley (Petroselinum hortense) was extensively purified. The enzyme behaved as a monomer with a molecular weight of approximately 60,000. A rabbit antiserum to the purified enzyme was obtained and used to determine the rates of 4-coumarate:CoA ligase synthesis under various conditions of induction, such as short term or continuous irradiation and treatment of the cells with an "elicitor" preparation from a fungal pathogen. In all cases, the time course of changes in the rate of synthesis, measured both in vivo and in vitro, was very similar for 4-coumarate:CoA ligase and a closely related enzyme, phenylalanine ammonia-lyase (EC 4.3.1.5). The results suggest that the mRNA activities encoding the two enzymes are regulated in a coordinated manner.

The lack of a phospholipid-exchange-protein activity in soluble fractions of Spinacia oleracea leaves.

When 14C-labelled liposomes prepared from Spinacia oleracea leaf lipids or 14C-labelled microsomal fraction ('microsomes') prepared from Spinacia oleracea leaf protoplasts were incubated with unlabelled intact chloroplasts, there was a considerable transfer of label to the chloroplasts. This transfer occurred in the absence of added protein, but was stimulated by soluble protein fractions from Spinacia oleracea leaves. The stimulation was heat-stable and decreased after dialysis of the protein fractions. Salt solutions, containing no protein, stimulated lipid transfer proportionally to their conductivity. In all cases, the lipid transfer was not protein-dependent, but rather resulted from the fusion of 14C- and 3H-labelled liposomes or microsomes with chloroplasts. It is proposed that this photosynthetic tissue contains no detectable lipid-exchange activity between liposomes, microsomes and chloroplasts and that lipid transfer between these organelles is achieved by non-protein-dependent means.

Subcellular localization of the starch degradative and biosynthetic enzymes of spinach leaves.

The subcellular localization of the starch biosynthetic and degradative enzymes of spinach leaves was carried out by measuring the distribution of the enzymes in a crude chloroplast pellet and soluble protein fraction, and by the separation on sucrose density gradients of intact organelles, chloroplasts, peroxisomes, and mitochondria of a protoplast lysate. ADP-Glucose pyrophosphorylase, starch synthase, and starch-branching enzymes are quantitatively associated with the chloroplasts. The starch degradative enzymes amylase, R-enzyme (debranching activity), phosphorylase, and D-enzyme (transglycosylase) are observed both in the chloroplast and soluble protein fractions, the bulk of the degradative enzyme activities reside in the latter fraction. Chromatography of a chloroplast extract on diethylaminoethyl-cellulose resolves the R- and D-enzymes from amylase and phosphorylase activities although the two latter enzyme activities coeluted. The digestion pattern of amylase with amylopectin as a substrate indicates an endolytic activity but displays properties unlike the typical alpha-amylase as isolated from endosperm tissue.

Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea.

This communication demonstrates that all de novo fatty acid biosynthesis in spinach leaf cells requires acyl carrier protein (ACP) and occurs specifically in the chloroplasts. Antibodies raised to purified spinach ACP inhibited at least 98% of malonyl CoA-dependent fatty acid synthesis by spinach leaf homogenates. Therefore, the presence of ACP in a compartment of the spinach leaf cell would serve as a marker for de novo fatty acid biosynthesis. A radioimmunoassay capable of detecting 10(15) mol (10(-11) g) of spinach ACP was developed to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach leaf protoplasts. All of the ACP of the leaf cell could be attributed to the chloroplast. Less than 1% of the ACP associated with chloroplasts resulted from binding of free ACP to chloroplasts. Of interest, ACP from Escherichia coli, soybean, and sunflower showed only partial crossreactivity with spinach ACP by the radioimmunoassay. These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the de novo synthesis of C16 and C18 fatty acids. These fatty acids are then transported into the cytoplasm for further modification and are either inserted into extrachloroplastic membrane lipids or returned to the chloroplast for insertion into lamellar membrane lipids.