Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of Poncirus trifoliata (L.) Raf.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.

A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifoliata (L.) Raf.

Resistance to citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. A dominant gene (Ctv) controlled CTV resistance in P. trifoliata. Twenty-one dominant PCR-based DNA markers were identified as linked to Ctv by bulked segregant analysis. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. Seven RFLP markers were then evaluated in 10 populations. Marker orders were consistent in all linkage maps based on data of single populations or on combined data of populations with similar segregation patterns. In a consensus map, the six closest marker loci spanned 5.3 cM of the Ctv region. Z16 cosegregated with Ctv. C19 and AD08 flanked Ctv at distances of 0.5 and 0.8 cM, respectively. These 3 markers were present as single copies in the Poncirus genome, and could be used directly for bacterial artificial chromosome library screening to initiate a walk toward Ctv. BLAST searches of the GenBank database revealed high sequence similarities between 2 markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata.

The chromosomes of Citrus and Poncirus species and hybrids: identification of characteristic chromosomes and physical mapping of rDNA loci using in situ hybridization and fluorochrome banding.

In situ hybridization of 18S-5.8S-25S rDNA probes labeled with biotin or rhodamine and 5S rDNA probes labeled with digoxigenin was used to locate rDNA sites on root-tip metaphase chromosomes of Citrus sinensis L. (2n = 2x = 18), Poncirus trifoliata L. Raf. (2n = 2x = 18), and Citrus x Poncirus hybrids (2n = 2x = 18). Counterstaining with the fluorochromes chromomycin A3 and DAPI uniquely identified many but not all chromosomes. C. sinensis had five 18S-25S rDNA sites, P. trifoliata had seven, and three different Citrus x Poncirus hybrids had five or six sites. Four 5S rDNA sites were detected, mostly linked to 18S-25S rDNA sites. Overall we observed high levels of chromosomal heterozygosity in all accessions examined.

Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange.

Transgenic Washington navel orange [Citrus sinensis (L.) Osbeck] plants were obtained using Agrobacterium-mediated transformation of seedling epicotyl tissue. An average of 45% (58 out of 128 segments) of the epicotyl segments produced shoots expressing the ß-glucuronidase (GUS)-intron reporter gene when using Agrobacterium strain C58 C1, compared to 29% (38 out of 128 segments) for EHA101-5 and 0% for LBA4404. Co-culture of 21-day-old Washington navel epicotyl stem segments gave greater transformation efficiency than co-culture of 35- or 56-day-old stem segments. After 6 weeks, regenerated shoots were micro-grafted in vivo onto seedling rootstocks of Carrizo citrange. Stable integration of the transgene sequence was confirmed by expression of the plant intron-containing GUS gene, PCR and Southern hybridization. The apomictic (non-zygotic) state of the transgenic plants was confirmed by isoenzyme and random amplified polymorphic DNA analyses. More than 50 transgenic plants have been obtained and are growing in the greenhouse.

Development of molecular markers linked to a gene controlling fruit acidity in citrus.

Fruit juice pH, titratable acidity, or citric acid content was measured in 6 populations derived from an acidless pummelo (pummelo 2240) (Citrus maxima (Burm.) Merrill). The acidless trait in pummelo 2240 is controlled by a single recessive gene called acitric. Using bulked segregant analysis, three RAPD markers were identified as linked to acitric. RAPD marker OpZ20410, which mapped 1.2 cM from acitric, was cloned and sequenced, and a sequence characterized amplified region (SCAR) marker (SCZ20) was developed. The SCZ20-410 marker allele that is linked to the acitric allele occurs only in pummelo 2240 and other pummelos, and therefore, this SCAR marker should be useful as a dominant or codominant marker for introgressing acitric into mandarins and other citrus species. Using the cloned OpZ20410 band as a hybridization probe revealed a codominant RFLP marker called RFZ20 that mapped 1.2 cM from acitric. Progeny homozygous (acac) for the acitric allele had citric acid content below 10 μM, the minimum level detectable by high pressure liquid chromatography. The citric acid content of fruit juice from progeny predicted to be heterozygous (Acac) for acitric by the above markers was about 30% lower than that of juice from individuals predicted to be homozygous (AcAc) for the normal acid allele. Markers OpZ20410, SCZ20, and RFZ20 were highly polymorphic among 59 citrus accessions, and using one or more of these markers would allow citrus breeders to select seedling progeny heterozygous for acitric in nearly all crosses between pummelo 2240 or its offspring and other citrus genotypes.

Molecular characterization of the mitochondrial citrate synthase gene of an acidless pummelo (Citrus maxima).

Pummelo (Citrus maxima [Burm.] Merrill) cDNAs encoding mitochondrial citrate synthase (mCS) were cloned by reverse transcription of juice-sac poly(A)+ mRNA, followed by Taq Polymerase-mediated amplification. The nucleotide sequence of the citrus gene (cit1) is 77% conserved relative to plant mRNAs for mCS. The encoded polypeptide includes a mitochondrial targeting signal at its amino terminus; all 20 putative active-site residues of the citrus enzyme are conserved. Southern hybridization showed that citrus cit1 is a single-copy gene. A polymorphism associated with cit1 did not cosegregate with fruit acidity indicating that acitric, the gene causing the acidless phenotype of pummelo 2240, is not an allele of cit1 locus. Quantitative detection of cit1 mRNA showed that transcript levels are not developmentally regulated in juice sacs; no differences were observed between high- and low-acid genotypes.

A genetic map of citrus based on the segregation of isozymes and RFLPs in an intergeneric cross.

Isozymes and restriction fragment length polymorphisms were used as markers in the construction of a genetic map of the citrus nuclear genome. The map was based on the segregation of 8 isozyme, 1 protein, and 37 RFLP loci in 60 progeny of a cross of two intergeneric hybrids, 'Sacaton' citrumelo (Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) and 'Troyer' citrange (C. sinensis (L.) Osbeck x P. trifoliata), often used as rootstocks. The map contains 38 of 46 studied loci distributed on ten linkage groups. A genome size of 1,700 cM was estimated from partial linkage data. Approximately 35% of the genome should be within 10 cM and 58% within 20 cM of the mapped markers. Eight loci in three linkage groups and 1 unlinked locus deviated significantly from Mendelian segregation.

Genetic analysis of Verticillium wilt tolerance in cotton using pedigree data from three crosses.

Three crosses and descendant generations were used in a field study of the inheritance of tolerance to Verticillium wilt, caused by Verticillium dahliae Kleb., in upland cotton (Gossypium hirsutum L.). The tolerant cultivar 'Acala SJC-1' was crossed to more susceptible parents, breeding line S5971 and cultivars 'Acala 4-42' and 'Deltapine 70'. Seven generations were evaluated for each cross: the two parents (P1 and P2), F1; F2, F3, and reciprocal backcrosses (B1 and B2). The genetic control of tolerance in these crosses appears to involve more than one gene, based on an unsatisfactory fit to expected phenotypic distributions for the generations under a single-locus model. An analysis of generation means indicated that pooled additive and pooled dominance effects over loci were adequate to explain the variation among generations for crosses of SJC-1 X S5971 and SJC-1 X DPL70. Tolerance in these crosses appeared to be controlled by recessive factors. For the SJC-1 X 4-42 cross, an adequate fit to a digenic epistatic model was not possible, and none of the genetic parameters except the F2 mean were significant. Heritabilities for tolerance to Verticillium wilt, determined from regressions of F3 progeny on F2 parents for the crosses of SJC-1 X S5971 and SJC-1 X DPL70, ranged from 0.12 to 0.28. Therefore, individual plant selection for improved tolerance is expected to be inefficient.

Catalytic properties of alcohol dehydrogenase isozymes specified by duplicate genes in the diploid plant Stephanomeria exigua.

The alcohol dehydrogenase (ADH) isozymes induced in flooded roots of the diploid plant Stephanomeria exigua are specified by tightly linked genes comprising a complex locus, Adh1. Individuals homozygous for a complex with two active genes which specify electrophoretically different subunits have three ADH-1 isozymes, two intragenic homodimers and an intergenic heterodimer. Individual isozymes were partially purified from plants homozygous for several different Adh1 complexes and apparent Km values for acetaldehyde, ethanol, NAD, and NADH and responses to temperature, pH, and two different alcohols were determined. The two homodimeric enzymes specified by a particular Adh1 complex generally differed in one or more of the properties studied, and in three of four cases, intergenic heterodimers differed significantly from intermediacy, often having lower Km values than either homodimer. None of the isozymes studied could be considered greatly divergent or defective. Constraints on evolution of duplicate genes which form intergenic heterodimers are considered.

Biochemical properties and level of expression of alcohol dehydrogenases in the allotetraploid plant Tragopogon miscellus and its diploid progenitors.

This study demonstrates that homoeologous genes in two diploid plant species that specify different amounts of an enzyme maintain the same relative level of expression in an allotetraploid derivative. The three predominant alcohol dehydrogenase (ADH) isozymes (DD, DP, PP) in seeds of the recently evolved allotetraploid plant Tragopogon miscellus (Compositae) are dimers specified by Adh3-D and Adh3-P genes derived from its diploid progenitors T. dubius and T. pratensis. Seeds of T. pratensis contain twice as much ADH activity as those of T. dubius, while T. miscellus is intermediate. The three isozymes were similar in a number of catalytic properties; the densitometric ratio of the isozymes purified from T. miscellus was 1 DD : 4DP : 4PP for both ADH activity and protein; and dissociation-reassociation of the DP enzyme gave a 1:2:1 ratio of the three isozymes. Therefore, the enzymes were similar in specific activity, but twice as many P as D subunits were present in active enzymes in T. miscellus, precisely the difference in activity between the parents. In T. miscellus, the specific activity of ADH and its activity per mg tissue are intermediate to those of the diploids, because relative expression of the Adh gene in each genome is not influenced by the presence of the other genome.

Alcohol Dehydrogenase in the Diploid Plant STEPHANOMERIA EXIGUA (Compositae): Gene Duplication, Mode of Inheritance and Linkage.

Study of the biochemical genetics of alcohol dehydrogenase (ADH) in the annual plant Stephanomeria exigua (Compositae) revealed that the isozymes are specified by a small family of tightly linked structural genes. One set of ADH isozymes (ADH-1) was induced in roots by flooding, and was also expressed in thickened unflooded tap roots, stems, ovaries and seeds. As in other plants, the enzymes are dimeric and form homo- and heterodimers. An electrophoretic survey of ADH-1 phenotypes in two natural populations revealed seven different ADH-1 homodimers in various phenotypes having one to eight enzyme bands. Genetic analysis of segregations from crosses involving 59 plants showed that the ADH-1 isozymes are inherited as a single Mendelian unit, Adh1. Adh1 is polymorphic for forms that specify one, two, or three different ADH-1 subunits (which combine to form homo- and heterodimers), and are expressed co-dominantly in all genotypic combinations. Staining intensity of enzymes extracted from various homozygous and heterozygous plants indicated that the different subunit types specified by Adh1 are produced in approximately equal amounts. These observations suggest that Adh1 is a compound locus consisting of one to several tightly linked (0 recombinants among 579 testcross progeny), coordinately expressed structural genes. The genes in the two triplications also occur in various duplicate complexes and thus could have originated via unequal crossing over. The ADH-2 isozyme found in pollen and seeds is apparently specified by a different gene, Adh2. Adh1 and Adh2 are tightly linked (0 recombinants among 81 testcross progeny).