Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind S C10-RNase in SI N. alata SC10SC10 and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia ؋ SI N. alata S C10SC10) hybrids with reduced levels of HT-protein continued to express SC10-RNase but failed to reject SC10-pollen. Control hybrids expressing both S C10-RNase and HT-protein showed a normal S-allelespecific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.
The genomic distribution of actin-related sequences in tomato was investigated using a cloned actin gene from soybean. Ten actin loci account for most of the hybridizing fragments observed with Southern analysis. Single loci were found on chromosomes 1, 3 and 10 and two loci on chromosome 4. One locus is linked to an unmapped isozyme marker, Sod-1. The four remaining actin loci are independent of each other and of any of the other markers tested. The number of actin loci in tomato (10) is greater than that estimated for soybean (8). As soybean is apparently a tetraploid and tomato a diploid, these results suggest that the number of actin loci has not been stable during the evolution of dicots. A number of these mapped loci lie in regions of the genome previously devoid of molecular markers and thus may be useful in basic and applied genetic research.
Three alleles of the self-incompatibility gene of Nicotiana alata have been cloned and sequenced. A comparison of the sequences shows a surprisingly low level of homology (56%) and the presence of defined regions of homology and variability. The homologous regions include the N-terminal sequence, most of the cysteine residues and glycosylation sites, as well as other blocks throughout the sequence. We interpret these conserved regions as "framework" and nonconserved regions as "hypervariable," following the terminology used to describe analogous regions in the IgG supergene family. The low level of overall homology forms the basis of a general method for isolating S-allele-specific cDNAs. Allele-specific antibodies can be generated using synthetic peptides corresponding to one of the variable regions. When applied to sections of the pistil, these antibodies label the intercellular matrix in the stigma and transmitting tissue of the style and the cell walls in the epidermis of the placenta. HindIII digestion of genomic DNA generates a characteristic pattern of S-gene fragments for each genotype. These restriction fragment length polymorphisms can be used to assign S-genotype to progeny arising from breeding experiments.
Homologies of tomato and pepper genes have been compared, and genetic linkage maps have been constructed based on a common set of cDNA clones and selected single-copy genomic clones. We report here that the gene repertoire of these two species is highly conserved, yet the linear order of the genes on the chromosomes has been greatly modified. Although the two species share the same number of centromeres, the chromosomal regions around those centromeres have undergone extensive rearrangements. Accompanying the extensive chromosome rearrangement has been a change in locus number for approximately 12% of the loci detected by random cDNA clones. Duplicated loci within each genome are normally found on different chromosomes and are not confined to one species, thus ruling out gene duplication as an explanation for the 4-fold higher DNA content of pepper. At least one of the duplications occurred since the divergence of tomato and pepper from their last common ancestor.The family Solanaceae contains many well-known plant species, including tomato, potato, eggplant, tobacco, pepper, andjimsonweed (Datura). The haploid DNA content of these species varies greatly: from 0.74 pg in tomato to 2.76 pg in pepper (1), yet they share the same basic chromosome number (x = 12). Since the basic chromosome number is generally conserved in this family, the question arises whether the number and order of genes are also conserved. In mammals there is good evidence for conservation of gene order even among species as divergent as cats and humans (2, 3).A linkage map of tomato (Lycopersicon esculentum) chromosomes has recently been constructed based largely on loci detected by hybridization to cDNA clones and selected single-copy genomic clones (4, 5). To gain a better understanding of the organization and evolution of plant chromosomes, we now report construction of a genetic linkage map of pepper (Capsicum spp.) based on hybridization to the same clones used to develop the tomato map. The use of these molecular probes provides a rapid method for identifying and mapping orthologous genes* in reproductively isolated species, a prerequisite to comparing gene number and order. MATERIALS AND METHODSThe majority of the clones used for mapping both tomato and pepper chromosomes were from a cDNA library derived from tomato leaf mRNA (4). The loci detected by hybridization to these tomato cDNA clones are designated CD], CD2, etc. Additional cDNA clones used for mapping were derived from pepper leaf mRNA, and the loci detected are designated with the symbol PC. Random single-copy tomato genomic clones (0.5-3.0 kilobases) found to hybridize to both tomato and pepper were also used as a source of probes, and these loci are designated TG. Clones of several known genes were also used in the comparative mapping study: small subunit ribulose bisphosphate carboxylase (Rbcs), chlorophyll a/b binding polypeptide (Cab), alcohol dehydrogenase (Adh), and the 45S ribosomal subunit (R45S). The Rbcs and Cab clones were selected from cDNA and phage lib...
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