Robert Bernatzky

Restriction fragment length polymorphism

In the 1960s, a group of enzymes were discovered in bacteria that could degrade incoming bacteriophage DNA and would ‘restrict’ their establishment in the cell [2,6]. These enzymes, known as restriction enzymes [19], have proved very valuable in modern manipulations of DNA. The first enzymes described (type I) require various cofactors and lack sequence specificity for their sites of cleavage. However, a second class (type II) were discovered that require only Mg2+ asa cofactor and have the distinct advantage that they recognize and cleave very specific sequences. Type II restriction enzymes are therefore capable of reducing complex DNA, such as plant nuclear DNA, into a population of fragments with discrete sizes. At least 475 restriction endonucleases have been described to date [24].

Breakdown of self-incompatibility in tetraploid Lycopersicon peruvianum: inheritance and expression of S-related proteins

Abstract Lycopersicon peruvianum displays gametophytic self-incompatibility (GSI). We have isolated self-compatible (SC) tetraploids of L. peruvianum from tissue-cultured leaves and have explored the expression and inheritance of their S-related proteins. The Srelated protein profiles of styles of SC tetraploids were indistinguishable from the diploid self-incompatible (SI) explant source based on SDS-PAGE. All progeny obtained from self-fertilization of two tetraploids were SC. Cloned cDNA sequences of the S-related proteins were used to determine the inheritance of this locus in these progeny through Southern hybridization. The allelic ratio, as determined from the intensity of DNA restriction fragments, was consistent with the predicted ratio if only pollen bearing two different alleles was successful in achieving fertilization. All progeny obtained had at least one copy of each allele, and individuals fully homozygous for either allele were not found, indicating that pollen grains bearing two identical alleles were inhibited. In addition, the level of expression of the S-related proteins in the progeny correlated with the allelic dosage at the DNA level. We demonstrate that the observed self-compatibility in the tetraploids was not caused by an alteration in the expression of S-related proteins.

Molecular diversity at the self-incompatibility locus is a salient feature in natural populations of wild tomato (Lycopersicon peruvianum)

A cDNA encoding a stylar protein was cloned from flowers of self-incompatible wild tomato (Lycopersicon peruvianum). The corresponding gene was mapped to the S locus, which is responsible for self-incompatibility. The nucleotide sequence was determined for this allele, and compared to other S-related sequences in the Solanaceae. The S allele was used to probe DNA from 92 plants comprising 10 natural populations of Lycopersicon peruvianum. Hybridization was conducted under moderate and permissive stringencies in order to detect homologous sequences. Few alleles were detected, even under permissive conditions, underscoring the great sequence diversity at this locus. Those alleles that were detected are highly homologous. Sequences could not be detected in self-incompatible Nicotiana alata, self-compatible L. esculentum (cultivated tomato) or self-compatible L. hirsutum. However, hybridization to an individual of self-incompatible L. hirsutum revealed a closely related sequence that maps to the S locus in this reproductively isolated species. This supports the finding that S locus polymorphism predates speciation. The extraordinarily high degree of sequence diversity present in the gametophytic self-incompatibility system is discussed in the context of other highly divergent systems representing several kingdoms.

Methods of Southern blotting and hybridization

At present, the technique that remains central to RFLP analysis is Southern blotting and hybridization [15]. Briefly, the procedure involves the enzymatic cleavage of DNA with restriction endonucleases, the separation of the resultant fragments by electrophoresis through an agarose gel and the transfer of the fragments from the gel to a membrane that binds nucleic acids. The fragments are transferred in a single-stranded state by treating the gel with alkali which disrupts the hydrogen bonding between the native double-stranded DNA. The membrane-bound DNA is then hybridized in solution to specific radio labelled DNA sequences (probes) that anneal to complementary sequences on the membrane. Through autoradiography, one or a few fragments can be visualized separately from the very large number of fragments that make up a complex genome.

S-related protein can be recombined with self-compatibility in interspecific derivatives ofLycopersicon

Stylar proteins involved in the self-incompatible (SI) response ofLycopersicon hirsutum have been identified and mapped to the locus that controls SI (S locus).L. esculentum, a self-compatible (SC) species of cultivated tomato, does not display these proteins. Hybrids between SCL. esculentum and SIL. hirsutum are self-sterile despite these individuals bearing pollen containing theS allele ofL. esculentum. In progeny derived from backcrossing the hybrids toL. esculentum, there was a strong correlation between the presence of theS allele fromL. hirsutum and self-infertility. However, this relationship was uncoupled in a number of backcross (BC) progeny. The SI response appeared to be nonexistent in two self-fertile BC individuals that were heterozygous for theS allele ofL. hirsutum, based on Mendelian segregation of a tightly linked DNA marker,CD15, in selfed progeny. Among these progeny self-fertile individuals that were homozygous for theL. hirsutum allele of the linked marker were also determined to be homozygous for anS-related protein ofL. hirsutum through test crosses withL. esculentum. Therefore, plants were produced that were homozygous for a functionalS allele but were self-fertile. This result and other evidence suggest that theS-related proteins are not sufficient to elicit a self-incompatible response inL. esculentum and that there is a mutation(s) inL. esculentum somewhere other than theS locus that leads to self-compatibility.

Genetic mapping and protein product diversity of the self-incompatibility locus in wild tomato (Lycopersicon peruvianum)

Phenotypic diversity of self-incompatibility (S) alleles within nine natural populations ofLycopersicon peruvianum was investigated. Only 7 incompatible responses were observed of a total of 276 unique combinations tested, on the basis of controlled pollinations, indicating the large number of alleles that exist within these populations. Molecular weight polymorphism for specific major stylar proteins observed on SDS-PAGE was also evident in two of the populations examined. Five proteins were shown to map to theS locus and to be associated with differentS alleles through controlled pollinations and segregation of the proteins. Two of theseS related proteins had been described previously in terms of spatial and temporal expression consistent with their involvement in self-incompatibility (Mauet al., Planta169, 184–191, 1986). A mapping population derived from a fully compatible cross was used to establish linkage of theS locus to two DNA markers,CD15 andTG184, that lie on chromosome 1. The order of the markers and estimates of map distances are given.

A nuclear sequence associated with self-incompatibility in Nicotiana alata has homology with mitochondrial DNA.

SummaryA 1.0-kb nuclear fragment located 5′ to a coding sequence associated with self-incompatibility in N. alata shows homology with mitochondrial chromosomal DNA on Southern blots. This sequence is also present in the mitochondrial DNA of two species of tomato, L. esculentum and L. pennellii, but shows no homology to mtDNA of Zea mays. The homologous mitochondrial fragment from N. alata was cloned and sequenced. A short region of 56 bp matches the nuclear sequence in 53/56 bp. Other matched but misaligned segments flank the 3′ end. The nuclear sequence is marked at the 5′ end by two 8 bp direct repeats. The function of the nuclear sequence is not known although, it is located 397 bp upstream from the site of transcription of the self-incompatibility gene. The mitochondrial sequence contains only limited open reading frames and the nuclear sequence has none. There is evidence that additional segments of the mitochondrial clone hybridize to other nuclear sequences. The exchange of sequences between the mitochondrial and nuclear genomes of plants is discussed.

The identification and characterization of a genetic marker linked to hypersensitivity to the cherry leafroll virus in walnut

Walnut blackline disease, caused by the walnut strain of the cherry leafroll virus, causes fatal necrosis of the graft union between susceptible, infected scions of Persian walnut (Juglans regia L.) and hypersensitive, resistant rootstocks. A backcross breeding program to transfer hypersensitivity to cherry leafroll virus from the Norther California black walnut (Juglans hindsii (Jeps.)), into Persian walnut was begun in 1987. Hypersensitivity to the virus is inherited as a single, dominant gene. The current procedures for identifying hypersensitive backcross progeny are slow and labor intensive. Bulks of DNA from backcrosses that were either hypersensitive or susceptible to cherry leafroll virus were compared using randomly amplified polymorphic DNA. One random decamer, sequence 5′-CTCCTGCCAA-3′ (OP-K15), produces a polymorphic fragment of about 720 bp that has about 7% recombination with hypersensitivity to cherry leafroll virus in our backcross populations. The polymorphic fragment was cloned and converted into a restriction fragment length polymorphism to demonstrate that it is a distinct, low-copy sequence.

Self-incompatibility is codominant in intraspecific hybrids of self-compatible and self-incompatible Lycopersicon peruvianum and L. hirsutum based on protein and DNA marker analysis

Self-compatibility was investigated separately in two species of tomato, Lycopersicon peruvianum and L. hirsutum. The codominant expression of self-compatibility (SC)/self incompatibility (SI) was established using intraspecific hybrids of SC and SI hybrids. In SC L. peruvianum, a major stylar protein of approximately 29 kDa cosegregates with self-compatibility in the progeny of SC/SI hybrids. The SC/SI hybrids are self-fertile, but only partially so, since the SI allele present in the hybrids is capable of eliminating certain genotypes in the resultant progeny. In L. hirsutum, the majority of hybrids between one accession of SI L. hirsutum f. hirsutum and one of SC L. hirsutum f. glabratum are self-fertile. Analysis of the progeny revealed that the SC and SI alleles are codominant in this species as well. A protein product for the SC allele is not obvious in style extracts of L. hirsutum f. glabratum. Segregating progeny from SC/SI hybrids of L. hirsutum were used to map the S locus against five RFLP markers on chromosome 1, and estimated map distances are given. In addition, evidence is presented that indicates that one of the DNA markers, CD15, is duplicated in L. hirsutum f. glabratum, and the duplication is not linked to the S locus.

Protein expression of a self-compatible allele from Lycopersicon peruvianum: introgression and behavior in a self-incompatible background

Lycopersicon peruvianum (wild tomato) is a gametophytic self-incompatible (SI) species. One natural population has been shown to harbor a self-compatible (SC) allele. A stylar protein associated with the self-compatibility allele has been elucidated using SDS-PAGE. The temporal and spatial expression of this protein is presented and compared with protein expression of two SI alleles. Hybrids containing the SC and SI alleles were used in a backcrossing program to introgress the SC allele into SI backgrounds in six independent lines. Controlled pollinations and SDS-PAGE were used to identify and select classes of progeny. After four backcross generations (approximately 97% recovery of the SI backgrounds) the SC allele still confers self-fertility in lines that contain this allele, providing evidence that the mutation to SC occurred at the S-locus and that the associated protein is likely responsible.