Linkage map construction in the outcrossing species Lolium perenne.

Genetic maps have proven useful tools in several fields of application. First of all, they allow to get insight into the genome organization of a species, and to compare the genome structures of different species. Genetic maps are also useful for the identification of genomic regions involved in physiological processes, and are valuable tools for the positional cloning of genes. In the framework of a project with the aim of identifying genomic regions involved in disease resistance in Lolium spp., we constructed genetic maps using different marker systems. In the ryegrass species L. perenne and L. multiflorum self-pollination is prevented by a very efficient self incompatibility system. This has clear implications in mapping studies: inbred lines and double haploids are difficult to produce, and if produced low seed yields are obtained. For this reason, we created several mapping populations by crossing two highly heterozygous unrelated plants. This implies that at any given locus, up to four different alleles might be segregating in the offspring. Different marker systems were used for the construction of the genetic maps. In first instance, AFLP was used as a high throughput marker system. This allowed to generate a high number of DNA-markers useful for map construction in a quick way. The drawback of the AFLP technique is twofold: AFLP markers are 'anonymous', and are dominant (not all alleles at a locus can be detected). Co-dominant marker systems, which allow to detect all alleles present at any locus, are much more informative and are required for the construction of detailed linkage maps in outcrossers. In this study, we used three co-dominant marker systems: SSR, STS and RFLP. The RFLP markers were generated using heterologous probes derived from other monocots, what should allow to analyze the syntenic relationships between ryegrass and other monocots. Using these markers generated with different techniques, a genetic map of ryegrass has been constructed suitable for QTL analysis and comparative genetics.

Stability and expression of transferred DNA in F1 tobacco transformants studied at various states of differentiation.

Grafts from the SR1 tobacco crown-gall lines NT1 (having a deletion eliminating part of the transferred (TL)-DNA auxin locus) and NT2 (having an IS60 insertion in gene 2 of this auxin locus) were cross-pollinated with pollen from nontransformed SR1 tobacco plants. One half of the resulting F1 progeny resembled the female parent ("transformed" NT1-like and NT2-like seedlings respectively) and one half resembled the male parent ("non-transformed" SR1-like seedlings). For three states of differentiation (callus, shoot, graft) all phenotypic markers of the transformed seedlings studied were identical to those of the transformed female parent. Most phenotypic markers of non-transformed seedlings corresponded with markers of the male parent. Unlike the SR1 male parent, however, the SR1-like seedlings showed the maternal traits hyperstyly and male sterility. These two traits were inherited by 100% of the F1 seedlings studied. Ninety percent of the non-transformed F2 seedlings were still male-sterile whereas in as much as 50-100% of the non-transformed F3 progeny, male fertility had been restored. The SR1-like F1 seedlings did not contain any T-DNA. At the level of restriction-fragment analysis the T-DNA structures of all 22 NT1-like seedlings examined were identical to the T-DNA structure of their female parent NT1. The steady-state level of transcripts 4 (cytokinin locus) and 6a/6b relative to transcript 3 (octopine-synthase locus) was less in shoots and grafts than in callus. Observed variation in shoot morphology among the twenty-two NT1-like seedlings was not correlated with T-DNA structure, organization and expression at the level of steady-state mRNA. The T-DNA structure of NT2 and its transformed seedlings deviated from regular border-to-border TL-DNA, in that it extended beyond the left border repeat.

Structure, organization and expression of transferred DNA in Nicotiana plumbaginifolia crown-gall tissues.

Data are provided which show that transferred DNA (T-DNA) present in Nicotiana plumbaginifolia crown-gall lines in most cases was scrambled and not intact. Both wild-type, and 'rooter'- and 'shooter'-type mutants of octopine-type Agrobacterium tumefaciens were used to infect N. plumbaginifolia plantlets, cultured in vitro. Resulting tumors were excised from the plantlets and cultured for more than three years. During subculturing the tumor lines were scored for the following phenotypic traits: phytohormone autonomous growth in vitro (Aut(+)), spontaneous shoot regeneration (Reg(+)), root deficiency of shoots (Rod(+)), octopine production (Ocs(+)) and mannopine and agropine production (Mas(+)Ags(+)). An unexpectedly large variety of phenotypes was observed. For instance, two out of three tumor lines induced on haploid plantlets by the rooter mutant LBA4210 regenerated shoots, a phenomenon which is not observed for octopine tobacco tumor lines. Fifty percent of the crown-gall lines studied did not contain octopine. Only one line out of six independent lines analyzed was found to have a 'regular' T-DNA structure. Occurrence of aberrant T-DNA structures was not correlated with the ploidy level of infected plantlets, nor with the T-region structure of the inciting bacterial strain. The pattern of TL-DNA transcripts was studied for one line and correlated well with the aberrant T-DNA structure detected. Segments of TR-DNA, having irregular structures as well, were detected in two out of the six lines studied. The scrambled nature of the TR-DNA explained the absence of mannopine and agropine in these two lines. In addition, it was observed that N. plumbaginifolia tissue lines which did not carry T-DNA, became readily phytohormone autotrophic (habituated) at an early stage in tissue culture.

T-DNA rearrangements due to tissue culture: somaclonal variation in crown gall tissues.

After three years of apparent stability in tissue culture, the single cell derived shooty crown gall line sNT1.013 produced a revertant shoot which had switched from non-rooting (Rod(+)) and octopine synthesizing (Ocs(+)) to Rod(-) Ocs(-), indicating that in this revertant TL-DNA genes 4 (causing the Rod(+) trait) and gene 3 (causing the Ocs(+) trait) had been inactivated. Southern blots revealed that the inactivation of these T-DNA genes was the result of a considerable rearrangement of DNA sequences, accompanied by deletions and possibly also by DNA amplifications. This study for the first time unambiguously proves that foreign genes which have been introduced via Agrobacterium tumefaciens can, at a low frequency, be inactivated after T-DNA integration because of reorganization of T-DNA sequences during tissue culture. This can be considered as an event of somaclonal variation.

Clones from a shooty tobacco crown gall tumor I: deletions, rearrangements and amplifications resulting in irregular T-DNA structures and organizations.

Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having a Tn1831 insertion in the auxin locus, were investigated for their T-DNA structure and expression. In addition to clones with the expected phenotype, i.e. phytohormone autonomy, regeneration of non-rooting shoots and octopine synthesis (Aut(+)Reg(+)Ocs(+) 'type I' clones), clones were obtained with an aberrant phenotype. Among these were the Aut(-)Reg(-)Ocs(+) 'type II' clones. Two shooty type I clones and three type II callus clones (all randomly chosen) as well as a rooting shoot regenerated from a type II clone via a high kinetin treatment, all had a T-DNA structure which differed significantly from 'regular' T-DNA structures. No Tn1831 DNA sequences were detected in these clones. The two type I clones were identical: they both contained the same highly truncated T-DNA segments. One TL-DNA segment of approximately 0.7 kb, originating form the left part of the TL-region, was present at one copy per diploid tobacco genome. Another segment with a maximum size of about 7 kb was derived from the right hand part of the TL-region and was present at minimally two copies. Three copies of a truncated TR-DNA segment were detected, probably starting at the right TR-DNA border repeat and ending halfway the regular TR-region. Indications have been obtained that at least some of the T-DNA segments are closely linked, sometimes via intervening plant DNA sequences. The type I clones harbored TL-DNA transcripts 4, 6a/b and 3 as well as TR-DNA transcript 0'. The type II clones harbored three to six highly truncated T-DNA segments, originating from the right part of the TL-region. In addition they had TR-DNA segments, similar to those of the type I clones. On Northern blots TR-DNA transcripts 0' and 1' were detected as well as the TL-DNA transcripts 3 and 6a/b and an 1800 bp hybrid transcript (tr.Y) containing gene 6b sequences. Possible origins of the observed irregularities in T-DNA structures are discussed in relation to fidelity of transformation of plant cells viaAgrobacterium.

Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization, T-DNA methylation and conditional expression of opine genes.

Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut(+)), shoot regeneration (Reg(+)) and octopine synthesis (Ocs(+))), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut(+)Reg(+) but shows little or no octopine synthesis activity (Ocs(-)). Subclones of TSO38, however, are either Ocs(-) or Ocs(+). Ocs(-) shoots become Ocs(+) under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs(-) subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs(+) and an Ocs(-) subclone of TSO38, which were negative for the presence of mannopine (Mas(-)) and agropine (Ags(-)), became Mas(+)Ags(+) after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs(-) line and in the Ocs(+) line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs(-) and an Ocs(+) TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs(+) and an Ocs(-) subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs(-) and the Ocs(+) TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.

Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences.

With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T(+)), also had acquired non-selected calf thymus carrier DNA sequences (C(+)), being integrated in their nuclear genomes. From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas(+)), shoots were grafted and mature, flowering plants (gNT4) were obtained. After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features. The mannopine locus segregated independently from the locus determining the crown gall phenotype. When screened for integrated ('transforming') foreign DNA sequences 97% of the NT4-like seedlings turned out to be C(+)T(+). Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C(+)T(+):C(-)T(+): C(+)T:C T SR1-like seedlings. Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes.

Characterization and partial purification of androgen receptors from ram seminal vesicles.

An androgen receptor has been demonstrated in the cytosol and in the nuclear fraction of ram seminal vesicles. The cytosol receptor was stabilized by sodium molybdate and 2 distinct [3H]methyltrienolone-binding proteins, one sedimenting at 9S and one sedimenting at 3S, could be demonstrated by sucrose-gradient centrifugation in the presence of 50 mM molybdate. The slower sedimenting form could be partially purified by ADP-sepharose chromatography. The purified receptor still sedimented at 3S after centrifugation on sucrose gradients containing either 0.6 M KCl or 50 mM molybdate. The receptor was destroyed by heating at 50 degrees C for 30 min and its complex with [3H]methyltrienolone dissociated slowly at low temperatures. The apparent equilibrium-dissociation constant (KD) for the purified receptor was: 3.8 x 10(-10) M. The relative affinities for different steroids decreased in the following sequence: 5 alpha-dihydrotestosterone greater than or equal to methyltrienolone greater than testosterone much greater than estradiol greater than R5020 greater than progesterone greater than diethylstilbestrol. The nuclear androgen receptor sedimented at 3S on sucrose gradients containing 0.6 M KCl. At pH 7.4 it behaved as an acidic protein with an electrophoretic mobility towards the anodic region of the agar gel. Because of the relatively large content of cytoplasmic and nuclear androgen receptors and the availability of large amounts of tissue the ram seminal vesicles could be a suitable source for large-scale purification of these receptors.