Distribution, inheritance and linkage relationships of ribosomal DNA spacer length variants in pea.

DNA restriction endonuclease fragment analysis is used to examine the genetic organization, inheritance and linkage associations of the ribosomal DNA in pea. The substantial variation observed in the length of the intergenic spacer region is shown to segregate in Mendelian fashion involving two independent genetic loci, designated Rrn1 and Rrn2. Linkage between Rrn1 and two marker loci on chromosome 4 establishes the approximate location of this tandem array. Rrn2 shows linkage with a set of isozyme loci which assort independently of other markers on all seven chromosomes. Combining these observations with previous cytological data, we suggest that Rrn2 and the isozyme loci linked to it constitute a new linkage group on chromosome 7. The general absence of spacer length classes common to both rRNA loci in any of the lines we examined indicates that little or no genetic exchange occurs between the nonhomologous nucleolar organizer regions.

Dark inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase in legumes: A biosystematic study.

2-carboxyarabinitol 1-phosphate (CA 1-P) is a naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Members of the Fabaceae exhibit a particularly wide range in the extent of CA 1-P accumulation during darkness and include Phaseolus vulgaris, whose dark/light regulation of Rubisco activity is principally achieved by synthesis/degradation of CA 1-P. An extensive survey of the degree of dark inhibition of Rubisco was undertaken for the subfamily Papilionoideae to elucidate evolutionary patterns in the occurrence of this regulatory mechanism. Seventy-five species from 21 tribes were examined. Dark inhibition of Rubisco was found in ancestral tribes such as the Sophoreae, but was substantially reduced or absent in representative species of three more recently evolved tribes, Cicereae, Hedysareae and Vicieae. We conclude that regulation of Rubisco by CA 1-P is neither of recent origin nor of restricted distribution among the Papilionoideae. On the contrary, it becomes lost or less pronounced only in a minority of the more evolutionarily advanced species in this important subfamily.

Plastid inheritance in Pisum sativum L.

Cultivar variability for levels of plastid DNA (cpDNA) in the germ cell line of germinated pea pollen has suggested the possibility of biparental plastid transmission. In order to examine this possibility further, RFLP markers were used to follow the transmission of cpDNA from parents to their F1 offspring. Results from these inheritance studies clearly indicate the presence of only maternal plastid markers in the F1 progeny of each cross examined, irrespective of the pollen cpDNA levels of the paternal parent. The same result is obtained for F1 progeny produced from crosses using pollen characterized by comparatively high cpDNA content, even when offspring are sampled at early developmental stages. Thus, there appears to be little correspondence between pollen cytological data indicating potential paternal plastid transmission and data from molecular marker studies confirming that P. sativum generally follows a uniparental-maternal mode of plastid inheritance. Insufficient F1 progeny were examined to exclude instances of trace biparentalism.

Cultivar variability for the presence of plastid DNA in pollen of Pisum sativum L.: implications for plastid transmission.

The mode of plastid transmission in the garden pea (Pisum sativum L.) was analyzed cytologically using the DNA-fluorochrome 4',6-diamidino-2-phenylindole (DAPI) in conjunction with epifluorescence microscopy. The reproductive cells of mature pollen obtained from 12 inbred lines and cv "Early Alaska" were examined for the presence or absence of DAPI-stained plastid DNA aggregates. Plastid DNA was detected in all 13 pea lines examined, although there was variability with regard to the percentage of pollen grains showing plastid DNA aggregates in generative cells (ranging from 3% in accession 82-12r to 65% in accession 82-14n). These cytological results may indicate genetic variability for plastid DNA inheritance in the garden pea.

Inheritance, organization, and mapping of rbcS and cab multigene families in pea.

DNA restriction endonuclease fragment patterns corresponding to both the rbcS and cab multigene families of pea are each shown to segregate as single Mendelian units in the F(2) progeny of two separate crosses. All of the observed variation in each of the multigene families is thus organized on the chromosome in a tightly linked complex. Linkage relationships between both multigene families and an array of morphological and isozyme markers establish the location of the rbcS and cab gene clusters on pea chromosomes 5 and 2, respectively. Our results, which indicate a high level of DNA restriction fragment length polymorphism in pea, suggest sufficient variation to permit the construction of a highly detailed linkage map.

A phylogenetic analysis of Pisum based on morphological characters, and allozyme and RAPD markers.

Cladistic analyses of 17 wild and cultivated pea taxa were performed using morphological characters, and allozyme and RAPD (random amplified polymorphic DNA) markers. Both branch-and-bound and bootstrap searches produced cladograms that confirmed the close relationships among the wild species and cultivars of Pisum proposed by a variety of systematic studies. Intraspecific rankings were supported for northern P. humile, southern P. humile, P. elatius and P. sativum, which together comprise a single-species complex. P. fulvum, while clearly the most divergent of the pea taxa, could also be assigned to the same species complex without violating the hierarchial logic of the cladogram. Its inclusion or exclusion depends on whether the level of interfertility it displays with other pea taxa or its overall morphological and chromosomal distinction are emphasized. As suggested by previous studies, northern P. humile was the most likely sister taxon to cultivated P. sativum; although, rigorous phylogenetic evaluation revealed a close genealogical affinity among P. elatius, northern P. humile and P. sativum. Despite their limited number, the 16 morphological characters and allozyme markers used precisely organized the pea taxa into established taxonomic groupings, perhaps in part reflecting the role morphology has played historically in pea classification. The RAPD data also generally supported these same groupings and provided additional information regarding the relationships among the taxa. Given that RAPDs are relatively quick and easy to use, are refractory to many environmental influences, can be generated in large numbers, and can complement traditional characters that may be limited in availability, they provide a valuable new resource for phylogenetic studies.

Phytochrome control of RNA levels in developing pea and mung-bean leaves.

We have examined phytochrome effects on the abundance of transcripts from several nuclear and chloroplast genes in buds of dark-grown pea seedlings and primary leaves of dark-grown mung-bean seedlings. Probes for nuclear-coded RNAs were selected from a library of cDNA clones and included those corresponding to the small subunit (SS) of ribulosebisphosphate carboxylase and a chlorophyll a/b binding protein (AB). Transcripts from chloroplast genes for RuBP carboxylase large subunit (LS) and a 32,000-dalton photosystem II polypeptide (PII) were assayed with cloned fragments of the chloroplast genome. In addition, we present data on transcripts from a number of other nuclear genes of unknown function, several of which change in abundance during light-induced development. Transcript levels were measured as a proportion of total RNA by a dot blot assay in which RNA from different tissues or stages is fixed to nitrocellulose and hybridized with (32)P-labeled probes prepared from cloned DNAs. Several patterns of induction can be seen. For example, although both SS and AB RNAs show positive, red/far-red reversible responses in both pea and mung bean, in pea buds the induction ratio for SS RNA is much higher than that for AB RNA, while just the reverse is true for mung-bean leaves. In addition, treatment with lowfluence red light produces full induction of the pea AB RNA, while SS RNA in the same tissue does not reach a maximum steady-state level until after about 24 h of supplementary high-intensity white light. In pea buds, chloroplast genes (LS, PII) also show clear responses to phytochrome, as measured by the steady-state levels of their RNA products. Chloroplast DNA levels (as a fraction of the total cellular DNA) show the same response pattern, which may indicate that in peas many of the light effects we see are related to a general stimulation of chloroplast development. In mung beans, the levels of plastid DNA and RNA are already quite high in the leaves of 7-d dark-grown seedlings, and light effects are much less pronounced. The results are consistent with the notion that chloroplast development is arrested at a later stage in dark-grown mung-bean leaves than in etiolated pea buds.

Structural analysis of the short length ribosomal DNA variant from Pisum sativum L. cv. Alaska.

The genomic clone, RRNpss1, representing the short ribosomal DNA length variant in Pisum sativum L. cv. Alaska, has been isolated and the 2859 bp intergenic spacer, along with the 25S rRNA 3' border and 18S rRNA 5' border, has been sequenced. The intergenic spacer contains nine tandem repeats, approximately 180 bp in length, which show greater than 80% sequence homology to each other. The RNA polymerase I transcription start site and a processing site, located 776 bp and 536 bp upstream of the 5' end of 18S rRNA, respectively, have been determined using S1 analysis. The region surrounding the +1 site shows strong homology between the positions -6 to +10 to the rDNA sites of initiation in radish, maize, and wheat. The sequence CATGCAAA is located 19 bp upstream of the site of initiation, and appears once within each subrepeat and twice more between the end of the subrepeat array and the site of initiation. A previously characterized HpaII site which shows developmental regulation of methylation is located 31 bp downstream of the site of initiation. Using RFLP linkage analysis, the short rDNA length variant of cv. Alaska is assigned to Chromosome 4 where it is genetically independent of the long rDNA length variant which is putatively assigned to Chromosome 7.