R. L. Phillips

DNA-based markers in plants

General preface. Preface. 1. Some concepts and new methods for molecular mapping in plants* B. Burr. 2. PCR-based marker systems R. Reiter. 3. Constructing a plant genetic linkage map with DNA markers N.D. Young. 4. Use of DNA markers in introgression and isolation of genes associated with durable resistance to rice blast D.-H. Chen, et al. 5. Mapping quantitative trait loci S.J. Knapp. 6. Comparative mapping of plant chromosomes A.H. Paterson, J.L. Bennetzen. 7. Breeding multigenic traits C.W. Stuber. 8. Information systems approaches to support discovery in agricultural genomics B.W.S. Sobral, et al. 9. Introduction: molecular marker maps of major crop species R.L. Phillips, I.K. Vasil. 10. Molecular marker analyses in alfalfa and related species E.C. Brummer, et al. 11. An integrated RFLP map of Arabidopsis thaliana* H.M. Goodman, et al. 12. An integrated map of the barley genome A. Kleinhofs, A. Graner. 13. DNA-based marker maps of Brassica, C.F. Quiros. 14. Molecular genetic map of cotton A.H. Paterson. 15. Maize molecular maps: Markers, bins, and database E.H. Coe, et al. 16. RFLP map of peanut H.T. Stalker, et al. 17. Phaseolus vulgaris - The common bean integration of RFLP and RAPD-based linkage maps C.E. Vallejos, et al. 18. RFLP map of the potato C. Gebhardt, et al. 19. Rice molecular map S.R. McCouch. 20. A framework genetic map of sorghum containing RFLP, SSR and morphological markers J.L. Bennetzen, et al. 21. RFLP map of soybean R.C. Shoemaker, et al. 22. Genetic mapping in sunflowers S.J. Knapp, et al. 23. The molecular map of tomato A. Frary, S.D. Tanksley. 24. Molecular-marker maps of the cultivated wheats and other Triticum species G.E. Hart. 25. Molecular marker linkage maps in diploid and hexaploid oat (Avena sp.) S.F. Kianian, et al. 26. A compilation of molecular genetic maps of cultivated plants O. Riera-Lizarazu, et al. List of Contributors. Subject Index.

Discovery of Transposable Element Activity Among Progeny of Tissue Culture—Derived Maize Plants

Tissue culture-derived plants of many species have often been observed to possess both genetic and cytogenetic abnormalities. A high frequency of structurally altered chromosomes in maize (Zea mays L.) plants regenerated from tissue culture led to the prediction that newly activated transposable elements could be detected in regenerated plants. Testcrosses of 1200 progeny from 301 regenerated maize plants confirmed that ten regenerated plants from two independent embryo cell lines contained an active Actransposable element. No active Ac elements were present in the explant sources. Recovery of transposable element activity in regenerated plants indicates that some tissue culture—derived genetic variability may be the result of insertion or excision of transposable elements, or both.

Genetic implications of somaclonal variation in plants

Publisher Summary This chapter discusses the genetic implications of somaclonal variation in plants. The chapter investigates the topic of somaclonal variation from two perspectives. The first perspective is an examination of the various categories of somaclonal variation and speculation on the underlying mechanisms, including a discussion of how seemingly unrelated phenomena may be interconnected. The second perspective is a description of the unusual genetic outcomes that would be predicted, and the implications of these types of variation for use of tissue culture-derived materials. Emphasis could be placed on the genetic changes that may be detected in regenerated plants and their progeny because these genetic changes are of utmost practical significance. Throughout the chapter, “somaclonal variation’’ is taken to mean any genetic, cytogenetic, or molecular changes produced during tissue culture or plant regeneration. Changes detected in the sexual progeny of regenerated plants are often considered to be “stable,” and contrasted with the “epigenetic” traits that are sometimes detected in tissue cultures or primary regenerants.

Cytological and molecular characterization of oat x maize partial hybrids

In cereals, interspecific and intergeneric hybridizations (wide crosses) which yield karyotypically stable hybrid plants have been used as starting points to widen the genetic base of a crop and to construct stocks for genetic analysis. Also, uniparental genome elimination in karyotypically unstable hybrids has been utilized for cereal haploid production. We have crossed hexaploid oat (2n=6x=42, Avena sativa L.) and maize (2n=2x=20, Zea mays L.) and recovered 90 progenies through embryo rescue. Fifty-two plants (58%) produced from oatxmaize hybridization were oat haploids (2n=3x=21) following maize chromosome elimination. Twenty-eight plants (31%) were found to be stable partial hybrids with 1–4 maize chromosomes in addition to a haploid set of 21 oat chromosomes (2n=21+1 to 2n=21+4). Ten of the ninety plants produced were found to be apparent chromosomal chimeras, where some tissues in a given plant contained maize chromosomes while other tissues did not, or else different tissues contained a different number of maize chromosomes. DNA restriction fragment length polymorphisms (RFLPs) were used to identify the maize chromosome(s) present in the various oat-maize progenies. Maize chromosomes 2, 3, 4, 5, 6, 7, 8, and 9 were detected in partial hybrids and chromosomal chimeras. Maize chromosomes 1 and 10 were not detected in the plants analyzed to-date. Furthermore, partial self-fertility, which is common in oat haploids, was also observed in some oat-maize hybrids. Upon selfing, partial hybrids with one or two maize chromosomes showed nearly complete transmission of the maize chromosome to give self-fertile maize-chromosome-addition oat plants. Fertile lines were recovered that contained an added maize chromosome or chromosome pair representing six of the ten maize chromosomes. Four independently derived disomic maize chromosome addition lines contained chromosome 4, one line carried chromosome 7, two lines had chromosome 9, one had chromosome 2, and one had chromosome 3. One maize chromosome-8 monosomic addition line was also identified. We also identified a double disomic addition line containing both maize chromosomes 4 and 7. This constitutes the first report of the production of karyotypically stable partial hybrids involving highly unrelated species from two subfamilies of the Gramineae (Pooideae — oat, and Panicoideae — maize) and the subsequent recovery of fertile oat-maize chromosome addition lines. These represent novel material for gene/ marker mapping, maize chromosome manipulation, the study of maize gene expression in oat, and the transfer of maize DNA, genes, or active transposons to oat.

DNA methylation and tissue culture-induced variation in plants

SummaryPlant cells growing in an artificial culture environment make numerous genetic mistakes. These alterations are manifested as increased frequencies of single-gene mutations, chromosome breakages, transposable element activations, quantitative trait variations, and modifications of normal DNA methylation patterns. Evidence is presented that indicates a high frequency of DNA hypomethylation as the result of the tissue culture process. Fifteen percent of the methylation changes appear to have been homozygous in the original regenerated plants. A hypothesis is advanced that relates DNA methylation to the variety of genetic alterations found among maize tissue culture regenerants and their progenies. The epigenetic nature of DNA methylation raises questions concerning the stability of tissue culture-induced changes in self-pollinations and crosses.

The nucleolus organizer region of maize (Zea mays L.): Chromosomal site of DNA complementary to ribosomal RNA

A maize genetic marker strain (designated as the 2NOR strain) was found to carry a cytologically-visible “duplication” of the nucleolar organizer region (NOR). The 2 NOR condition is a simply inherited cytological marker which is transmitted normally through mega- and microsporogenesis and is associated when homozygous with a nucleolus approximately 64% greater in volume than that of two representative normal inbred lines of maize.—Utilizing DNA · rRNA hybridization techniques and the 2 NOR strain plus two unrelated inbred lines with normal NORs, the chromosomal site of DNA complementary to rRNA was shown to be localized in the NOR. To our knowledge, this is the first report on the localization of rDNA cistrons in the NOR of plants. An estimate was made of 17,000 rDNA cistrons per diploid nucleus for normal maize.—The 2 NOR strain was shown to possess approximately twice as many rDNA cistrons (34,000) as normal maize. The usefulness of this 2 NOR condition for plant protein improvement programs is being investigated.

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

SummaryPrevious experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R1 progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R1 progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.

The nucleolus organizer region of maize (Zea mays L.)

Maize plants were produced partially triploid for the heterochromatic segment of the nucleolus organizer region (NOR) or partially triploid or tetraploid for the site giving rise to the secondary constriction of the NOR. These partially hyperploid plants were characterized in terms of chromosome and/or nucleolar constitution by light microscopy at pachytene, diakinesis, and quartet stages of microsporogenesis. DNAs of the various partially hyperploid plants and appropriate controls were extracted and hybridized with 3H-rRNA. The heterochromatic segment of the NOR was found to contain most of the rRNA cistrons, but has little or no interaction with the nucleolus. In contrast with the heterochromatic segment, the site giving rise to the secondary constriction contains few rRNA cistrons but is active in nucleolar formation as viewed at pachytene, diakinesis and quartet stages.

Identification of a Maize Neocentromere in an Oat-Maize Addition Line

We report a neocentromere event on maize chromosome 3 that occurred due to chromosome breakage. The neocentromere lies on a fragment of the short arm that lacks the primary centromere DNA elements, CentC and CRM. It is transmitted in the genomic background of oat via a new centromere (and kinetochore), as shown by immunolocalization of the oat CENH3 protein. Despite normal transmission of the maize fragment in most progeny, neocentromeres appear to vary in size within the same tissue, as shown by fluorescent measurements. A secondary truncation in one line lowered mitotic transmission to 3% and precipitously reduced the size of the chromosome. The results support the view that neocentromere formation is generally associated with major genomic disturbances such as wide species crosses or deletion of an existing centromere. The data further suggest that new centromeres may undergo a period of instability that is corrected over a period of several generations.

Do We Understand Somaclonal Variation

Tissue culture is clearly a mutagenic procedure. Although some progenies of regenerated plants may be highly productive and appear normal in all measured traits, most have detectable variation not present in the controls. The tissue culture process itself appears to be inducing these alterations as the cells proliferate in a relatively undifferentiated state. Probably all forms of mutational events that can occur in nature also happen in tissue cultured cells. However, certain mutational events appear to occur in exceedingly high frequencies in plant tissue cultures. These events may vary in frequency among species, genotypes, explant source, media, etc., but, in general, elevated mutational events appear to occur regardless of the specific cultured materials or conditions. The unusually frequent events include the following: 1) Single gene mutations; 2) Transposable element activation; 3) Quantitative trait variation; and 4) Chromosome breakage. In this paper, we will propose a hypothesis attempting to relate these four seemingly different kinds of heritable variation.