Richard D. Thompson

Development and mapping of SSR markers for maize.

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.

The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene.

The maize locus, Opaque‐2, controls the expression in developing endosperm of structural genes encoding a family of storage proteins, the 22 kd zeins, and an abundant albumin, termed b‐32. It is shown that the promoter of the b‐32 gene is activated in vivo in the presence of the O2 gene product and that the information necessary for this activation resides in a 440 bp DNA fragment containing five O2 binding sites (GATGAPyPuTGPu). Two of these sites are embedded in copies of the ‘endosperm box’, a motif thought to be involved in endosperm‐specific expression, which is also represented in 22 kd zein promoters. The O2 protein is also shown to be capable of binding in vitro and activating in vivo, its own promoter.

The O2 gene which regulates zein deposition in maize endosperm encodes a protein with structural homologies to transcriptional activators

The structure of the zein regulatory gene Opaque 2 of Zea mays has been determined by sequence analysis of genomic and cDNA clones. The size of O2 mRNA is 1751 bp [poly(A) tail not included] containing a major open reading frame (ORF) of 1380 bp preceded by three short ORFs of 3, 21 and 20 amino acid residues. The main ORF comprises 1362 bp and is composed of six exons ranging in size from 465 to 61 bp and five introns of 678 bp to 83 bp. A putative protein 454 amino acids long was derived by the theoretical translation of the genomic sequences corresponding to exons. The opaque 2 protein contains a domain similar to the leucine zipper motif identified in DNA binding proteins of animal protooncogenes such as fos, jun and myc, and in the transcriptional activators GCN4 and C/EBP. The region of 30 amino acid residues next to the leucine repeats towards the N terminus is rich in basic amino acids and is also homologous to a domain present in fos, jun and GCN4. Moreover, in the carboxy terminal region an amino acid motif closely resembling a metal binding domain is present.

Identification of a transposon-like insertion in a Glu-1 allele of wheat

SummaryThe Glu-1 locus, present on the long arms of the group 1 chromosomes of wheat, codes for a group of storage protein polypeptides termed high molecular weight (HMW) subunits of glutenin. Hexaploid wheat varieties carry a ‘silent’ Glu-1y allele on chromosome 1A, no polypeptide being attributable to this locus. When two such alleles from different varieties were compared, one was found to contain an 8 kb insertion of DNA, termed Wis-2, interrupting the coding sequence. The insertion site is flanked by a 5 bp duplication. The two ends of Wis-2 contain similar sequences over 500 bp long and its termini contain almost the same short sequences but in opposite orientation. These terminal sequences are related to those of several ‘retroposon’-type transposable elements found in other organisms.

Characterization of the multigene family coding for HMW glutenin subunits in wheat using cDNA clones

SummarycDNA clones encoding wheat HMW glutenin subunits have been isolated from a cDNA bank made to poly A+ RNA from developing wheat endosperm var. Chinese Spring. One such clone, pTag 1290, has enabled us to identify the HMW glutenin mRNA species. The DNA sequence of this clone has been partially determined and it contains several tandem DNA repeats. The sequence is discussed in relation to the generation of the HMW glutenin subunit gene family. Analysis of the organization of the HMW glutenin sequences in the wheat genome revealed that the genes encoding HMW glutenin subunits exist in low copy number and are located on the long arm of each of the homoeologous group 1 chromosomes.

Molecular characterization of BET1, a gene expressed in the endosperm transfer cells of maize.

A cDNA clone, BET1 (for basal endosperm transfer layer), was isolated from a cDNA bank prepared from 10-days after pollination (DAP) maize endosperm mRNA. BET1 mRNA was shown to encode a 7-kD cell wall polypeptide. Both the mRNA and protein were restricted in their distribution to the basal endosperm transfer layer and were not expressed elsewhere in the plant. BET1 expression commenced at 9 DAP, reached a maximum between 12 and 16 DAP, and declined after 16 DAP. The initial accumulation of the BET1 polypeptide reached a plateau by 16 DAP and declined thereafter, becoming undetectable by 20 DAP. The antibody raised against the BET1 protein reacted with a number of polypeptides of higher molecular mass than the BET1 monomer. Most of these were present in cytosolic fractions and were found in nonbasal cell endosperm extracts, but three species appeared to be basal cell specific. This result and the reactivity of exhaustively extracted cell wall material with the BET1 antibody suggest that a fraction of the protein is deposited in a covalently bound form in the extracellular matrix. We propose that the BET1 protein plays a role in the structural specialization of the transfer cells. In addition, BET1 provides a new molecular marker for the development of this endosperm domain.

Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum

SummaryAllelic complexity is a key feature of self-incompatibility (S) loci in gametophytic plants. We describe in this report the allelic diversity and gene structure of the S locus in Solanum tuberosum revealed by the isolation and characterization of genomic and cDNA clones encoding S-associated major pistil proteins from three alleles (S1, Sr1, S2). Genomic clones encoding the S1 and S2 proteins provide evidence for a simple gene structure: Two exons are separated by a small intron of 113 (S1) and 117 by (S2). Protein sequences deduced from cDNA clones encoding S1 and Sr1 proteins show 95% homology. 15 of the 25 residues that differ between these S1and Sr1alleles are clustered in a short hypervariable protein segment (amino acid positions 44–68), which corresponds in the genomic clones to DNA sequences flanking the single intron. In contrast, these alleles are only 66% homologous to the S2allele, with the residues that differ between the alleles being scattered throughout the sequence. DNA crosshybridization experiments identify a minimum of three classes of potato S alleles: one class contains the alleles S1, Sr1and S3, the second class S2and an allele of the cultivar Roxy, and the third class contains at present only S4. It is proposed that these classes reflect the origin of the S alleles from a few ancestral S sequence types.

Molecular cloning of the o2-m5 allele of Zea mays using transposon marking

SummaryThe deposition of zein protein in maize endosperm is under the control of several regulatory loci. The isolation of DNA sequences corresponding to Opaque-2 (O2), one of such loci, is described in this paper. The mutable allele, o2-m5 was first induced moving the Ac transposable element present at the wx-m7 allele to the O2 locus. Genetic data suggest that a functional Ac element is responsible for the observed somatic mutability of o2-m5. The isolation of genomic clones containing flanking sequences corresponding to the O2 gene was possible by screening an o2-m5 genomic libary with a probe corresponding to internal Ac sequences usually absent in the defective element Ds. Out of 27 clones isolated with homology to the central part of Ac element, only clones 6IP and 21IP generated a 2.5 kb internal fragment size of an active Ac element when digested with PvuII restriction enzyme. A sequence representing a XhoI fragment of 0.9 kb lying, in the 6IP clone, adjacent to the Ac elements, was subcloned and utilized to prove that it corresponded to a part of the O2 gene. To obtain this information we made use of: (1) DNAs from several reversions originating from the unstable (o2mk-(r) allele, which, when digested with SstI, showed a correct 3.4 kb fragment typical of non-inserted alleles of the O2 locus; and (2) recessive alleles of the O2 locus which were devoid of a 2.0 kb mRNA, present on the contrary in the wild type and in other zein regulating mutants different from O2.

Analysis of the gliadin multigene loci in bread wheat using nullisomic-tetrasomic lines

SummaryThe polypeptides specified by mRNAs hybridizing to several wheat storage protein cDNAs were determined by one-and two-dimensional gel electrophoresis of hybrid-selected translation products. Some of the polypeptides could be assigned to chromosomes on the basis of results gained from two-dimensional fractionation of the in vitro translation products of poly A+ RNA from nullisomic-tetrasomic lines of wheat. cDNA clones belonging to different hybridization groups contained sequences related to different gliadin polypeptide types. In order to determine the chromosomal location and copy number of homologous sequences in the wheat genome, selected cDNA clones were hybridized to restriction endonucleasedigested wheat DNA. The cDNA clones hybridized to sequences derived either from the group 1 chromosomes (γ-3 gliadin and pTag 544 type) or from the group 6 chromosomes (α/β, pTag 53 type). The α/β type sequences are present in 25–35 copies per haploid genome and pTag 544 type sequences in 10–15 copies per haploid wheat genome. Partial sequencing of some of the cDNAs revealed low level homology between the different gliadin cross-hybridization groups, a high-molecular-weight glutenin cDNA sequence and a clone encoding the barley storage protein B-hordein. The significance of these findings is discussed with respect to the probable ancestral relations between wheat endosperm storage protein genes.

Molecular analysis of γ-gliadin gene families at the complex Gli-1 locus of bread wheat(T. aestivum L. )

SummaryA cDNA clone (pTag1436) carrying a complete coding sequence for a γ-gliadin polypeptide has been identified and sequenced. By hybridisation to size fractionated poly A+ RNA from wheat nullisomic-tetrasomic lines, homologous transcripts from the Gli-A1, Gli-B1 and Gli-D1 loci were identified. These mRNAs differed from those complementary to a low molecular weight (LMW) glutenin cDNA clone. Hybridization of pTag1436 to digested wheat DNA produced a pattern of fragments unrelated to that obtained using a LMW glutenin cDNA probe. These results indicate that the γ-gliadin and LMW glutenin families, although both located at the Gli-1 loci, are distinct by hybridisation.