Stable and unstable mutations in aberrant ratio stocks of maize

Aberrant Ratio (AR) stocks of maize were tested for transposition activity. Lines exhibiting AR and homozygous for the dominant alleles at the Sh Bz and Wx loci in the short arm of chromosome 9 were crossed as males to a sh bz wx tester. Among a population of 346,201 kernels, eight mutations of sh and two of bz were recovered. Eight of the ten mutations survived and none was as vigorous as its normal sibs. At least five of the sh mutants appear to be unstable in F₂ and subsequent generations. An unexpected observation was the high incidence of somatic loss of chromosome 9 markers (Sh Bz and Wx), indicating chromosome breakage or nondisjunction. Southern blot hybridization analysis of the sh alterations indicate that all but one mutant are associated with structural DNA rearrangements at the shrunken locus. Possible mechanisms by which these alterations arose are discussed.     

The frequency of transposition of the maize element Activator is not affected by an adjacent deletion

Summary The maize mutable allele bz-m2 (Ac), which arose from insertion of the 4.6 kb Ac element in the bz (bronze) locus, gives rise to stable bz (bz-s) derivatives that retain an active Ac element closely linked to bz. In the derivative bz-s:2114 (Ac), the Ac element is recombinationally inseparable from bz and transposes to unlinked sites at a frequency similar to that in the progenitor allele bzm2 (Ac). Both alleles have been cloned and sequenced. The bz-s:2114 (Ac) mutation retains Ac at the original site of insertion, but has lost a 789 pb upstream bz sequence adjacent to the insertion, hence the stable phenotype. The 8 bp target site direct repeat flanking the Ac insertion in the bz-m2 (Ac) allele is deleted in bz-s: 2114 (Ac), yet the Ac element is not impaired in its ability to transpose. The only functional Ac element in bz-s:2114 (Ac) is the one at the bz locus: in second-cycle derivatives without Ac activity, the loss of Ac activity correlated with the physical loss of the Ac element from the bz locus. The deletion endpoint in bz-s: 2114 (Ac) corresponds exactly with the site of insertion of a Ds element in a different bz mutation, which suggests that there may be preferred integration sites in the genome and that the deletion originated as the consequence of an abortive transposition event. Finally, we report two errors in the published Ac sequence.     

The Waxy Locus in Maize III. Effect of Structural Heterozygosity on Intragenic Recombination and Flanking Marker Assortment

The effect of heterozygosity for structural rearrangements on recombination between two wx heteroalleles (C and 90) and the pattern of flanking markers in the resultant Wx gametes has been examined. The rearrangements are Tp9, an insertional translocation in which a segment of chromosome 3 has been inserted into the short arm of chromosome 9 close to the wx locus; In9a, a long pericentric inversion with wx in the inverted segment; and Rearr 9, a complex rearrangement of chromosome 9. Heterozygosity for rearrangements decreases the frequency of Wx gametes to varying degrees.—Heterozygosity for Tp9 enhances the proportion of Wx gametes that are apparent convertants and allows the conclusion that such gametes do not normally arise from an exchange in the wx locus plus a second exchange distal to wx. Heterozygosity for In9a markedly decreases the frequency of Wx gametes that are recombinant for outside markers but does not decrease the frequency of convertants.—Heterozygosity for Rearr 9 permits a low frequency of Wx gametes, all of which are apparent convertants.—A high proportion of the convertants have the flanking markers that entered the cross with C so recombination is polarized in normal homologs and in heterozygotes for all rearrangements.     

Allelic diversification at the <Emphasis Type="Italic">wx</Emphasis> locus in landraces of Asian rice

To examine continuous variation of amylose levels in Asian rice (Oryza sativa) landraces, the five putative alleles (Wx ^a , Wx ⁱⁿ , Wx ^b , Wx ^op , and wx) at the wx locus were investigated in near-isogenic lines (NILs). Apparent amylose levels ranged from 0.5 to 29.9% in the NILs, showing a positive relation with the levels of Wx gene product, granule-bound starch synthase (GBSS) as well as the enzymatic activity per milligram starch granule. Only opaque (Wx ^op ) accessions had an enzymatic activity per GBSS that was reduced to half the level of the others. Nucleotide sequences in the Wx gene were compared among 18 accessions harboring the five different alleles. Each of the Wx alleles had a unique replacement, frame-shift or splice donor site mutation, suggesting that these nucleotide changes could be reflected in phenotype alterations. A molecular phylogenetic tree constructed using the Wx gene indicated that ssp. japonica forms a distinct clade, whereas ssp. indica forms different clades together with the wild progenitor. Unexpectedly, the wx allele of 160 (indica from Taiwan) joined the japonica lineage; however, comparisons using linked genes for two Taiwanese accessions revealed that the wx gene was the product of gene flow from japonica to indica. Therefore, the japonica lineage frequently included Wx ⁱⁿ , Wx ^b and wx, while Wx ^a and Wx ^op were found in the other lineages, strongly suggesting that allelic diversification occurred after divergence of the two subspecies. The present results were discussed in relation to the maintenance of agronomically valuable genes in various landraces.     

Genetic Fine Structure of the BRONZE Locus in Maize

The bronze (bz) locus in maize, located in the short arm of chromosome 9 (9S), is the structural gene for the anthocyanin biosynthetic enzyme UFGT. The gene has been cloned and its physical map has been oriented relative to the centromere of 9S. We report here the genetic fine structure mapping of several biochemically characterized EMS-induced bz-E mutations, derived from the Bz-W22 isoallele, and Ds insertion bz-m mutations, derived from the Bz-McC isoallele. Two UFGT(-), CRM(+ ) mutants (bz-E2 and bz-E5), which genetically identify coding sequences in the gene, and three UFGT(-), CRM(- )bz-E mutants were mapped against the Ds insertion mutants bz-m1 and bz-m2(DI) by selecting Bz intragenic recombinants from heterozygotes of the type bz-E/bz-m . The exclusive occurrence of one recombinant outside marker class allowed the unambiguous placement of the mutants in a genetic fine structure map. Peculiarly, the two CRM(+)bz-E mutants lie upstream of the three CRM(-)bz-E mutants and at a considerable genetic distance. The UFGT allozymes encoded by the progenitor alleles Bz-W22 and Bz-McC differ in two properties, thermal stability and activity. The sites responsible for these properties were mapped as unselected markers among the Bz intragenic recombinants. The thermal stability site, which also identifies a coding region of the gene, mapped very close to the CRM(+)bz-E mutant sites. The site responsible for variation in activity, which probably identifies a region involved in regulation of expression of the bz locus, mapped at the 5' or proximal end of the locus. It was found to be inseparable from the Ds insertion in bz-m1 that lies very close to the 5' end of the transcribed region.-Evidence was obtained that the insertion of Ds within the bz gene has a suppressing effect on intragenic recombination. Additional data are also presented supporting our observation that Ds affects the pattern of intragenic recombination at bz.-Based on the total genetic length of the bz gene and on the physical size of the transcribed region, we estimate that one unit of recombination at bronze corresponds to 14 kb of DNA. This estimate is more than 100 times smaller than the average value for the whole genome and implies that there may be regions, such as bronze, that serve as hotspots for recombination.     

Cloning of the Bz2 locus of Zea mays using the transposable element Ds as a gene tag

Summary The Bz2 locus of Zea mays has been cloned, utilizing the presence of the transposable element Dissociation (Ds) at the locus as a gene tag. The Ds element inserted in the bz2-m allele was identified among many members of the Ac/Ds family in a Southern blot analysis of a population segregating for bz2-m and Bz2. After cloning a DNA fragment from the bz2-m allele, sequences flanking the Ds insertion were shown to be Bz2-specific and were used to isolate a homologous fragment from a wild-type Bz2 line. The Ds insertion in the bz2-m allele was found to be a Ds2 element identical to the Ds insertion in adh1-2F11.     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

Location dependent,intragenic recombination in maize

With the use of chromosome interchanges, the waxy (wx) locus on chromosome 9 has been relocated to various positions in the maize genome. Four wx alleles, wx ^C, wx ^B, wx ⁹⁰, and wx ^H21, were crossed to six chromosome translocation stocks (four with break points proximal to wx and, two distal to wx). Of the 26 possible homozygous translocation heteroallelic combinations, the results of eight are available in this report. In most instances, the frequencies of wx intragenic recombination of the rearranged chromosomes were lower than that of the control. A significant difference in degree of reduction in recombination values is found for different heteroallelic combinations at the same location and in one instance for the same heteroallelic combination at a different chromosome position. The linear order of the 4 wx mutants within the wx cistron is wx ^C-wx ^H21-wx ⁹⁰ (wx ^B). Additional effects from both genetic background and seasonal factors of the different plantings also are observed.Journal Paper No. J-6906 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1335.     

Studies on the Mx transposable element system in maize recovered from X-irradiated stocks

Summary The unstable mutant bz-x3m arose in a plant subjected to X-irradiation. The element at the bronze locus is non-autonomous and recombination data indicate that an autonomous element is tightly linked. The autonomous element has been designated Mx (mobile element induced by X-rays) and the non-autonomous element, rMx (responder to Mx). Linkage data indicate that a second Mx lies near the end of the short arm of chromosome 9; in one plant, an Mx that is unlinked was detected. Distinguishing characteristics of bz-x3m are a large window of time in endosperm development during which somatic reversions can arise and a wide range in the frequency at which they occur; these features are heritable. With increasing doses of bz-x3m and Mx, the window expands and the frequency range increases. In kernels containing the bz-x3m allele and the tightly linked Mx, breakage occurs in chromosome 9 distal to the C locus, resulting in breakage-fusion-bridge patterns for endosperm markers that lie proximal to the break. The frequency of breaks and the developmental time at which they occur exhibit the same dosage effect as the somatic reversions of the bz-x3m allele. These observations suggest that an rMx (designated rMxBr) that causes chromosome breakage is positioned distal to the C locus. At the molecular level, the bz-x3m allele is associated with a 0.5 kb increase in fragment size in DNA samples digested with BglII, EcoRI, HindIII and PstI; in germinal revertants, the fragment size returns to that of the progenitor.     

Detection of allelic variation at the Wx locus with single-segment substitution lines in rice (Oryza sativa L.)

Apparent amylose content (AAC) is a key determinant of eating and cooking quality in rice and it is mainly controlled by the Wx gene which encodes a granule-bound starch synthase (GBSS). In this study, sixteen single-segment substitution lines harboring the Wx gene from 16 different donors and their recipient HJX74 were used to detect the naturally occurring allelic variation at the Wx locus. The AAC in the materials varied widely and could be grouped into glutinous, low, intermediate, and two high AAC sub-classes, high I (24.36?C25.20%) and high II (25.81?C26.19%), under different experimental environments, which showed a positive correlation with the enzymatic activity of GBSS. One insertion/deletion (InDel) and three single nucleotide polymorphisms in the Wx gene were detected and their combinations resulted in the variation of five classes of AAC. Based on the results of AAC phenotypes, GBSS activities and cDNA sequences, five Wx alleles, wx, Wx ^t, Wx ^g1, Wx ^g2, and Wx ^g3, were identified, two of which, Wx ^g2 and Wx ^g3, are separated for the first time in this study. Under different cropping seasons, the AAC differed significantly for the Wx ^t and Wx ^g1 alleles, with higher AAC in the fall season than in the spring season, but did not differ significantly for the wx, Wx ^g2, and Wx ^g3 alleles. In conclusion, the present results might contribute to our understanding of the naturally occurring allelic variation at the Wx locus and will facilitate the improvement of rice quality by marker-assisted selection.     

Weitere Untersuchungen zu Komplementarit?t und Nicht-Autonomie Deraugenfarb-MutantenMA-L undMA-LBZ vonDrosophila melanogaster

Summary Data found byGlassman (1959, 1960) in compound females of the genotypev bz/v f B x ma-l (wherebz stands forma-l ^bz ) showed complementation between these alleles in both eye color and activity of the enzyme, xanthine dehydrogenase, the latter however being incomplete. These results have been reexamined and extended, using compounds of purebz andma-l stocks as well as various heterozygotes between these stocks and the mutantrosy. Complementation in the red eye pigments (drosopterines), though incomplete in newly emerged compound femalesma-l/bz andbz/ma-l becomes firmly established in older flies, where the red pigments reach the amount present in wildtype eyes. Isoxanthopterin however, as well as enzyme activity do not exceed approximately 10% of the level typical for wildtype. These features of slow and incomplete complementation are very probably due to the interaction ofma-l andbz; they do not occur in a series of other related heterozygotes.Transplantation experiments revealed non-autonomy of bothma-l andbz with regard to the formation of drosopterines in the eyes and isoxanthopterin in the testes.The results are discussed with respect to the allelsm of the two mutants.

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Ausgeführt mit Unterstützung des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung. Herrn Prof. Dr.E. Hadorn danke ich herzlich für die Förderung dieser Arbeit, herrn Prof. Dr.U. Leupold für kritische Diskussion.     

Identification of SNPs in the<Emphasis Type="Italic"> waxy</Emphasis> gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice

Common non-waxy (Wx) rice cultivars contain two different alleles at the waxy locus, designated Wx ^a and Wx ^b, which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wx ^a allele was predominant in non-waxy indica cultivars, whereas the Wx ^b allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wx ^a from Wx ^b have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AGGT to AGTT) at a donor site of the first intron within the Wx gene. In the case of waxy (wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wx ^a or Wx ^b alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wx ^a allele (AGGT) but not in the Wx ^b allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wx ^a- and Wx ^b-derived alleles, but that the Wx ^b-derived allele was predominant, and its distribution was independent of indica-japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples.Communicated by J. Heslop-Harrison     

Evidence that the two sucrose synthetase genes in maize are related

Summary Evidence is presented that the sucrose synthetase coding sequence at the Shrunken locus is distantly related to the sequence encoding a second, minor sucrose synthetase present in maize endosperm. Three doubly mutant sh bz strains lacking at least part of the Sh coding sequence produce an antigenically cross-reactive protein having the same electrophoretic mobility as the Sh-encoded, 92-kD sucrose synthetase monomer, but differing in primary structure. An mRNA is present in endosperm of mutants with deletions at the Sh locus that is weakly homologous to the Sh coding sequence and encodes a 92-kD protein precipitable with antiserum to sucrose synthetase. We conclude that the genes encoding the two different proteins are related.     

Mutation loci and intragenic selection marker of the granule-bound starch synthase gene in waxy maize

Four pairs of specific PCR primers have been designed on the basis of the sequence of the granule-bound starch synthase gene (GBSS; dominant non-waxy gene Wx) and used to amplify its homologous sequence from thirteen waxy and two non-waxy inbred lines. Results from electrophoresis indicated that the recessive waxy gene was wx, derived from the dominant non-waxy gene Wx by mutation at its 3′ end. The sequence of the mutated 3′ end was amplified by the TAIL-PCR technique. Sequence alignment showed that the mutation of the wx gene was caused by transposition of the aldehyde dehydrogenase gene rf2. Two pairs of specific primers were designed on the basis of the sequence difference between the dominant gene Wx and its mutated recessive allele wx and used as intragenic selection markers to identify individual plants of genotypes WxWx, Wxwx, and wxwx by PCR amplification from the segregating population of the F₂ generation crossed between waxy and non-waxy inbred lines. Iodine solution staining and starch component assay showed that all the 35 F₂ plants identified as genotype WxWx produced non-waxy kernels of the F₃ generation and that all 33 F₂ plants identified as genotype wxwx produced waxy kernels of the F₃ generation. This result can be used to improve the selection efficiency of waxy maize breeding and for selection of other single genes and major polygenes.     

Transposable element Ds at the shrunken locus in Zea mays

Summary Maize DNAs isolated from wild type and from mutants caused by the insertion of transposable genetic element Ds at the gene encoding endosperm sucrose synthase (Sh) are compared in Southern blotting experiments by hybridization to Sh-cDNA cloned in pBR322. Differences observed between the DNAs of the wild type and the mutants indicate the presence of additional DNA at the Sh locus and/or DNA alterations that have occurred subsequent to the insertion of Ds. A double mutant exhibiting the recessive phenotype of both sh and the closely linked gene bz lacks DNA hybridizing to the probe and may be a deletion.