Comparison of the waxy locus sequence from a non-waxy strain and two waxy mutants of spontaneous and artificial origins in barley

Molecular characterization of 3 alleles of the waxy gene from a non-waxy strain "Shikoku hadaka No. 84" (SH84), an indigenous waxy strain "Mochimugi D" (MMD), and an artificial waxy mutant strain "Shikoku hadaka No. 97" (SH97) of barley (Hordeum vulgare ssp. vulgare) was performed via a PCR direct sequencing strategy. The 3 haplotypes were analyzed in terms of single nucleotide polymorphisms, insertion/deletion mutations, and simple sequence repeat polymorphisms. In comparison with the barley non-waxy gene sequence deposited in the public DNA database, 110 polymorphic sites were found in the 5,190-bp sequenced region of the non-waxy strain SH84. A 418-bp deletion in the 5' non-coding sequence was identified in the indigenous waxy strain MMD. Except for the deletion in the promoter region, the spontaneous mutant wax allele and non-waxy allele were identical. Such highly conserved sequences provide evidence for the recent occurrence of a deletion event in the cultivated barley gene pool. Compared to the original variety SH84, induced waxy mutant SH97 had a base substitution of a C to T in the exon 5, which converting Gln-89 of the wild-type gene into a stop codon, suggesting the involvement of a nonsense-mediated mRNA decay. These results will be helpful for understanding the mechanism of the variable amylose content in waxy cultivars of cereal species.     

A novel codominant marker for selection of the null Wx-B1 allele in wheat breeding programs

Waxy protein (granule-bound starch synthase I) is a key enzyme in the synthesis of amylose in endosperm tissue. The amylose content of wheat flour plays a significant role in determining Japanese udon noodle quality. Most wheat cultivars suitable for producing udon noodles have a low amylose level due to a lack of Wx-B1 protein conditioned by null Wx-B1 alleles. It was previously determined that the entire coding region of the wheat Wx-B1 gene is deleted in the most common null allele. However, the extent and breakpoints of the deletion have not been established. In this study, the position of the 3′ deletion breakpoint was refined by mapping with PCR-based markers. Using information from this analysis, a chromosome walk was initiated and the DNA sequence flanking the deletion breakpoints was obtained. The deletion included a 3,872 bp region downstream from the termination codon of Wx-B1 gene. Based on similarity with T. monococcum sequences, it was estimated that approximately 60 kb upstream of the Wx-B1 gene was also deleted. Using this sequence information, a codominant marker for the identification of the Wx-B1 null allele was developed. This marker can unambiguously identify heterozygous plants, which will accelerate the selection of partial waxy mutants carrying the Wx-B1 null allele.     

Microsatellites in starch-synthesizing genes in relation to starch physicochemical properties in waxy rice ( Oryza sativa L.)

Rice starch is composed of amylose and amylopectin. Amylose content, an important determinant of rice starch quality, is primarily controlled by the waxy gene, encoding granule-bound starch synthase (GBSS). The starch branching enzyme (SBE) and soluble starch synthase (SSS) play major roles in the synthesis of amylopectin. Microsatellite polymorphisms in the three genes, the wx gene encoding granule-bound starch synthase I, the SBE gene encoding starch branching enzyme I and the SSS gene encoding soluble starch synthase I, were studied for 56 accessions of waxy rice ( Oryza sativa L.). Four (CT)(n) microsatellite alleles, (CT)(16), (CT)(17), (CT)(18) and (CT)(19), at the wx locus were detected in this set of waxy rice, of which (CT)(17) was the most frequent. Three (CT)(n) microsatellite allele classes were found at the SBE locus, (CT)(8) or (CT)(10) together with an insertion sequence of CTCTCGGGCGA, and (CT)(8) alone without the insertion. There were multiple microsatellites clustered at the SSS locus. However, these alleles can also be grouped into three classes, i.e. the allele class SSS-A = (AC)(2) em leader TCC(TC)(11) em leader (TC)(5)C(ACC)(11), the allele class SSS-B = (AC)(3) em leader TCT(TC)(6) em leader (TC)(4)C(ACC)(9), and the allele class SSS-C = (AC)(3) em leader TCT(TC)(6) em leader (TC)(4)C(ACC)(8). The analyses of starch physicochemical properties among different microsatellite genotypes indicated that the waxy rice group with the (CT)(19) allele, the SBE-A allele and the SSS-B allele was quite different from other groups. Nine out of 15 accessions with a high gelatinization temperature (GT) belonged to the wx (CT)(19) group, all of them belonged to the SBE-A group and 13 of them belonged to the SSS-B group. These microsatellites might be useful in marker-assisted breeding for the improvement of rice grain quality.     

The genes encoding granule-bound starch synthases at the waxy loci of the A, B, and D progenitors of common wheat.

Three genes encoding granule-bound starch synthase (wx-TmA, wx-TsB, and wx-TtD) have been isolated from Triticum monococcum (AA), and Triticum speltoides (BB), by the polymerase chain reaction (PCR) approach, and from Triticum tauschii (DD), by screening a genomic DNA library. Multiple sequence alignment indicated that the wx-TmA, wx-TsB, and wx-TtD genes had the same extron and (or) intron structure as the previously reported waxy gene from barley. The lengths of the three wx-TmA, wx-TsB, and wx-TtD genes were 2834 bp, 2826 bp, and 2893 bp, respectively, each covering 31 bp in the untranslated leader and the entire coding region consisting of 11 exons and 10 introns. The three genes had identical lengths of exons, except exonl, and shared over 95% identity with each other within the exon regions. The majority of introns were significantly variable in length and sequence, differing mainly in length (1-57 bp) as a result of insertion and (or) deletion events. The deduced amino acid sequence from these three genes indicated that the mature WX-TMA, -TSB, and -TTD proteins contained the same number of amino acids, but differed in predicted molecular weight and isoelectric point (pI) due to amino acid substitutions (13-18). The predicted physical characteristics of the WX proteins matched the respective proteins in wheat very closely, but the match was not perfect. Furthermore the exon5 sequences of the wx-TmA, wx-TsB, and wx-TtD genes were different from a cDNA encoding a waxy gene of common wheat previously reported. The striking difference was that an insertion of 11 amino acids occurred in the cDNA sequence that could not be observed in the exons of the A, B, and D genes. It was noted, however, that the 3' end of intron4 of these genes could account for the additional 11 amino acids. The sequence information from the available waxy genes identified the intron4-exon5-intron5 region as being diagnostic for sequence variation in waxy. The sequence variation in the waxy genes provides the basis for primer design to distinguish the respective genes in common wheat, and its progenitors, using PCR.     

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.     

Molecular basis of the Cd36 chromosomal deletion underlying SHR defects in insulin action and fatty acid metabolism

The human insulin resistance syndromes—type 2 diabetes, obesity, combined hyperlipidemia, and essential hypertension—are genetically complex disorders whose molecular basis is largely unknown. The spontaneously hypertensive rate (SHR) is a model of these human syndromes. In the SHR/NCrlBR strain, a chromosomal deletion event that occurred at the Cd36 locus during the evolution of this SHR strain has been proposed as a cause of defective insulin action and fatty acid metabolism. In this study, three copies of the Cd36 gene, one transcribed copy and two pseudogenes, were identified in normal rat strains, but only a single gene in SHR/NCrlBR. Analysis of SHR genomic sequence localized the chromosomal deletion event between intron 4 of the normally transcribed copy of the gene and intron 4 of the second pseudogene. The deletion led to the creation of a single chimeric Cd36 gene in SHR/NCrlBR. The boundaries of the recombination/deletion junction identified within intron 4 were surrounded by long interspersed nuclear elements (LINEs) and DNA topoisomerase I recognition sequences. An 8-bp deletion at the intron 14/exon 15 boundary of the second pseudogene abolishes the putative splice acceptor site and is the cause of an aberrant 3′ UTR previously observed in SHR/NCrlBR. We conclude that in SHR/NCrlBR, the complex trait of insulin resistance and defective fatty acid metabolism is caused by Cd36 deficiency, resulting from a chromosomal deletion caused by unequal recombination. This demonstrates that chromosomal deletions caused by unequal recombination can be a cause of quantitative or complex mammalian phenotypes. Received: 7 September 2001 / Accepted: 3 October 2001     

Microsatellites and a single-nucleotide polymorphism differentiate apparentamylose classes in an extended pedigree of US rice germ plasm

 The Waxy gene (Wx) encodes the granule-bound starch synthase responsible for the synthesis of amylose in rice (Oryza sativa). Recently, a polymorphic microsatellite sequence closely linked to the Wx gene was reported. To determine whether polymorphism in this sequence correlates with variation in apparent amylose content, we tested an extended pedigree of 92 current and historically important long-, medium- and short-grain US rice cultivars representing the efforts of many breeders over more than 80 years. Seven Wx microsatellite alleles were identified which together explained 82.9% of the variation in apparent amylose content of the 89 non-glutinous rice cultivars tested. Similar results were also obtained with 101 progenyof a cross between low- and intermediate-amylose breeding lines. An additional, unique microsatelliteallele, (CT)₁₆, was detected in one glutinous cultivar,CI 5309. However, the other glutinous cultivars,Calmochi 101 and Tatsumi mochi, were in the (CT)₁₇ class along with three other cultivars that contained15–16.5% amylose. We sequenced a 200-bp PCR-amplified fragment containing the CT microsatellite and the putative 5′ splice site of the Wx leader intron from a subset of 42 cultivars representing all eight microsatellite alleles. All of the cultivars with 18% or less amylose had the sequence AGTTATA at the putative leader intron 5′ splice site, while all cultivars with a higher proportionof amylose had AGTTATA. This single nucleotidesubstitution could also be assayed by AccI digestion of the amplified fragment. Overall, this single nucleotide polymorphism could explain 79.7% of the variation in the apparent amylose content of the 89 non-glutinous cultivars tested. Interestingly, cultivars in the (CT)₁₉ microsatellite classes that differed substantially in amylose content still showed the correlation between this G-T polymorphism and apparent amylose content. The G-T polymorphism at this site was not, however, able to explain the very low amylose contents of the three glutinous cultivars tested, all of which had the sequence AGTTATA. Received: 31 July 1996 / Accepted: 22 November 1996     

Sequence of the structural gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele

Summary The genomic sequence of the potato gene for starch granule-bound starch synthase (GBSS; waxy protein) has been determined for the wild-type allele of a monoploid genotype from which an amylose-free (amf) mutant was derived, and for the mutant part of the amf allele. Comparison of the wild-type sequence with a cDNA sequence from the literature and a newly isolated cDNA revealed the presence of 13 introns, the first of which is located in the untranslated leader. The promoter contains a G-box-like sequence. The deduced amino acid sequence of the precursor of GBSS shows a high degree of identity with monocot waxy protein sequences in the region corresponding to the mature form of the enzyme. The transit peptide of 77 amino acids, required for routing of the precursor to the plastids, shows much less identity with the transit peptides of the other waxy preproteins, but resembles the hydropathic distributions of these peptides. Alignment of the amino acid sequences of the four mature starch synthases with the Escherichia coli glgA gene product revealed the presence of at least three conserved boxes; there is no homology with previously proposed starch binding domains of other enzymes involved in starch metabolism. We report the use of chimeric constructs with wild-type and amf sequences to localize, via complementation experiments, the region of the amf allele in which the mutation resides. Direct sequencing of polymerase chain reaction products confirmed that the amf mutation is a deletion of a single AT basepair in the region coding for the transit peptide. Premature termination of translation as a result of this frameshift mutation results in a small peptide. However, a protein reacting with anti-GBSS serum, slightly larger than the wild-type mature GBSS, can be detected in a membrane fraction from amylose-free tubers. A possible explanation for this phenomenon will be discussed.     

Major histocompatibility complex class IIB allele polymorphism and its association with resistance/susceptibility to Vibrio anguillarum in Japanese flounder (Paralichthys olivaceus)

The full length of major histocompatibility complex (MHC) class IIB cDNA was cloned from a Chinese population of Paralichthys olivaceus by homology cloning and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The MHC IIB genomic sequence is 1,864 bp long and consists of 34-bp 5′UTR, 741-bp open reading frame, 407-bp 3′UTR, 96-bp intron1, 392-bp intron2, 85-bp intron3, and 109-bp intron4. Phylogenetic analysis showed that the putative MHC class IIB amino acid of the Chinese P. olivaceus shared 28.3% to 85.4% identity with that of the reported MHC class IIB in other species. A significant association between MHC IIB polymorphism and disease resistance/susceptibility was found in Chinese P. olivaceus. Thirteen different MHC IIB alleles were identified among 411 clones from 84 individuals. Among the 280 (268) nucleotides, 32 (11.4%) nucleotide positions were variable. Most alleles such as alleles a, b, c, d, e, f, j, k, i, m were commonly found in both resistant and susceptible stock. Via χ² test, allele d was significantly more prevalent in individuals from susceptible stock than from resistant stock, and their percentages were 23.80% and 7.14%, respectively. In addition, allele g occurred in 9 and allele h in 4 of 42 resistant individuals that were not present in the susceptible stock; their percentages were 21.4% and 9.52%, respectively. Although allele l was found only in 8 individuals from the susceptible stock, its percentage is 19.05%.     

The altered pattern of amylose accumulation in the endosperm of low-amylose barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I with a deletion in the 5'-non-coding region

Reasons for the variable amylose content of endosperm starch from waxy cultivars of barley (Hordeum vulgare) were investigated. The mature grains of most such cultivars contain some amylose, although amounts are much lower than in wild-type cultivars. In these low-amylose cultivars, amylose synthesis starts relatively late in grain development. Starch granules in the outer cell layers of the endosperm contain more amylose than those in the center. This distribution corresponds to that of granule-bound starch synthase I (GBSSI), which is more severely reduced in amount in the center of the endosperm than in the outer cell layers, relative to wild-type cultivars. A second GBSSI in the barley plant, GBSSIb, is not detectable in the endosperm and cannot account for amylose synthesis in the low-amylose cultivars. The change in the expression of GBSSI in the endosperm of the low-amylose cultivars appears to be due to a 413-bp deletion of part of the promoter and 5'-untranslated region of the gene. Although these cultivars are of diverse geographical origin, all carry this same deletion, suggesting that the low-amylose cultivars have a common waxy ancestor. Records suggest a probable source in China, first recorded in the 16th century. Two further families of waxy cultivars have no detectable amylose in the endosperm starch. These amylose-free cultivars were selected in the 20th century from chemically mutagenized populations of wild-type barley. In both cases, 1-bp alterations in the GBSSI gene completely eliminate GBSSI activity.     

Diversification of the rice Waxy gene by insertion of mobile DNA elements into introns.

The waxy (wx) gene of Oryza glaberrima was cloned, and its nucleotide sequence was determined. A waxy mutant of O. glaberrima showing a glutinous phenotype was found to contain a substitution mutation generating a termination codon in the coding region of the wx gene. The Wx sequence of O. glaberrima was different from that of Oryza sativa by substitutions and insertions/deletions, among which only a few substitutions occurred in several exons not to severely alter the amino acid sequence of the Wx protein. The most striking difference observed in introns was a 139-bp deletion (or insertion) in intron 10 of O. glaberrima (or O. sativa). In O. sativa, 125 bp of the 139-bp sequence was flanked by direct repeats of a 14-bp sequence. A sequence homologous to the 125-bp sequence was found in the region preceding exon 2; this sequence was also flanked by direct repeats of another 14-bp sequence. This result and the observation that the 125-bp sequence was interspersed in rice genomes indicate that they are SINEs (short interspersed elements) in the plant system. We also identified a DNA sequence with long terminal inverted repeats in intron 13 of both O. glaberrima and O. sativa. This sequence was present in multiple copies in rice genomes, suggesting that it is a transposable element. These results obtained suggest that mobile DNA elements have diversified the rice Waxy gene by inserting into introns, each of which may originally have a length of about 100 bp.     

Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5′-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast λEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae. Received: 20 November 1996 / Accepted: 25 February 1997     

Human ferrochelatase: a novel mutation in patients with erythropoietic protoporphyria and an isoform caused by alternative splicing

Erythropoietic protoporphyria (EPP), attributable to deficiency of ferrochelatase activity (FECH), is characterised mainly by cutaneous photosensitivity. To define the molecular defect in two EPP-affected siblings and their parents in a Swiss family, ferrochelatase cDNA was amplified by the polymerase chain reaction (PCR) and subjected to sequence analysis. A 5-bp deletion (T580G584) was identified on one allele of the ferrochelatase gene in both patients and their mother. Screening of the mutation among family members by RsaI digestion of PCR-amplified genomic DNA revealed autosomal dominant inheritance associated with abnormal protoporphyrin concentration and enzyme activity. We also isolated ferrochelatase cDNAs containing a 18-bp insertion (part of the intron 2 sequence) between exons 2 and 3; this corresponded to six extra amino acids (YESNIR) inserted between Arg-65 and Lys-66 of the known ferrochelatase. This isoform was identified initially in mRNAs derived from both alleles of the ferrochelatase gene in one patient. Its existence was confirmed in six additional EPP patients, in five out of seven controls, and in four different cell lines (fibroblast, muscle, hepatoma and myelogenous leukaemia). This isoform, roughly 20% of the total ferrochelatase mRNA, was generated through splicing at a second donor site in intron 2 and its presence was not linked to EPP.     

Molecular analysis of the gene encoding a rice starch branching enzyme

Summary The sequence of a rice gene encoding a starch branching enzyme (sbe1) shows extreme divergence from that of the rice gene, that is homologous to bacterial glycogen branching enzyme (sbe2). sbe1 is expressed abundantly and specifically in developing seeds and maximally in the middle stages of seed development. This expression pattern completely coincides with that of the waxy gene, which encodes a granule-bound starch synthase. Three G-box motifs and consensus promoter sequences are present in the 5 flanking region of sbe1. It encodes a putative transit peptide, which is required for transport into the amyloplast. A 2.2 kb intron (intron 2) precedes the border between the regions encoding the transit peptide and the mature protein, and contains a high G/C content with several repeated sequences in its 5 half. Although only a single copy of sbe1 is present in the rice genome, Southern analysis using intron 2 as a probe indicates the presence of several homologous sequences in the rice genome, suggesting that this large intron and also the transit peptide coding region may be acquired from another portion of the genome by duplication and insertion of the sequence into the gene.     

The mouse Clc1/myotonia gene: ETn insertion, a variable AATC repeat, and PCR diagnosis of alleles

Myotonias are muscle diseases in which the function of the muscular chloride channel ClC-1 is impaired. Null alleles of the corresponding Clc1 gene on mouse chromosome (Chr) 6 provide animal models for human myotonias. It was shown that the allele adr (Clc1 ^adr ) is due to an insertion of an ETn type transposon that is transcribed and leads to multiple splicing events; the allele mto (Clc1 ^adr-mto ) involves a stop codon near the N-terminus. We have determined the genomic organization of the mouse Clc1 gene and the sequence requirements for the transposon insertion in the Clc1 ^adr allele. The mouse Clc1 gene is composed of 23 exons, ranging from 39 to 372 bp, and spans approximately 23 kb of genomic DNA. The exon/intron organization is highly homologous to that of the human CLCN1 gene; the homology of the coding sequence is 97% to rat and 89% to human. In the adr allele the ETn transposon is inserted into intron 12, the largest intron. Whereas the 5′ and 3′ LTR sequences of the ETn transposon are homologous to those reported for other insertional mutations of the mouse, no consensus motif for an insertion target site could be defined. On the basis of flanking sequences, we provide duplex PCR diagnoses for the adr, adr-mto, and wild-type alleles of Clc1. Close to the 3′ end of intron 12, a tetranucleotide repeat (AATC)_n was found that is polymorphic between mouse species Mus musculus, M. molossinus, M. castaneus, and M. spretus, and can thus be used for chromosomal mapping studies. Received: 13 October 1996 / Accepted: 10 May 1997     

Mutations in the agouti (ASIP), the extension (MC1R), and the brown (TYRP1) loci and their association to coat color phenotypes in horses (Equus caballus)

Coat color genetics, when successfully adapted and applied to different mammalian species, provides a good demonstration of the powerful concept of comparative genetics. Using cross-species techniques, we have cloned, sequenced, and characterized equine melanocortin-1-receptor (MC1R) and agouti-signaling-protein (ASIP), and completed a partial sequence of tyrosinase-related protein 1 (TYRP1). The coding sequences and parts of the flanking regions of those genes were systematically analyzed in 40 horses and mutations typed in a total of 120 horses. Our panel represented 22 different horse breeds, including 11 different coat colors of Equus caballus. The comparison of a 1721-bp genomic fragment of MC1R among the 11 coat color phenotypes revealed no sequence difference apart from the known chestnut allele (C901T). In particular, no dominant black (E ^D) mutation was found. In a 4994-bp genomic fragment covering the three putative exons, two introns and parts of the 5′- and 3′-UTRs of ASIP, two intronic base substitutions (SNP-A845G and C2374A), a point mutation in the 3′-UTRs (A4734G), and an 11-bp deletion in exon 2 (ADEx2) were detected. The deletion was found to be homozygous and completely associated with horse recessive black coat color (A ^a /A ^a ) in 24 black horses out of 9 different breeds from our panel. The frameshift initiated by ADEx2 is believed to alter the regular coding sequence, acting as a loss-of-function ASIP mutation. In TYRP1 a base substitution was detected in exon 2 (C189T), causing a threonine to methionine change of yet unknown function, and an SNP (A1188G) was found in intron 2. Received: 22 November 2000 / Accepted: 07 February 2001     

A GC cluster repeat is a hotspot for mit- macro-deletions in yeast mitochondrial DNA.

Summary In a random collection of mit^– mutations of the yeast strain 777-3A we find that deletions are exceptionally frequent in the OXI3 gene, a large mosaic gene coding for subunit I of cytochrome oxidase. About 10% of all oxi3 ^– mutants carry the same macro-deletion, del-A, extending from the 5 non-translated leader of OXI3 to intron 5b of this gene. Determination of the respective wild-type sequences and of the del-A junction sequence revealed that the end-points of the deletion are in two GC clusters with 31 by sequence identity which are located at a distance of 11.3 kb. We speculate that not only the sequence identity of the two GC clusters but also the palindromic structure of these putatively mobile elements of yeast mitochondrial DNA (mtDNA) plays a role in deletion formation.