Gene identification and allele-specific marker development for two allelic low phytic acid mutations in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate) is an important anti-nutritional component in cereal and legume grains. PA forms of phosphorus (P) and its salts with micronutrient cations, such as iron and zinc, are indigestible in humans and non-ruminant animals, and hence could affect food/feed nutritional value and cause P pollution of ground water from animal waste. We previously developed a set of low phytic acid (LPA) rice mutants with the aim to increase their nutritional quality. Among them, one line, i.e., Os-lpa-XQZ-1 (hereafter lpa 1-2), was identified to have a mutation allelic to the KBNT lpa 1-1 mutation (hereafter lpa 1-1), which was already delimited to a 47-kb region on chromosome 2. In this study, we searched the candidate gene for these two allelic LPA mutations using T-DNA insertion mutants, mutation detection by CEL I facilitated mismatch cleavage, and gene sequencing. The TIGR locus LOC_Os02g57400 was revealed as the candidate gene hosting these two mutations. Sequence analysis showed that the lpa 1-1 is a single base pair substitution mutation, while lpa 1-2 involves a 1,475-bp fragment deletion. A CAPS marker (LPA1_CAPS) was developed for distinguishing the lpa 1-1 allele from lpa 1-2 and WT alleles, and InDel marker (LPA1_InDel) was developed for differentiating the lpa 1-2 allele from lpa 1-1 and WT ones. Analysis of two populations derived from the two mutants with wild-type varieties confirmed the complete co-segregation of these two markers and LPA phenotype. The LOC_Os02g57400 is predicted to encode, through alternative splicing, four possible proteins that are homologous to the 2-phosphoglycerate kinase reported in hyperthermophilic and thermophilic bacteria. The identification of the LPA gene and development of allele-specific markers are of importance not only for breeding LPA varieties, but also for advancing genetics and genomics of phytic acid biosynthesis in rice and other plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Differential Effects of the KitW Mutation with Other Kit Allelic Combinations on Male Sterility Among Inter- or Intrasubspecific Hybrids in Mice

The combination of the Kit ^W mutation and Kit ^S allele from Mus spretus leads to male hybrid sterility. The effects of other combinations between Kit ^W and Kit ^M from Mus m. molossinus or Kit ^N from Mus m. musculus on male reproductive ability were examined in this study. The Kit ^W/Kit ^M and Kit ^W/Kit ^N males were fertile and showed the normal pattern of spermatogenesis in most seminiferous tubules. There were two amino acid substitutions in the protein deduced from the cDNA sequence coded by the Kit ^M allele sequence and three in the Kit ^M allele compared with the protein from the + Kit allele of C57BL mice. These amino acid exchanges had no effect on the fertility of Kit ^W/Kit ^M and Kit ^W/Kit ^N males. Therefore, comparing the sequence data from cDNA coded by Kit ^M and Kit ^N alleles with that for the Kit ^S allele, we concluded that one or more amino acid exchanges in the extracellular domain would be the cause of male hybrid sterility in the Kit ^W/Kit ^S combination; these substitutions are Phe to Ser at position 72, Thr to Ala at 95, Ser to Arg at 101, Leu to Pro at 123, and Ile to Met at 1303     

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.     

High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley

An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A new inbred strain JF1 established from Japanese fancy mouse carrying the classic piebald allele

A new inbred strain JF1 (Japanese Fancy Mouse 1) was established from a strain of fancy mouse. Morphological and genetical analysis indicated that the mouse originated from the Japanese wild mouse, Mus musculus molossinus. JF1 has characteristic coat color, black spots on the white coat, with black eyes. The mutation appeared to be linked to an old mutation piebald (s). Characterization of the causative gene for piebald, endothelin receptor type B (ednrb), demonstrated that the allele in JF1 is same as that of classic piebald allele, suggesting an identical origin of these two mutants. Possibly, classic piebald mutation was introduced from the Japanese tame mouse, which was already reported at the end of the 1700s. We showed that JF1 is a useful strain for mapping of mutant genes on laboratory strains owing to a high level of polymorphisms in microsatellite markers between JF1 and laboratory strains. The clarified genotypes of JF1 for coat color are ``aa BB CC DD ss'. Received: 30 May 1997 / Accepted: 26 August 1997     

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.     

Ultra‐deep Illumina sequencing accurately identifies MHC class IIb alleles and provides evidence for copy number variation in the guppy (Poecilia reticulata)

We address the bioinformatic issue of accurately separating amplified genes of the major histocompatibility complex (MHC) from artefacts generated during high‐throughput sequencing workflows. We fit observed ultra‐deep sequencing depths (hundreds to thousands of sequences per amplicon) of allelic variants to expectations from genetic models of copy number variation (CNV). We provide a simple, accurate and repeatable method for genotyping multigene families, evaluating our method via analyses of 209 b of MHC class IIb exon 2 in guppies (Poecilia reticulata). Genotype repeatability for resequenced individuals (N = 49) was high (100%) within the same sequencing run. However, repeatability dropped to 83.7% between independent runs, either because of lower mean amplicon sequencing depth in the initial run or random PCR effects. This highlights the importance of fully independent replicates. Significant improvements in genotyping accuracy were made by greatly reducing type I genotyping error (i.e. accepting an artefact as a true allele), which may occur when using low‐depth allele validation thresholds used by previous methods. Only a small amount (4.9%) of type II error (i.e. rejecting a genuine allele as an artefact) was detected through fully independent sequencing runs. We observed 1–6 alleles per individual, and evidence of sharing of alleles across loci. Variation in the total number of MHC class II loci among individuals, both among and within populations was also observed, and some genotypes appeared to be partially hemizygous; total allelic dosage added up to an odd number of allelic copies. Collectively, observations provide evidence of MHC CNV and its complex basis in natural populations.     

Forward and reverse genetics of rapid-cycling Brassica oleracea

Seeds of rapid-cycling Brassica oleracea were mutagenized with the chemical mutagen, ethylmethane sulfonate. The reverse genetics technique, TILLING, was used on a sample population of 1,000 plants, to determine the mutation profile. The spectrum and frequency of mutations induced by ethylmethane sulfonate was similar to that seen in other diploid species such as Arabidopsis thaliana. These data indicate that the mutagenesis was effective and demonstrate that TILLING represents an efficient reverse genetic technique in B. oleracea that will become more valuable as increasing genomic sequence data become available for this species. The extensive duplication in the B. oleracea genome is believed to result in the genetic redundancy that has been important for the evolution of morphological diversity seen in today’s B. oleracea crops (broccoli, Brussels sprouts, cauliflower, cabbage, kale and kohlrabi). However, our forward genetic screens identified 120 mutants in which some aspect of development was affected. Some of these lines have been characterized genetically and in the majority of these, the mutant trait segregates as a recessive allele affecting a single locus. One dominant mutation (curly leaves) and one semi-dominant mutation (dwarf-like) were also identified. Allelism tests of two groups of mutants (glossy and dwarf) revealed that for some loci, multiple independent alleles have been identified. These data indicate that, despite genetic redundancy, mutation of many individual loci in B. oleracea results in distinct phenotypes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection of single nucleotide mutations in wheat using single strand conformation polymorphism gels

We show that single strand conformation polymorphism (SSCP) analysis, using the mutation detection enhancement (MDE^TM) matrix, is efficient at detecting sequence polymorphisms in PCR amplicons. Four independent wheat genomic fragments were amplified from two contrasting templates and sequenced. The allelic fragments were differentiated at 1–6 single nucleotide positions, but MDE-SSCP was able to unequivocally distinguish each allelic pair. The approach is therefore considered a powerful way of identifying single nucleotide polymorphisms (SNPs) without extensive amplicon sequencing.     

Mapping of the chicken cleft primary palate mutation on chromosome 11 and sequencing of the 4.9 Mb linked region

An embryonic lethal mutation in chicken named cleft primary palate (cpp) is inherited in an autosomal recessive mode and results in a severely truncated upper beak. In this study, genotyping and sequencing techniques were employed to advance our genetic and genomic knowledge of the mutation’s chromosomal location, candidate region and possible causative element using a congenic inbred line. Herein, the candidate region for the cpp developmental mutation was established as a ca. 5.1 Mb region of chicken chromosome 11 (GGA 11) through the use of a 600K Affymetrix SNP array. The SNPs identified from this array linked to cpp were used to genotype individuals from the congenic inbred line over several generations and thereby fine-map the causative region resulting in an approximately 200 kb size reduction. This candidate region (4.9 Mb) was sequenced via capture array in a cohort of 24 individuals, including carriers, mutants and their wild type (wt) siblings. Interestingly, the GGA 11 region for cpp encompasses the predicted centromere location and is thus unlikely to be highly disrupted by further recombination. Here we report on the variation unique to the cpp mutation, i.e. single-nucleotide variants and insertions or deletions. Although the candidate region contains several genes of interest with regard to the cpp phenotype, only one cpp-linked variant was predicted to have a significant physiological effect by causing a frameshift mutation in ESRP2, which has a role in tissue-specific splicing during development.     

Altered ivermectin pharmacology and defective visual system in <Emphasis Type="Italic">Drosophila</Emphasis> mutants for histamine receptor HCLB

The Drosophila gene hclB encodes a histamine-gated chloride channel, which can be activated by the neurotoxin ivermectin when expressed in vitro. We have identified two novel hclB mutants, carrying either a missense mutation (P293S, allele hclB ^T1 ) or a putative null mutation (W111*, allele hclB ^T2 ), as well as a novel splice form of the gene. In survival studies, hclB ^T1 mutants were more sensitive to ivermectin than wild-type, whereas hclB ^T2 were more resistant. Electroretinogram recordings from the two mutants exhibited enlarged peak amplitudes of the transient components, indicating altered synaptic transmission between retinal photoneurons and their target cells. Ivermectin treatment severely affected or completely suppressed these transient components in an allele-specific manner. This suppression of synaptic signals by ivermectin was dose-dependent. These results identify HCLB as an important in vivo target for ivermectin in Drosophila melanogaster, and demonstrate the involvement of this protein in the visual pathway. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Chocolate coated cats: TYRP1 mutations for brown color in domestic cats

Brown coat color phenotypes caused by mutations in tyrosinase-related protein-1 (TYRP1) are recognized in many mammals. Brown variations are also recognized in the domestic cat, but the causative mutations are unknown. In cats, Brown, B, has a suggested allelic series, B > b > b^l. The B allele is normal wild-type black coloration. Cats with the brown variation genotypes, bb or bb^l, are supposedly phenotypically chocolate (aka chestnut) and the light brown genotype, b^lb^l, are supposedly phenotypically cinnamon (aka red). The complete coding sequence of feline TYRP1 and a portion of the 5′ UTR was analyzed by direct sequencing of genomic DNA of wild-type and brown color variant cats. Sixteen single nucleotide polymorphisms (SNPs) were identified. Eight SNPs were in the coding regions, six are silent mutations. Two exon 2 on mutations cause amino acid changes. The C to T nonsense mutation at position 298 causes an arginine at amino acid 100 to be replaced by the opal (UGA) stop codon. This mutation is consistent with the cinnamon phenotype and is the putative light brown, b^l, mutation. An intron 6 mutation that potentially disrupts the exon 6 downstream splice-donor recognition site is associated with the chocolate phenotype and is the putative brown, b, mutation. The allelic series was confirmed by segregation and sequence analyses. Three microsatellite makers had significant linkage to the brown phenotype and two for the TYRP1 mutations in a 60-member pedigree. These mutations could be used to identify carriers of brown phenotypes in the domestic cat.     

Identification of a Van der Woude syndrome mutation in the cleft palate 1 mutant mouse

Mutations in Interferon Regulatory Factor 6 (IRF6) have been identified in two human allelic syndromes with cleft lip and/or palate: Van der Woude (VWS) and Popliteal Pterygium syndromes (PPS). Furthermore, common IRF6 haplotypes and single nucleotide polymorphisms (SNP) alleles are strongly associated with nonsyndromic clefting defects in multiple ethnic populations. Mutations in the mouse often provide good models for the study of human diseases and developmental processes. We identified the cleft palate 1 (clft1) mouse mutant in a forward genetic screen for phenotypes modeling human congenital disease. In the clft1 mutant, we have identified a novel missense point mutation in the mouse Irf6 gene, which confers an amino acid alteration that has been found in a VWS family. Phenotypic comparison of clft1 mutants to previously reported Irf6 mutant alleles demonstrates the Irf6^clft1 allele is a hypomorphic allele. The cleft palate seen in these mutants appears to be due to abnormal adhesion between the palate and tongue. The Irf6^clft1 allele provides the first mouse model for the study of an etiologic IRF6 missense mutation observed in a human VWS family. genesis 48:303–308, 2010. © 2010 Wiley‐Liss, Inc.     

Sequence diversity and molecular evolution of the merozoite surface antigen 2 of plasmodium falciparum

Eleven new alleles of the Plasmodium falciparum merozoite surface antigen 2 (MSA2) from Papua New Guinea were analyzed by direct sequencing of polymerase chain reaction (PCR) products. We have used the sequence information to trace the molecular evolution of MSA2. The repeats of ten alleles belonging to the 3D7 allelic family differed considerably in size, nucleotide sequence, and repeat copy number. In the repeat region of these new alleles, codon usage was extremely biased with an exclusive use of NNT codons. Another new allele sequenced belonged to the FC27 family and confirmed the family-specific conserved structure of 96 and 36 bp repeats. In order to assess sequence microheterogeneity within samples defined as the same genotype by restriction fragment length polymorphism (RFLP), we have analyzed single-strand conformation polymorphism (SSCP) of different samples of the most frequent allele (D10 of the FC27 family) in the study population. No sequence heterogeneity could be detected within the repeat region. Based on analysis of the repeat regions in both allelic families, we discuss the hypothesis of a different evolutionary strategy being represented by each of the allelic families. Received: 8 February 1995 / Accepted: 24 March 1997     

Mutation rate in the hypervariable VNTR g3 (D7S22) is affected by allele length and a flanking DNA sequence polymorphism near the repeat array.

The hypervariable human minisatellite locus D7S22 (g3) is highly polymorphic. The allelic distribution in D7S22 features a size clustering of the alleles and a comparably low allelic diversity among small alleles. This reduced diversity could reflect a situation where some alleles are less likely to mutate than others. Several factors could explain such an effect, including allele size, variation in repeat composition, and allelic differences in nearby cis-acting elements affecting the mutation rate. We have characterized 40 de novo mutations found on Southern blots in a large amount of paternity-testing material. There is a significant excess of paternal mutations, and small size changes are most frequent. Mutation rate is affected by allele length, with highest rates in larger alleles. Alleles of the family groups with D7S22 mutations and 50 small alleles were analyzed by nucleotide sequencing. Two hundred thirty-six base pairs of the immediate flanking region upstream of the repeat array were PCR amplified and screened for point mutations by DNA sequencing of the PCR products. Two base substitution polymorphisms were identified: one C/G transversion and one A/G transition, 54 bp and 173 bp upstream of the repeat array, respectively. There is a significant association between mutation and occurrence of 54C, while association is not obvious between mutation rate and the 173A/G variants. There is a marked association between different flanking haplotypes and allele size, and within the smallest allele-size group, all alleles had the 54G/173A haplotype. Both allele size and allelic state at site 54 remain associated with mutation rate when the other factor is controlled. Possible mechanisms behind the variation in mutation rate in D7S22 are discussed.     

<Emphasis Type="Italic">kitb</Emphasis>, a second zebrafish ortholog of mouse <Emphasis Type="Italic">Kit</Emphasis>

The large numbers of duplicated pairs of genes in zebrafish compared to their mammalian counterparts has lead to the notion that expression of zebrafish co-orthologous pairs in some cases can together describe the expression of their mammalian counterpart. Here, we explore this notion by identification and analysis of a second zebrafish ortholog of the mammalian Kit receptor tyrosine kinase (kitb). We show that in embryos, kitb is expressed in a non-overlapping pattern to that of kita, in the anterior ventral mesoderm, Rohon-beardRohon–Beard neurons, the otic vesicle, and trigeminal ganglia. The expression pattern of kita and kitb in zebrafish together approximates that of Kit in mouse, with the exception that neither zebrafish kit gene is expressed in primordial germ cells, a site of kit expression in the mouse embryo. In addition, zebrafish kita is expressed in a site of zebrafish primitive hematopoiesis but not required for blood development, and we fail to detect kitb expression in sites of zebrafish hematopoiesis. Thus, the expression and function of zebrafish kit genes cannot be described as a simple partition of the expression and function of mouse Kit. We discuss the possibility that these unaccounted for expression domains and functions are derived from more ancestral gene duplications and partitioning instead of the relatively recent teleost teleost-specific duplication. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms (PCR-RFLP) and Electrophoretic Assays for the Mouse Obese (Lepob) Mutation

Three polymerase chain reaction-based assays for the mouse Lep^ob mutation are described: Dde I site created by the C←T transversion characterizing Lep^ob enables positive detection of the mutant allele; positive detection of the wild-type Lep allele is achieved by the use of primer sequence which introduces an A←C substitution, creating an Msp I site in the normal allele; and an electrophoretic assay which positively identifies the hetero-zygote.     

Immunoglobulin constant kappa gene alleles in twelve strains of mice

Genomic DNA for the immunoglobulin (Ig) constant kappa Igk-C gene region was amplified by the polymerase chain reaction (PCR) method and sequenced from twelve commonly used inbred mouse strains. PCR products were used directly as templates in dideoxy-DNA-sequencing, a method which avoids the sequencing errors caused by Taq polymerase, since no cloning step is required. In restriction fragment length polymorphism (RFLP) studies the SJL mouse strain has been shown to belong to a Igk-C allogroup different from other common inbred mouse strains. The BALB/c Igk-C region was sequenced earlier, but our Igk-C sequences clarify the situation and confirm the existence of three Igk-C alleles in inbred mice (Mus musculus domesticus). Mice belonging to the kappa (Igk) haplotype e (SJL) have allele c of the Igk-C gene. The strains belonging to the kappa haplotype [a albino strain, K subline (AKR), PL and d (C58)] have allele a, and all other eight strains belonging to three different Igk haplotypes (b, c, and f) use allele b of the gene. Allele b has at least one (possibly two) nucleotide differences from allele a in the Igk-C region, but five compared to allele c. The allelic sequences also predict two allotypic kappa polypeptide chains among twelve inbred strains. Alleles a and b encode identical polypetides, but allele c (SJL) has a conserved lysine to arginine substitution in residue 142.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X67002-X67012.