Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1) by denaturing high performance liquid chromatography

Background  

R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365~366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant.     

Association of rare chymotrypsinogen C (<Emphasis Type="Italic">CTRC</Emphasis>) gene variations in patients with idiopathic chronic pancreatitis

Extensive genetic studies of chronic pancreatitis over the past decade have highlighted the importance of a tightly regulated balance between activation and inactivation of trypsin within the pancreas to disease susceptibility and resistance. The recent identification of chymotrypsin C (CTRC) as enzyme Y, which was proposed to protect the pancreas by degrading prematurely activated trypsinogen within the pancreas 20 years ago, made CTRC an excellent candidate gene for disease-association studies. Here, we analyzed all eight exons of the CTRC gene for conventional genetic variants and copy number variations (CNVs) by direct sequencing and quantitative fluorescent multiplex PCR, respectively, in a total of 287 French white patients (idiopathic × 216; familial × 42; hereditary × 29). While no CNVs were found in any of the 287 subjects, 20 conventional variations including a nonsense mutation (p.W55X), a microdeletion mutation (p.K247_R254del) and nine missense mutations were found in the 216 patients with idiopathic chronic pancreatitis (ICP). Except for two common polymorphisms, all the remaining 18 mutational events represent rare variations, with a minor allele frequency of 0–0.3% in the control population. All these rare variants were always found more frequently in the ICP patients than in the controls, and their combined frequency in the ICP patients (26/216; 12.0%) is significantly different from that in the controls (4/350; 1.1%) (OR = 11.8 [3.9–40.6]), χ ² = 31.58, P < 10⁻⁶). This genetic finding, when considered in the perceived role of CTRC in eliminating prematurely activated trypsin, indicated that CTRC is a new pancreatitis susceptibility gene.     

Identification and linkage mapping of complementary recessive genes causing hybrid breakdown in an intraspecific rice cross

One outcome of hybrid breakdown is poor growth, which we observed as a reduction in the number of panicles per plant and in culm length in an F₂ population derived from a cross between the genetically divergent rice (Oryza sativa L.) cultivars ‘Sasanishiki’ (japonica) and ‘Habataki’ (indica). Quantitative trait locus (QTL) analysis of the two traits and two-way ANOVA of the detected QTLs suggested that the poor growth was due mainly to an epistatic interaction between genes at QTLs located on chromosomes 2 and 11. The poor growth was likely to result when a plant was homozygous for the ‘Habataki’ allele at the QTL on chromosome 2 and homozygous for the ‘Sasanishiki’ allele at the QTL on chromosome 11. The results suggest that the poor growth found in the F₂ population was due to hybrid breakdown of a set of complementary genes. To test this hypothesis and determine the precise chromosomal location of the genes causing the hybrid breakdown, we performed genetic analyses using a chromosome segment substitution line, in which a part of chromosome 2 from ‘Habataki’ was substituted into the genetic background of ‘Sasanishiki’. The segregation patterns of poor growth in plants suggested that both of the genes underlying the hybrid breakdown were recessive. The gene on chromosome 2, designated hybrid breakdown 2 (hbd2), was mapped between simple sequence repeat markers RM3515 and RM3730. The gene on chromosome 11, hbd3, was mapped between RM5824 and RM1341. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular pathology and evolutionary and physiological implications of pancreatitis-associated cationic trypsinogen mutations

Since the identification in 1996 of a "gain of function" missense mutation, R122H, in the cationic trypsinogen gene (PRSS1) as a cause of hereditary pancreatitis, continued screening of this gene in both hereditary and sporadic pancreatitis has found more disease-associated missense mutations than expected. In addition, functional analysis has yielded interesting findings regarding their underlying mechanisms resulting in a gain of trypsin. A critical review of these data, in the context of the complicated biogenesis and complex autoactivation and autolysis of trypsin(ogen), highlights that PRSS1 mutations cause the disease by various mechanisms depending on which biochemical process they affect. The discovery of these mutations also modifies the classical perception of the disease and, more importantly, reveals fascinating new aspects of the molecular evolution and normal physiology of trypsinogen. First, activation peptide of trypsinogen is under strong selection pressure to minimize autoactivation in higher vertebrates. Second, the R122 primary autolysis site has further evolved in mammalian trypsinogens. Third, evolutionary divergence from threonine to asparagine at residue 29 in human cationic trypsinogen provides additional advantage. Accordingly, we tentatively assign, in human cationic trypsinogen, the strongly selected activation peptide as the first-line and the R122 autolysis site as the second-line of the built-in defensive mechanisms against premature trypsin activation within the pancreas, respectively, and the positively selected asparagine at residue 29 as an "amplifier" to the R122 "fail-safe" mechanism.     

中国遗传性胰腺炎患者胰蛋白酶原基因多位点杂合突变及其临床特征

用基因产物直接测序法对2个遗传性胰腺炎家系中胰腺炎患者(共有4例成员)的胰蛋白酶原基因(cationic trypsinogen,PRSS1)5个外显子进行测序,并分析其各自的临床特征.在4例胰腺炎患者中均出现了PRSS1基因杂合突变,但两家系PRSS1基因突变的位点不同,且临床表现差异较大,其中家系1出现6例糖尿病患者且发病年龄较家系2明显延迟,平均发病年龄为29岁,分析其PRSS1基因发现3号外显子336位碱基存在G→A杂合性突变,为中性突变,表达的氨基酸从赖氨酸(Lys)→赖氨酸(Lys),同时在同一外显子的361位碱基还存在另一个G→A杂合性突变,造成121位的丙氨酸(Ala)被苏氨酸(Thr)所取代,胰蛋白酶原的空间结构发生改变,其与抑制因子的结合位点消失,"保护失败"而产生有活性的胰蛋白酶,造成胰腺自身的消化.而家系2未发现糖尿病患者,其胰腺炎患者的血清肿瘤标志物不增高,先证者(Ⅲ8)在胰腺炎发病过程中表现为CD4 T/CD8 Tcell和乙肝表面抗体(anti-HBs)随病程进展逐渐降低,而Ⅲ7不表现出此现象,分析其PRSS1基因发现3号外显子361位碱基同样存在G→A(c.361G→A)突变,而且在415位还存在一个杂合性突变点T→A(c.415T→A),其中c.415T→A不存在于Ⅲ7.胰蛋白酶原基因存在多种形式的突变,而且与临床表型相关.     

A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1

Here we report an approach to generate a knock-in mouse model using an ‘ends-out’ gene replacement vector to substitute the murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly, examination of mutant ES cell clones and mice revealed that site-specific homologous recombination was mimicked in three independently generated ES cell clones by bidirectional extension of the vector homology arms using the endogenous mParp-1-flanking sequences as templates. This was followed by adjacent integration of the targeting vector, thus leaving the endogenous mParp-1 locus functional. A related phenomenon termed ‘ectopic gene targeting’ has so far only been described for ‘ends-in’ integration-type vectors in non-ES cell gene targeting. We provide reliable techniques to detect such ectopic gene targeting which represents an unexpected caveat in mouse genetic engineering that should be considered in the design and validation strategy of future gene knock-in approaches. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transfer of a Dwarf Gene from Brassica rapa to Oilseed B. napus, Effects on Agronomic Traits, and Development of a ‘Perfect’ Marker for Selection

Lodging can be a serious problem in oilseed Brassica napus. Dwarf genes have been used to reduce lodging in other crops, and their use in wheat and rice was a major factor in the success of the ‘Green Revolution’. We previously reported on a single, semi-dominant, gibberellin insensitive dwarf mutant of B. rapa (Brrga1-d), and provided evidence for homology of this gene to the wheat ‘Green Revolution’ dwarf gene. In this paper, we report using interspecific hybridization of B. rapa and B. oleracea and embryo rescue to resynthesize B. napus containing the Brrga1-d dwarf gene. The dwarf gene was backcrossed into two parents of a commercial hybrid combination and evaluated as inbred and hybrid lines in field experiments. The Brrga1-d gene reduced plant height and lodging in inbred and hybrid lines of B. napus, even when present as a single dose in heterozygous genotypes. Seed yields of inbred lines homozygous for Brrga1-d were reduced compared to near isogenic wild-type inbreds, presumably due to damage by insects caused by a slight delay in flowering time. However, yields of hybrids that were homozygous or heterozygous for Brrga1-d were similar to those of near isogenic wild-type hybrids. In addition, we report on a ‘perfect’ PCR marker for selection of the Brrga1-d gene that is based on detecting the nucleotide mutation causing the dwarf phenotype.     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

Resistance to <Emphasis Type="Italic">Erysiphe necator</Emphasis> in the grapevine ‘Kishmish vatkana’ is controlled by a single locus through restriction of hyphal growth

Vitis vinifera ‘Kishmish vatkana’, a cultivated grapevine from Central Asia, does not produce visible symptoms in response to natural or artificial inoculation with the fungus Erysiphe necator Schwein., the casual agent of powdery mildew. ‘Kishmish vatkana’ allowed pathogen entry into epidermal cells at a rate comparable to that in the susceptible control Vitis vinifera ‘Nimrang’, but was able to limit subsequent hyphal proliferation. Density of conidiophores was significantly lower in ‘Kishmish vatkana’ (33.6 ± 8.7 conidiophores mm⁻²) than in ‘Nimrang’ (310.5 ± 24.0 conidiophores mm⁻²) by 120 h after inoculation. A progeny of 310 plants from a ‘Nimrang’ × ‘Kishmish vatkana’ cross were scored for the presence or absence of visible conidiophores throughout two successive seasons. Phenotypic segregation revealed the presence of a single dominant allele termed Resistance to Erysiphe necator 1 (REN1), which was heterozygous in ‘Kishmish vatkana’. A bulked segregant analysis was carried out using 195 microsatellite markers uniformly distributed across the entire genome. For each marker, association with the resistance trait was inferred by measuring in the bulks the ratio of peak intensities of the two alleles inherited from ‘Kishmish vatkana’. The phenotypic locus was assigned to linkage group 13, a genomic region in which no disease resistance had been reported previously. The REN1 position was restricted to a 7.4 cM interval by analyzing the 310 offspring for the segregation of markers that surrounded the target region. The closest markers, VMC9H4-2, VMCNG4E10-1 and UDV-020, were located 0.9 cM away from the REN1 locus. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore conclude that expression of WT or mutant human PRSS1 in murine acinar cells induces apoptosis and is sufficient to promote spontaneous pancreatitis, which is enhanced in response to cellular insult.     

A survey of unequal crossover systems and their mathematical properties

We present a model of gene duplication by means of unequal crossover (UCO) where the probability of any given pairing between homologous sequences scales as a penalty factor p ^z ≤ 1, with z the number of mismatches due to asymmetric sequence alignment. From this general representation, we derive several limiting case models of UCO, some of which have been treated elsewhere in the literature. One limiting case is random unequal crossover (RUCO), obtained by setting p = 1 (corresponding to equiprobable pairings at each site). Another limiting case scenario (the ‘Krueger-Vogel’ model) proposes an optimal ‘endpoint’ alignment which strongly penalizes both overhang and deviations from endpoint matching positions. For both of these scenarios, we make use of the symmetry properties of the transition operator (together with the more general UCO properties of copy number conservation and equal parent-offspring mean copy number) to derive the stationary distribution of gene copy number generated by UCO. For RUCO, the stationary distribution of genotypes is shown to be a negative binomial, or alternatively, a convolution of geometric distributions on ‘haplotype’ frequencies. A different type of model derived from the general representation only allows recombination without overhang (internal UCO or IntUCO). This process has the special property of converging to a single copy length or a distribution on a pair of copy lengths in the absence of any other evolutionary forces. For UCO systems in general, we also show that selection can readily act on gene copy number in all of the UCO systems we investigate due to the perfect heritability (h ² = 1) imposed by conservation of copy number. Finally, some preliminary work is presented which suggests that the more general models based on misalignment probabilities seem to also converge to stationary distributions, which are most likely functions of parameter value p. An erratum to this article is available at .     

Identification of a novel splicing mutation and 1-bp deletion in the 17α-hydroxylase gene of Japanese patients with 17α-hydroxylase deficiency

We report studies of two unrelated Japanese patients with 17α-hydroxylase deficiency caused by mutations of the 17α-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17α-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient’s mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17α-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation. Received: 15 November 1997 / Accepted: 15 March 1998     

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.     

Genetic analysis of resistance to blackspot (Diplocarpon rosae) in tetraploid roses

  Diplocarpon rosae is the causal agent of rose blackspot, one of the most severe diseases of field-grown roses. The genetics of resistance to this pathogen was investigated in crosses between tetraploid rose genotypes. The hybrid breeding line 91/100-5, which exhibits a broad resistance to all isolates tested so far, was selfed to produce an F₂ population, backcrossed to the susceptible tetraploid variety ‘Caramba’ and crossed to the susceptible varieties ‘Heckenzauber’, ‘Pariser Charme’ and ‘Elina’. Infection experiments resulted in segregation ratios consistent with the presence of a single dominant resistance locus in the duplex configuration in the hybrid 91/100-5. This suggests, together with previous data on the race structure of the fungus, a “gene-for-gene” type of interaction in the pathosystem Diplocarpon/Rosa. We propose to designate this gene Rdr1, which is the first resistance gene described in the genus Rosa. The advantages and limitations of such an interaction type for future rose breeding programmes and for marker-assisted selection strategies are discussed. Received: 29 August 1997 / Accepted: 19 September 1997     

Resistance to Phytophthora fragariae var. fragariae in strawberry: the Rpf2 gene

  Phytophthora fragariae var. fragariae is the causal agent of red stele (red core) root rot in strawberry (Fragaria spp.). The inheritance of resistance to one isolate of this fungus was studied in 12 segregating populations of F.×ananassa derived from crosses between four resistant cultivars (‘Climax’, ‘Redgauntlet’, ‘Siletz’, and ‘Sparkle’) and three susceptible cultivars (‘Blakemore’, ‘Glasa’, and ‘Senga’ Sengana’). The analysis clearly supports the hypothesis of a single segregating dominant resistance gene. It is proposed that this gene be designated Rpf2. Received 12 November 1996 / Accepted: 22 November 1996     

QTL analysis for aphid resistance and growth traits in apple

The rosy apple aphid (Dysaphis plantaginea), the leaf-curling aphid (Dysaphis cf. devecta) and the green apple aphid (Aphis pomi) are widespread pest insects that reduce growth of leaves, fruits and shoots in apple (Malus × domestica). Aphid control in apple orchards is generally achieved by insecticides, but alternative management options like growing resistant cultivars are needed for a more sustainable integrated pest management (IPM). A linkage map available for a segregating F₁-cross of the apple cultivars ‘Fiesta’ and ‘Discovery’ was used to investigate the genetic basis of resistance to aphids. Aphid infestation and plant growth characteristics were repeatedly assessed for the same 160 apple genotypes in three different environments and 2 consecutive years. We identified amplified fragment length polymorphism (AFLP) markers linked to quantitative trait loci (QTLs) for resistance to D. plantaginea (‘Fiesta’ linkage group 17, locus 57.7, marker E33M35–0269; heritability: 28.3%), and to D. cf. devecta (‘Fiesta’ linkage group 7, locus 4.5, marker E32M39–0195; heritability: 50.2%). Interactions between aphid species, differences in climatic conditions and the spatial distribution of aphid infestation were identified as possible factors impeding the detection of QTLs. A pedigree analysis of simple sequence repeat (SSR) marker alleles closely associated with the QTL markers revealed the presence of the alleles in other apple cultivars with reported aphid resistance (‘Wagener’, ‘Cox’s Orange Pippin’), highlighting the genetic basis and also the potential for gene pyramiding of aphid resistance in apple. Finally, significant QTLs for shoot length and stem diameter were identified, while there was no relationship between aphid resistance and plant trait QTLs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation,Characterization, and Expression Analysis of an <Emphasis Type="Italic">SKP1-</Emphasis>like Gene from ‘Shatian’ Pummelo (<Emphasis Type="Italic">Citrus grandis</Emphasis> Osbeck)

Plant s-phase kinase-associated protein 1 (SKP1) genes have diverse functions in plant developmental and physiological activities. Herein, we described a novel SKP1 gene, designated as CgSKP1, from ‘Shatian’ pummelo (Citrus grandis Osbeck). The cDNA sequence of CgSKP1 was 603 bp and contained an open reading frame of 477 bp. Genomic sequence of the CgSKP1 gene contained two exons and one intron. The predicted amino acid sequence of this gene is consisted of 158 amino acids with theoretical proteins size of 17.9 kDa. CgSKP1 had high identity with SKP1 genes from other plant species within two conserved region. Full-length cDNAs were also amplified and cloned from six citrus varieties, with 95% nucleotide identity and about 98% amino acid similarity among them. Gel blot analysis suggested that CgSKP1 existed as a single locus in the ‘Shatian’ pummelo genome. The expression of CgSKP1 was gradually increased during flower developmental stages in ‘Shatian’ pummelo. Moreover, expression analysis by RT-PCR, qRT-PCR and in situ hybridization of CgSKP1 showed that it was highly expressed in the leaf, petal, anther and ovary, but lowly in the style. These findings indicated that CgSKP1 was closely related to ‘Shatian’ pummelo flower development.     

Steroidal glycoalkaloid content of potato,tomato and their somatic hybrids

Summary Analyses of leaves and ‘tubers’ from somatic hybrids of potato and tomato (‘pomato’ with plastids of potato, ‘topato’ with plastids of tomato) produced by fusion of protoplasts from liquid cultures of dihaploid potato and mesophyll of tomato revealed the presence of the two major potato glycoalkaloids (α-solanine and α-chaconine) as well as the tomato glycoalkaloid (αtomatine). The total alkaloid content of leaves was greater than that of ‘tubers’ and similar to levels in the foliage of parent plants. However, glycoalkaloids were more abundant in hybrid ‘tubers’ than in normal potato tubers by a factor of 5–15. In hybrid foliage, approximately 98% of the alkaloid present was of potato origin whereas in ‘tubers’ the reverse was the case, with tomatine comprising 60–70% of the total alkaloid. The similarities in alkaloid content and ratios between the pomato and the topato lines indicate that plastomes do not influence the biosynthesis and distribution of these alkaloids. The results indicate that major secondary metabolites may prove useful for assessing the hybrid nature of such plants.     

Linkage maps of grapevine displaying the chromosomal locations of 420 microsatellite markers and 82 markers for <Emphasis Type="Italic">R</Emphasis>-gene candidates

Genetic maps functionally oriented towards disease resistance have been constructed in grapevine by analysing with a simultaneous maximum-likelihood estimation of linkage 502 markers including microsatellites and resistance gene analogs (RGAs). Mapping material consisted of two pseudo-testcrosses, ‘Chardonnay’ × ‘Bianca’ and ‘Cabernet Sauvignon’ × ‘20/3’ where the seed parents were Vitis vinifera genotypes and the male parents were Vitis hybrids carrying resistance to mildew diseases. Individual maps included 320–364 markers each. The simultaneous use of two mapping crosses made with two pairs of distantly related parents allowed mapping as much as 91% of the markers tested. The integrated map included 420 Simple Sequence Repeat (SSR) markers that identified 536 SSR loci and 82 RGA markers that identified 173 RGA loci. This map consisted of 19 linkage groups (LGs) corresponding to the grape haploid chromosome number, had a total length of 1,676 cM and a mean distance between adjacent loci of 3.6 cM. Single-locus SSR markers were randomly distributed over the map (CD = 1.12). RGA markers were found in 18 of the 19 LGs but most of them (83%) were clustered on seven LGs, namely groups 3, 7, 9, 12, 13, 18 and 19. Several RGA clusters mapped to chromosomal regions where phenotypic traits of resistance to fungal diseases such as downy mildew and powdery mildew, bacterial diseases such as Pierce’s disease, and pests such as dagger and root-knot nematode, were previously mapped in different segregating populations. The high number of RGA markers integrated into this new map will help find markers linked to genetic determinants of different pest and disease resistances in grape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A chimeric <Emphasis Type="Italic">Rfo</Emphasis> gene generated by intergenic recombination cosegregates with the fertility restorer phenotype for cytoplasmic male sterility in radish

In this work, we have identified a chimeric pentatricopeptide repeat (PPR)-encoding gene cosegregating with the fertility restorer phenotype for cytoplasmic male sterility (CMS) in radish. We have constructed a CMS-Rf system consisting of sterile line ‘9802A2’, maintainer line ‘9802B2’ and restorer line ‘2007H’. F₂ segregating population analysis indicated that male fertility is restored by a single dominant gene in the CMS-Rf system described above. A PPR gene named Rfoc was found in the restorer line ‘2007H’. It cosegregated with the fertility restorer in the F₂ segregating population which is composed of 613 fertile plants and 187 sterile plants. The Rfoc gene encodes a predicted protein 687 amino acids in length, comprising 16 PPR domains and with a putative mitochondrial targeting signal. Sequence alignment showed that recombination between the 5′ region of Rfob (EU163282) and the 3′ region of PPR24 (AY285675) resulted in Rfoc, indicating a recent unequal crossing-over event between Rfo and PPR24 loci at a distance of 5.5 kb. The sterile line ‘9802A2’ contains the rfob gene. In the F₂ population, Rfoc and rfob were observed to fit a segregation ratio 1:2:1 showing that Rfoc was allelic to Rfo. Previously we have reported that a fertile line ‘2006H’, which carries the recessive rfob gene, is able to restore the male fertility of CMS line ‘9802A1’ (Wang et al. in Theor Appl Genet 117:313–320, 2008). However, here when conducting a cross between the fertile line ‘2006H’ and CMS line ‘9802A2, the resulting plants were male sterile, which shows that sterile line ‘9802A2’ possesses a different nuclear background compared to ‘9802A1’. Based on these results, the genetic model of fertility restoration for radish CMS is also discussed.