QTLs for resistance to preharvest sprouting in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Grain quality of rye is often negatively affected by sprouting - a complex trait with a poorly understood genetic background and strong interaction with weather conditions. The aim of this report was to detect the main quantitative trait loci (QTLs) underlying preharvest sprouting resistance in rye, measured as a percentage of sprouted kernels after spraying spikes with water for 7 days. Simple and composite interval mapping, carried out in 3 environments on 94 F3 and F4 families of the cross between sprouting-susceptible (541) and sprouting-resistant (Ot1-3) inbred lines, revealed 5 QTLs located on chromosome arms 1RL, 2RL, 5RL, 6RL and 7RL. The significance of these QTLs was additionally proved by disruptive selection carried out on 5000 F2 plants of the 541 x Ot1-3 cross and continued to the F5 generation of recombinant inbred lines (RIL), which strongly affected allele frequencies at linked marker loci. Resistance to preharvest sprouting showed dominant inheritance except for QPhs.uas-7R.1 (recessive) and QPhs.uas-1R.1 (additive). Results of the present study suggest that introgression of 4-5 QTLs, identified in line Ot1-3, should substantially reduce sprouting risk in rye varieties.     

Co-expression and inheritance of foreign genes in transformants obtained by direct DNA transformation of tobacco protoplasts

Summary A plasmid containing two marker genes for expression in plants was constructed. This 16 kb vector, pCT1T3, contains an intact nopaline synthase gene and a chimaeric gene consisting of the promoter and terminator regions from cauliflower mosaic virus gene VI and a structural gene, aminoglycoside phosphotransferase (APH(3′)II), from the bacterial transposon Tn5. After transformation of tobacco mesophyll protoplasts with this plasmid, several kanamycin-resistant transformants were obtained. Intensive studies on the drug tolerance of growth and differentiation of the transformants showed that the chimaeric gene was stably expressed. Of 17 independent transformants, 3 (about 18%) expressed the two marker genes, regardless of the state of differentiation, as did individual plants regenerated from the same callus. Multiple copies of the inserted DNA were found in some transformants. Viable seeds were produced by 12 out of 15 independent transformants. Seeds obtained by self-pollination were germinated on medium containing kanamycin sulphate. With the exception of one clone, resistant seedlings with green leaves and sensitive seedlings with white leaves were found to segregate in a 3:1 ratio. This suggests that the inheritance of the inserted gene is Mendelian. A reciprocal cross between the transformants and wild-type tobacco also showed nuclear transmission of the APH(3′)II gene. This was consistently maintained in a subclone of the same transformant derived from the same callus line. Stable inheritance of the single dominant character was also seen among seeds formed in several different flower pods of the same individual plants. Two clones were also found to synthesize nopaline in addition to expressing APH(3′)II. Analysis of the progeny obtained by self-crosses of such transformants revealed the simultaneous expression of these two enzymes, indicating that the two marker genes are linked on the same chromosome.     

无选择标记和载体骨干序列的Xa21转基因水稻的获得

利用双右边界T-DNA载体通过根癌农杆菌介导法将水稻白叶枯病广谱抗性基因Xa21导入杂交稻重要恢复系C418中。T0代共获得27个独立转基因株系,通过田间抗性鉴定与PCR分析,有17个株系的Xa21基因分子鉴定为阳性,且对白叶枯病原菌P6生理小种具有抗性。通过对17个株系的后代植株进行田间抗性鉴定,分子标记辅助选择及Southern杂交分析,结果显示4个株系的T1代植株中能分离出无潮霉素标记基因的Xa21转基因植株。无选择标记Xa21转基因株系的获得率为15%。PCR检测还表明,这些无选择标记的Xa21转基因植株不带有载体骨架序列。通过对转基因后代进一步的抗性鉴定与PCR辅助选择,获得了无选择标记和载体骨架序列的转基因Xa21纯合的抗白叶枯病水稻。     

Mutations leading to nuclear restoration of fertility in S male-sterile cytoplasm in maize

Summary Among the offspring of crosses involving S male-sterile shrunken-2 inbred lines and their corresponding isogenic maintainer lines a number of exceptional male-fertile plants were identified. Some of these were plants with entirely fertile tassels but most were chimeras involving both sterile and fertile tassel elements. The majority of male-fertile exceptional plants, upon crossing with male-sterile testers, produced male-sterile test-cross progeny, indicating that the male-fertile trait is not pollen transmissible. However, there were four separate instances, involving three of the inbred lines, in which the crosses with S male-sterile testers produced male-fertile progeny, indicating that the newly arisen male-fertile trait is pollen transmissible. In three of these cases, the male fertility can be traced to a single plant in essentially male-sterile families. The fourth evidently involved a change in a maintainer plant whose progeny thereafter segregated for the ability to restore S sterile cytoplasm. In all cases, the results of progeny tests are consistent with the gametophytic pattern of restoration associated with S male-sterile cytoplasm.The male-fertile exceptions described here can be accounted for formally as mutations at one or more restorer gene loci in the nucleus. Taking account of the fact that mutations of restorer genes have not been reported previously in maize, and that four such changes were encountered in the same strains in which we have identified other male-fertile exceptions involving change in the cytoplasm, we have suggested a common basis for the two kinds of events. According to this scheme, given the first appearance, by whatever process, of the male-fertile element in sterile cytoplasm, it may become established and continue to propagate either in the cytoplasm or in the nucleus. In the former case, the change registers as cytoplasmic and the new strain has the characteristics of a maintainer which transmits the male-fertile trait through the egg, but not the sperm; in the latter case, the change occurs in the nucleus and the new strain, now behaving as a restorer, transmits male fertility through both egg and sperm.Dedicated to Dr. M. M. Rhoades on the occasion of his 70th birthday.Research supported in part by CSRS grant 177-15-04, PL 89–106, and by NSF GB 29488.     

Development of enhancer trap lines for functional analysis of the rice genome

Enhancer trapping has provided a powerful strategy for identifying novel genes and regulatory elements. In this study, we adopted an enhancer trap system, consisting of the GAL4/VP16-UAS elements with GUS as the reporter, to generate a trapping population of rice. Currently, 31 443 independent transformants were obtained from two cultivars using Agrobacterium-mediated T-DNA insertion. PCR tests and DNA blot hybridization showed that about 94% of the transformants contained T-DNA insertions. The transformants carried, on average, two copies of the T-DNA, and 42% of the transformants had single-copy insertions. Histochemical assays of approximately 1000 T0 plants revealed various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. The expression pattern of the reporter gene in T1 families corresponded well with the T0 plants and segregated in a 3 : 1 Mendelian ratio in majority of the T1 families tested. The frequency of reporter gene expression in the enhancer trap lines was much higher than that in gene trap lines reported previously. Analysis of flanking sequences of T-DNA insertion sites from about 200 transformants showed that almost all the sequences had homology with the sequences in the rice genome databases. Morphologically conspicuous mutations were observed in about 7.5% of the 2679 T1 families that were field-tested, and segregation in more than one-third of the families fit the 3 : 1 ratio. It was concluded that GAL4/VP16-UAS elements provided a useful system for enhancer trap in rice.     

Heterogeneous inbred family (HIF) analysis: a method for developing near-isogenic lines that differ at quantitative trait loci

Abtract  Analysis of near-isogenic lines (NILs) that differ at quantitative trait loci (QTL) can be an effective approach for the detailed mapping and characterization of individual loci. Although NILs are useful for genetic and physiological studies, the time and effort required to develop these lines have limited their use. Here we describe a procedure to identify NILs for any region of the genome that can be analyzed with molecular or other genetic markers. The procedure utilizes molecular markers to identify heterogeneous inbred families (HIFs) that segregate for a genomic region of interest. Each HIF is isogenic at the majority of loci in the genome, but NILs differing for markers linked to QTL of interest can be extracted from segregating families. The application of this procedure is described for two QTL associated with seed weight in sorghum. A population of 98 HIFs was screened with two RAPD markers from different linkage groups that were associated with seed weight. Three segregating families were identified for each marker. The progeny of these HIFs were characterized for the segregation of seed weight and other yield components and for markers flanking each QTL. NILs derived from each HIF had significantly different seed weights confirming the presence of at least two loci that influence seed weight in sorghum. Received: 16 September 1996 / Accepted: 25 April 1997     

A new recombined double low restorer line for the Ogu-INRA cms in rapeseed (Brassica napus L.)

A major objective of breeders using the Ogu-INRA cytoplasmic male sterility (cms) system in rapeseed (Brassica napus L.) is to obtain double low restorer lines with a shorter introgression and a good agronomic value. The development of low glucosinolate content (low GC) restorer lines often occurs through the deletion of a part of the introgression. One of these lines has lost the radish Pgi-2 allele expression, without recovering that of the rapeseed Pgi-2 allele. This line shows a defect in the meiotic transmission of the restorer gene Rfo and a very poor agronomic value. We initiated a programme to force non-spontaneous recombination between this Rfo-carrying introgression and the rapeseed homologous chromosome from a low GC B. napus line. Gamma ray irradiation was used to induce chromosome breakage just prior meiosis aiming at just such a recombination. Low GC cms plants were crossed with the pollen of irradiated plants that were heterozygous for this introgression. The F₂ families were scored for their vigour, transmission rate of Rfo and female fertility. One family of plants, R2000, showed an improved behaviour for these three traits. This family presented a unique combination of molecular markers when compared to other rapeseed restorers analysed, which suggests that the recombination event allowed the recovery of B. oleracea genetic information that was originally replaced by the radish introgression in the original restorers. This resulted in a duplicated region (originating from radish and B. oleracea) on the chromosome carrying the introgression in the R2000 family.Electronic Supplementary Material Supplementary material is available for this article at     

Sexual transmission of transposed activator elements in transgenic tomatoes

The transmission of transposed Ac elements in progeny derived by self-pollination of ten transformed tomato plants has been examined by Southern hybridization analysis. We show that six of these primary transformants have transmitted a transposed Ac to at least one progeny. One of the families was segregating for at least two different insertion events. In five of ten families, progeny were detected that contained a transposed Ac but no donor T-DNA sequences, indicating that a recombination event occurred between the original and new Ac insertion site. Somatic transposition of Ac as late as the R2 generation is evidenced. One family contained an empty donor site fragment but Ac was not detected in either the parent or progeny, indicating Ac was lost in this population early in regeneration. While four of ten families were segregating for aberrant phenotypes, there was no evidence that the mutated gene was linked to a transposed Ac.     

Glycerol kinase deficiency: evidence for complexity in a single gene disorder

Glycerol kinase deficiency (GKD) occurs as part of an Xp21 contiguous gene syndrome or as isolated GKD. The isolated form can be either symptomatic with episodic metabolic and central nervous system (CNS) decompensation or asymptomatic with hyperglycerolemia and glyceroluria only. To better understand the pathogenesis of isolated GKD, we sought individuals with point mutations in the GK coding region and measured their GK enzyme activities. We identified six individuals with missense mutations: four (N288D, A305V, M428T, and Q438R) among males who were asymptomatic and two (D198G, R405Q) in individuals who were symptomatic. GK activity measured in lymphoblastoid cell lines or fibroblasts was similar for the symptomatic and the asymptomatic individuals. Mapping of the individuals' missense mutations to the three-dimensional structure of Escherichia coli GK revealed that the symptomatic individuals' mutations are in the same region as a subset of the mutations among the asymptomatic individuals, adjacent to the active-site cleft. We conclude that, like many other disorders, GK genotype does not predict GKD phenotype. We hypothesize that the phenotype of an individual with GKD is a complex trait influenced by additional, independently inherited genes.     

Genetics of qualitative traits in domesticated chia (Salvia hispanica L.)

In Salvia hispanica L., several changes in qualitative characters, including seed coat color, stem pigmentation, and shattering, have evolved with cultivation and domestication. Three F(2) segregating generations from crosses between wild and domesticated parents were scored for three qualitative traits. A single recessive gene, designated scc, was found to govern the white seed characteristic. A single dominant gene, designated SSP, was found to control striated stem pigmentation. A complete dominance of open calyx over closed calyx was observed in F(1) generations and small numbers of plants with closed calyxes were observed in F(2) generations, not conforming to Mendelian ratios. For this non-shattering trait, a complementation test was conducted between two lines representative of geographically and morphologically divergent domesticated varieties. Complementary gene action was not observed in any F(1) plants, and all F(2) plants were homogeneous with respect to the trait, suggesting the same genetic control for non-shattering among domesticated varieties. An analysis of limited data for linkage of SSP and scc indicated that the two loci segregate independently.     

Dominant inheritance of isolated hypermethioninemia is associated with a mutation in the human methionine adenosyltransferase 1A gene.

Methionine adenosyltransferase (MAT) I/III deficiency, characterized by isolated persistent hypermethioninemia, is caused by mutations in the MAT1A gene encoding MAT(alpha)1, the subunit of major hepatic enzymes MAT I ([alpha1]4) and III([alpha1]2). We have characterized 10 MAT1A mutations in MAT I/III-deficient individuals and shown that the associated hypermethioninemic phenotype was inherited as an autosomal recessive trait. However, dominant inheritance of hypermethioninemia, also hypothesized to be caused by MAT I/III deficiency, has been reported in two families. Here we show that the only mutation uncovered in one of these families, G, is a G-->A transition at nt 791 in exon VII of one MAT1A allele that converts an arginine at position 264 to a histidine (R264H). This single allelic R264H mutation was subsequently identified in two hypermethioninemic individuals in an additional family, C. Family C members were also found to inherit hypermethioninemia in a dominant fashion, and the available affected members analyzed carried the single allelic R264H mutation. Substitution of R-264 with histidine (R264H, the naturally occurring mutant), leucine (R264L), aspartic acid (R264D), or glutamic acid (R264E) greatly reduced MAT activity and severely impaired the ability of the MAT(alpha)1 subunits to form homodimers essential for optimal catalytic activity. On the other hand, when lysine was substituted for R-264 (R264K), the mutant alpha1 subunit was able to form dimers that retain significant MAT activity, suggesting that amino acid 264 is involved in intersubunit salt-bridge formation. Cotransfection studies show that R264/R264H MAT(alpha)1 heterodimers are enzymatically inactive, thus providing an explanation for the R264H-mediated dominant inheritance of hypermethioninemia.     

Hybrid genes in the analysis of transformation conditions. 3. Temporal/spatial fate of NPTII gene integration,its inheritance and factors affecting these processes in Nicotiana plumbaginifolia

Freshly isolated haploid mesophyll protoplasts of Nicotiana plumbaginifolia were transformed for kanamycin resistance. In 38% of the 224 transformants analysed, transmission of the NPTII gene occurred as a homozygous trait, while 62% of the transformants were heterozygous for the trait. In the first case, the foreign DNA integration predominantly (95%) resulted in monogenic inheritance. The second group was characterized by a significant (46%) proportion of multiple insertions. However, there was no clear-cut difference in the integration pattern between the two groups. Furthermore, transformation rates were increased by 4- to 10-fold when transformed diploid protoplasts were treated with UV light or with 3-aminobenzamide. The number of insertion sites was also increased by these treatments. These results shed further light on the fate of the foreign DNA in transformed plants and on means to control or manipulate the integration event(s).     

Leveraging eQTLs to identify individual-level tissue of interest for a complex trait

Genetic predisposition for complex traits often acts through multiple tissues at different time points during development. As a simple example, the genetic predisposition for obesity could be manifested either through inherited variants that control metabolism through regulation of genes expressed in the brain, or that control fat storage through dysregulation of genes expressed in adipose tissue, or both. Here we describe a statistical approach that leverages tissue-specific expression quantitative trait loci (eQTLs) corresponding to tissue-specific genes to prioritize a relevant tissue underlying the genetic predisposition of a given individual for a complex trait. Unlike existing approaches that prioritize relevant tissues for the trait in the population, our approach probabilistically quantifies the tissue-wise genetic contribution to the trait for a given individual. We hypothesize that for a subgroup of individuals the genetic contribution to the trait can be mediated primarily through a specific tissue. Through simulations using the UK Biobank, we show that our approach can predict the relevant tissue accurately and can cluster individuals according to their tissue-specific genetic architecture. We analyze body mass index (BMI) and waist to hip ratio adjusted for BMI (WHRadjBMI) in the UK Biobank to identify subgroups of individuals whose genetic predisposition act primarily through brain versus adipose tissue, and adipose versus muscle tissue, respectively. Notably, we find that these individuals have specific phenotypic features beyond BMI and WHRadjBMI that distinguish them from random individuals in the data, suggesting biological effects of tissue-specific genetic contribution for these traits.     

T-DNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone

Many genes are thought to be expressed during the haploid phase in plants, however, very few haploid-specific genes have been isolated so far. T-DNA insertion mutagenesis is a powerful tool for generating mutations that affect gametophyte viability and function, as disruption of a gene essential for these processes should lead to a defect in the transmission of the gametes. Mutants can therefore be screened on the basis of segregation distortion for a reporter resistance gene contained in the T-DNA. We have screened the Versailles collection of Arabidopsis transformants for 1:1 Kan^R:Kan^S segregation after selfing, focussing on gametophyte mutations which show normal transmission through one gametophyte and cause lethality or dysfunction of the other. Only 1.3% (207) of the 16,000 lines screened were scored as good candidates. Thorough genetic analysis of 38 putative T-DNA transmission defect lines (Ttd) identified 8 defective gametophyte mutants, which all showed 0 to 1% T-DNA transmission through the pollen. During the screen, we observed a high background of low-penetrance mutations, often affecting the function of both gametophytes, and many lines which were likely to carry chromosomal rearrangements. The reasons for the small number of retained lines (all male gametophytic) are discussed, as well as the finding that, for most of them, residual T-DNA transmission is obtained through the affected gametophyte.     

Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.)

Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion.     

Molecular and genetic analysis of transgenic rice plants expressing the maize ribosome-inactivating protein b-32 gene and the herbicide resistance bar gene

We have previously transformed rice (Oyrza sativa L.) with the maize ribosome-inactivating protein b-32 gene (Zmcrip3a) and the phosphinothricin resistance gene (bar). In the present study, Southern blot hybridization analysis of 56 primary fertile transformants resulted in distinct band patterns, indicating that all the transformants had been generated by independent integration events and 30% of them contained a single copy of the transgene. Segregation analysis of 15 R0 plants revealed that transgene was stably transmitted to their progenies and Southern blot band patterns of R1 progenies remained the same as the corresponding parents, suggesting that all the loci of multiple integration events are genetically linked. Also, in most of the lines, physical presence of the b-32 transgene co-segregated with the phosphinothricin- resistant phenotype, confirming that the transgene is behaving as a normal locus in the genome. However, some of R1 seedlings that contained multiple copies of the transgene became sensitive to phosphinothricin, indicating that its expression was silenced. Immunoblot analysis demonstrated that b-32 protein was produced and the levels of expression differed in different lines, estimated to be 0.5–1% of total soluble protein in the leaf tissues. In addition, the transgene-encoded protein was preferentially processed in germinating seeds and young leaves of R2 transgenic plants in a way similar to that in maize kernels, suggesting that the processing mechanism is conserved in the germination stage between rice and maize. This revised version was published online in June 2006 with corrections to the Cover Date.     

导入β—1,3—葡聚糖酶及几丁质酶基因的转基因可育油菜及其？…

利用农杆菌转化法,将组成型表达β－１,３－葡聚糖酶及必丁质酶基因的双价值植物表达载体ｐＢＬＧＣ转化优质甘蓝型油菜品种Ｈ１６５,并得到了抗卡那毒素（Ｋａｎａｍｙｃｉｎ,Ｋａｎ）的再生植株。我们对所得到的抗Ｋａｎ的再生苗进行了初步分子生物学检测,结果表明,在Ｋ１５（Ｋａｎ １５ｍｇ／Ｌ）培养基上的绿苗中有３０％为ＰＣＲ阳性植株,而在Ｋ２５（Ｋａｎ １０ｍｇ／Ｌ）培养基上的绿苗有５３％的阳性率。对部分Ｐ