Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

Effects of Endogenous Salicylic Acid During Calcium Deficiency-Induced Tipburn in Chinese Cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

By cultivating tipburn-susceptible plants in modified Hoagland’s medium containing of gradient exogenous calcium (Ca²⁺), we have shown that Ca²⁺ deficiency is one of the main causes of tipburn in Chinese cabbage (Brassica rapa L. ssp. pekinensis). The effect of endogenous plant Ca²⁺ concentrations on tipburn was also studied in a doubled haploid (DH) population consisting of 100 individuals, but no correlation was found. We then examined the expression of 12 Ca²⁺ transporter genes that function in cytosolic Ca²⁺ homeostasis in both tipburn-susceptible and tipburn-resistant plants under normal and tipburn-inducing conditions. Expression patterns for most of these genes differed between the two types of plants. Salicylic acid (SA) accumulated in response to conditions of calcium deficiency in our study, and both total SA and SA β-glucoside (SAG) in tipburn-susceptible plants was ～3-fold higher than it was in resistant plants following Ca²⁺ deficiency treatment. Also, the changes observed in SA levels correlated well with cell death patterns revealed by trypan blue staining. Therefore, we speculate that the cytoplasmic Ca²⁺ fluctuation-induced downstream signaling events, as well as SA signaling or other biological events, are involved in the plant defense response to tipburn in Chinese cabbage.     

Identification and fine mapping of a stay-green gene (<Emphasis Type="Italic">Brnye1</Emphasis>) in pakchoi (<Emphasis Type="Italic">Brassica campestris</Emphasis> L. ssp. <Emphasis Type="Italic">chinensis</Emphasis>)

Key message

Using bulked segregant analysis combined with next-generation sequencing, we delimited the Brnye1 gene responsible for the stay-green trait of nye in pakchoi. Sequence analysis identified Bra019346 as the candidate gene.

Abstract

“Stay-green” refers to a plant trait whereby leaves remain green during senescence. This trait is useful in the cultivation of pakchoi (Brassica campestris L. ssp. chinensis), which is marketed as a green leaf product. This study aimed to identify the gene responsible for the stay-green trait in pakchoi. We identified a stay-green mutant in pakchoi, which we termed “nye”. Genetic analysis revealed that the stay-green trait is controlled by a single recessive gene, Brnye1. Using the BSA-seq method, a 3.0-Mb candidate region was mapped on chromosome A03, which helped us localize Brnye1 to an 81.01-kb interval between SSR markers SSRWN27 and SSRWN30 via linkage analysis in an F₂ population. We identified 12 genes in this region, 11 of which were annotated based on the Brassica rapa annotation database, and one was a functionally unknown gene. An orthologous gene of the Arabidopsis gene AtNYE1, Bra019346, was identified as the potential candidate for Brnye1. Sequence analysis revealed a 40-bp insertion in the second exon of Bra019346 in nye, which generated the TAA stop codon. A candidate gene-specific Indel marker in 1561 F₂ individuals showed perfect cosegregation with Brnye1 in the nye mutant. These results provide a foundation for uncovering the molecular mechanism of the stay-green trait in pakchoi.

     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.     

Accumulation and detoxification of cadmium in <Emphasis Type="Italic">Brassica pekinensis</Emphasis> and <Emphasis Type="Italic">B. chinensis</Emphasis>

The effect of excessive Cd on the growth and metal uptake by leafy vegetables Brassica chinensis L. (cv. Wuyueman) and Brassica pekinensis (Lour.) Rupr. (cv. Qingyan 87-114) were studied in hydroponic solution culture. The Cd concentration higher than 10 μM significantly decreased the root elongation and leaf chlorophyll contents of both plant species. The shoots of B. pekinensis had significantly higher concentrations of total and water-soluble Cd than B. chinensis. The roots of both species accumulated more Cd than the shoots in all the Cd treatments. Most of the Cd in the roots was found in the cell walls. The shoot/root ratio of Cd concentrations in B. pekinensis was always greater than that in B. chinensis. But, the concentration and proportion of Cd in the cell walls in B. chinensis were higher than that in B. pekinensis. Cadmium treatments also increased the concentrations of total non-protein thiols (NPT) in the shoots of the both species. A significant correlation was found between the concentrations of soluble Cd and NPT in plant shoots.     

Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

A number of clubroot resistant (CR) Chinese cabbage cultivars have been developed in Japan using resistant genes from CR European fodder turnips (B. rapa ssp. rapifera). Clubroot resistance in European fodder turnips are known to be controlled by the combined action of several dominant resistance genes. We have developed three Chinese cabbage clubroot-resistant doubled haploid (DH) lines-T136-8, K10, and C9-which express resistance in different manners against two isolates of Plasmodiophora brassicae, M85 and K04. Depending on the isolates, we identified two CR loci, CRk and CRc. CRk was identified by quantitative trait loci (QTL) analysis of an F(2) population derived from a cross between K10 and Q5. This locus showed resistance to both isolates and is located close to Crr3 in linkage group R3. The other locus, CRc was identified by QTL analysis of an F(2) population derived from a cross between C9 and susceptible DH line, 6R. This locus was mapped to linkage group R2 and is independent from any published CR loci. We developed sequence-tagged site markers linked to this locus.     

A high-resolution karyotype of <Emphasis Type="Italic">Brassica rapa</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis> revealed by pachytene analysis and multicolor fluorescence in situ hybridization

A molecular cytogenetic map of Chinese cabbage (Brassica rapa ssp. pekinensis, 2n=20) was constructed based on the 4-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 m to 3.30 m. Five 45S and three 5S rDNA loci identified were assigned to different chromosomes. The C11-350H loci were located on all the mitotic metaphase chromosomes, except chromosomes 2 and 4. The pachytene karyotype consisted of two metacentric (chromosomes 1 and 6), five submetacentric (chromosomes 3, 4, 5, 9 and 10), two subtelocentric (chromosomes 7 and 8), and one acrocentric (chromosome 2) chromosome(s). The mean lengths of ten pachytene chromosomes ranged from 23.7 m to 51.3 m, with a total of 385.3 m, which is 17.5-fold longer than that of the mitotic metaphase chromosomes. In the proposed pachytene karyotype, all the chromosomes of B. rapa ssp. pekinensis can be identified on the basis of chromosome length, centromere position, heterochromatin pattern, and the location of the three repetitive sequences. Moreover, the precise locations of the earlier reported loci of 5S rDNA, 45S rDNA, and Chinese cabbage tandem DNA repeat C11-350H were established using McFISH analysis. We also identified a 5S rDNA locus on the long arm of pachytene bivalent 7, which could not be detected in the mitotic metaphase chromosomes in the present and earlier studies. The deduced karyotype will be useful for structural and functional genomic studies in B. rapa.     

Induction of male sterile cabbage using a tapetum-specific promoter from<Emphasis Type="Italic"> Brassica campestris</Emphasis> L. ssp.<Emphasis Type="Italic"> pekinensis</Emphasis>

The anther (tapetum)-specific gene BcA9 was isolated from Chinese cabbage, Brassica campestris L. ssp. pekinensis cv. Jangwon, using the Arabidopsis tapetum-specific A9 gene as a probe. The DNA and amino acid sequences of the coding region of the BcA9 gene showed high homology with A9 genes from Arabidopsis and B. napus. However, the DNA sequences of the 5 noncoding (promoter) region were different, except for the sequence from –281 to –89. To test the specific activity of this promoter, a plant expression vector, pGR011, was constructed by fusing the BcA9 promoter and the cytotoxic diphtheria toxin A-chain (DTx-A) gene. Several transgenic plants from cabbage, B. oleracea ssp. capitata, were obtained by way of Agrobacterium-mediated transformation. Southern blot analysis indicated that the tapetum-specific BcA9 promoter and DTx-A gene were successfully integrated into the genome of the transgenic cabbage. Under the control of the BcA9 promoter, expression of the cytotoxic DTx-A gene in the tapetal cells of the transgenic plants resulted in male sterile cabbages. Microscopic examination revealed that pollen grains in anthers of the male sterile cabbages had not developed normally, but the vegetative growth and phenotype showed no difference compared to wild-type plants.Abbreviations At Arabidopsis thaliana - Bc Brassica camepstris - Bn Brassica napus - DTx-A Diphtheria toxin A-chain gene - hpt Hygromycin phosphotransferase - PCR Polymerase chain reaction - SDS Sodium dodecyl sulfate - SSC Sodium chloride-sodium citrate bufferThis revised version was published online September 2003 with corrections to Figure 6.Communicated by I.S. Chung     

Genetic and molecular variability of a<Emphasis Type="Italic">Turnip mosaic virus</Emphasis> population from horseradish (<Emphasis Type="Italic">Cochlearia armoracia</Emphasis> L.)

Variability and genetic structure of a novel Turnip mosaic virus (TuMV) population from horseradish (Cochlearia armoracia L.) were examined. Over 60 horseradish plants were tested to identify a total of 28 TuMV isolates, constituting the Cochlearia ARmoracia (CAR) TuMV population. Two subgroups of the CAR TuMV isolates could be distinguished: subgroup N did not infect oilseed rape (Brassica napus var. oleifera) cv. Westar plants, while subgroup A infected these plants systemically. Two types of infection of oilseed rape plants were induced by inoculation with the CAR TuMV isolates: systemic mosaic infection and systemic necrotic lesions. The complete sequences of isolates CAR37 (subgroup N) and CAR37A (subgroup A) were determined and compared. The sequences of HC-Pro and CP genes of CAR37 and CAR37A and other isolates of TuMV from other countries were compared to provide some insight into their relatedness. CAR37A, initially regarded as a variant, proved to be very different from CAR37. Re-sequencing after repeated passages confirmed the genetic stability of both isolates.     

Changes in leaf tissue of <Emphasis Type="Italic">Carica papaya</Emphasis> during single and mixed infections with <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> and <Emphasis Type="Italic">Papaya mosaic virus</Emphasis>

Papaya (Carica papaya L.) is susceptible to viral diseases caused by Papaya mosaic virus (PapMV) and Papaya ringspot virus (PRSV), which limit fruit production and affect economic yield. The symptoms produced by both the viruses are similar in early stages of infection and include vein and leaf chlorosis, which develop into mosaic at later stages of infection when leaf lamina can get reduced in size and distorted with a shoe-string aspect. Digital image analyses, such as fractal dimension (FD) and lacunarity (λ) were used here to examine papaya tissue after single and mixed infections of PRSV and PapMV. Morphological changes, such as hypoplasia, hyperplasia, and neoplasia are described in tissues with viral infections. Furthermore, we quantified these changes and suggest three ranges of tissue damage according to their λ values in rank 1 (0.01 to 0.39), rank 2 (0.4 to 0.79), and rank 3 (0.8 to 1). Our analyses suggest that in synergism and antagonism there is a competition of both viruses to occupy the same mesophyll cells, as indicated by their intermediate values of lacunarity.     

The symptom difference induced by <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in tobacco plants containing the <Emphasis Type="Italic">N</Emphasis> gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for large-scale producion of transgenic chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp.<Emphasis Type="Italic">pekinensis</Emphasis>) plants for insertional mutagenesis

In order to better utilize insertional mutagenesis and functional genomics in Chinese cabbage, we have developed an improved transformation system that more efficiently produces a large number of transgenic plants. Hypocotyl explants were inoculated withAgrobacterium tumefaciens LBA4404. This strain harbors tagging vector pRCV2, which contains a hygromycin-resistance gene, an ampicillin resistance gene, and a bacterial replication origin within the T-DNA. Transformation efficiency was highest when the explants were first co-cultivated for 3 d in a medium supplemented with 5 mg L^-1 acetosyringone, then transferred to a 0.8% agar selection medium containing 10 mg L^-1 hygro-mycin. In addition, maintaining a low pH in the co-cultivation medium was critical to enhancing transformation frequency. A total of 3369 transgenic plants were obtained, with efficiencies ranging from 2.89% to 5.00%. Southern blot analysis and T, progeny tests from 120 transgenic plants confirmed that the transgenes were stably inherited to the next generation. We also conducted plasmid rescue and inverse PCR with some transformants, based on their phenotype, to demonstrate the applicability of T-DNA tagging in Chinese cabbage. The tagged sequences were then analyzed.     

Integrated analysis of leaf morphological and color traits in different populations of Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

Key message

QTLs and candidate gene markers associated with leaf morphological and color traits were identified in two immortalized populations of Brassica rapa, which will provide genetic information for marker-assisted breeding.

Abstract

Brassica rapa is an important leafy vegetable consumed worldwide and morphology is a key character for its breeding. To enhance genetic control, quantitative trait loci (QTLs) for leaf color and plant architecture were identified using two immortalized populations with replications of 2 and 4 years. Overall, 158 and 80 QTLs associated with 23 and 14 traits were detected in the DH and RIL populations, respectively. Among them, 23 common robust-QTLs belonging to 12 traits were detected in common loci over the replications. Through comparative analysis, five crucifer genetic blocks corresponding to morphology trait (R, J&U, F and E) and color trait (F, E) were identified in three major linkage groups (A2, A3 and A7). These might be key conserved genomic regions involved with the respective traits. Through synteny analysis with Arabidopsis, 64 candidate genes involved in chlorophyll biosynthesis, cell proliferation and elongation were co-localized within QTL intervals. Among them, SCO3, ABI3, FLU, HCF153, HEMB1, CAB3 were mapped within QTLs for leaf color; and CYCD3;1, CYCB2;4, AN3, ULT1 and ANT were co-localized in QTL regions for leaf size. These robust QTLs and their candidate genes provide useful information for further research into leaf architecture with crop breeding.