Development of INDELs markers in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) using re-sequencing data

Insertions/deletions (INDELs), a type of abundant length polymorphisms in the plant genomes, combine the characteristics of both simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNP), and thus can be developed as desired molecular markers for genetic studies and crop breeding. There has been no large-scale characterization of INDELs variations in Brassica napus yet. In this study, we identified a total of 538,691 INDELs in size range of 1–10 bp by aligning whole-genome re-sequencing data of 23 B. napus inbred lines (ILs) to the B. napus genome sequence of ‘Darmor-bzh.’ Of these, 104,190 INDELs were uniquely mapped on the pseudochromosomes of the reference genome. A set of 595 unique INDELs of 2–5 bp in length was selected for experimental validation in the 23 ILs. Of these INDELs, 530 (89.01 %) produced a single PCR product and were single locus. A total of 523 (87.9 %) INDELs were found polymorphic among the 23 ILs. A genetic linkage map containing 108 single-locus INDELs and 89 anchor SSR markers was constructed using 188 recombinant ILs. The majority of INDELs markers on the linkage map showed consistency with the pseudochromosomes of the B. napus cultivar ‘Darmor-bzh.’ The INDELs variations and markers reported here will be valuable resources in future for genetic studies and molecular breeding in oilseed rape.     

Pollen-mediated intraspecific gene flow from herbicide resistant oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The cultivation of genetically modified (GM) herbicide resistant oilseed rape (Brassica napus) has increased over the past few years. The transfer of herbicide resistance genes via pollen (gene flow) from GM crops to non-GM crops is of relevance for the realisation of co-existence of different agricultural cultivation forms as well as for weed management. Therefore the likelihood of pollen-mediated gene flow has been investigated in numerous studies. Despite the difficulty to compare different experiments with varying levels of outcrossing, we performed a literature search for world-wide studies on cross-fertilisation in fully fertile oilseed rape. The occurrence and frequency of pollen-mediated intraspecific gene flow (outcrossing rate) can vary according to cultivar, experimental design, local topography and environmental conditions. The outcrossing rate from one field to another depends also on the size and arrangement of donor and recipient populations and on the ratio between donor and recipient plot size. The outcrossing levels specified in the presented studies are derived mostly from experiments where the recipient field is either surrounding the donor field (continuous design) or is located as a patch at different distances from the donor field (discontinuous design). Reports of gene flow in Brassica napus generally show that the amount of cross-fertilisation decreases as the distance from the pollen source increases. The evidence given in various studies reveals that the bulk of GM cross-fertilisation occurs within the first 10 m of the recipient field. The removal of the first 10 m of a non-transgenic field facing a GM crop might therefore be more efficient for reducing the total level of cross-fertilisation in a recipient sink population than to recommend separation distances. Future experiments should investigate cross-fertilisation with multiple adjacent donor fields at the landscape level under different spatial distributions of rapeseed cultivars and different cropping systems. The level of cross-fertilisation occurring over the whole field is mainly important for co-existence and has not been investigated in agricultural scale experiments until now. Potential problems with herbicide resistant oilseed rape volunteers arising from intraspecific gene flow can be largely solved by the choice of suitable cultivars and herbicides as well as by soil management.     

Association mapping of quantitative resistance for <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Stem canker caused by the fungus Leptosphaeria maculans is a major disease of Brassica napus. Quantitative resistance factors appear to be important components for effective and durable control of this pathogen. Quantitative trait loci (QTL) for stem canker resistance have previously been identified in the Darmor variety. However, before these QTL can be used in marker-assisted selection (MAS) to breed resistant varieties, they must be validated in a wide range of genetic backgrounds. We used an association mapping approach to confirm the markers located within the QTL previously identified in Darmor and establish their usefulness in MAS. For this, we characterized the molecular diversity of an oilseed rape collection of 128 lines showing a large spectrum of responses to infection by L. maculans, using 72 pairs of primers for simple sequence repeat and other markers. We used different association mapping models which either do or do not take into account the population structure and/or family relatedness. In all, 61 marker alleles were found to be associated with resistance to stem canker. Some of these markers were associated with previously identified QTL, which confirms their usefulness in MAS. Markers located in regions not harbouring previously identified QTL were also associated with resistance, suggesting that new QTL or allelic variants are present in the collection. All of these markers associated with stem canker resistance will help identify accessions carrying desirable alleles and facilitate QTL introgression.     

Defence reactions in the apoplastic proteome of oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> var. <Emphasis Type="Italic">napus</Emphasis>) attenuate <Emphasis Type="Italic">Verticillium longisporum</Emphasis>growth but not disease symptoms

Background

Verticillium longisporum is one of the most important pathogens of Brassicaceae that remains strictly in the xylem during most stages of its development. It has been suggested that disease symptoms are associated with clogging of xylem vessels. The aim of our study was to investigate extracellular defence reactions induced by V. longisporum in the xylem sap and leaf apoplast of Brassica napus var. napus in relation to the development of disease symptoms, photosynthesis and nutrient status.

Results

V. longisporum (strain VL43) did not overcome the hypocotyl barrier until 3 weeks after infection although the plants showed massive stunting of the stem and mild leaf chlorosis. During this initial infection phase photosynthetic carbon assimilation, transpiration rate and nutrient elements in leaves were not affected in VL43-infected compared to non-infected plants. Proteome analysis of the leaf apoplast revealed 170 spots after 2-D-protein separation, of which 12 were significantly enhanced in response to VL43-infection. LS-MS/MS analysis and data base searches revealed matches of VL43-responsive proteins to an endochitinase, a peroxidase, a PR-4 protein and a β-1,3-glucanase. In xylem sap three up-regulated proteins were found of which two were identified as PR-4 and β-1,3-glucanase. Xylem sap of infected plants inhibited the growth of V. longisporum.

Conclusion

V. longisporum infection did not result in drought stress or nutrient limitations. Stunting and mild chlorosis were, therefore, not consequences of insufficient water and nutrient supply due to VL43-caused xylem obstruction. A distinct array of extracellular PR-proteins was activated that might have limited Verticillium spreading above the hypocotyl. In silico analysis suggested that ethylene was involved in up-regulating VL43-responsive proteins.

     

The effect of <Emphasis Type="Italic">INDEHISCENT</Emphasis> point mutations on silique shatter resistance in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>)

Key message

This study elucidates the influence of indehiscent mutations on rapeseed silique shatter resistance. A phenotype with enlarged replum-valve joint area and altered cell dimensions in the dehiscence zone is described.

Abstract

Silique shattering is a major factor reducing the yield stability of oilseed rape (Brassica napus). Attempts to improve shatter resistance often include the use of mutations in target genes identified from Arabidopsis (Arabidopsis thaliana). A variety of phenotyping methods assessing the level of shatter resistance were previously described. However, a comparative and comprehensive evaluation of the methods has not yet been undertaken. We verified the increase of shatter resistance in indehiscent double knock-down mutants obtained by TILLING with a systematic approach comparing three independent phenotyping methods. A positive correlation of silique length and shatter resistance was observed and accounted for in the analyses. Microscopic studies ruled out the influence of different lignification patterns. Instead, we propose a model to explain increased shattering resistance of indehiscent rapeseed mutants by altered cell shapes and sizes within the contact surfaces of replum and valves.

     

Amino acid contents and transport in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under different nitrogen conditions

Oilseed rape (Brassica napus L.) needs very high nitrogen fertilizer inputs. Significant amounts of this nitrogen are lost during early leaf shedding and are a source of environmental and economic concern. The objective of this study was to investigate whether the remobilization of leaf amino acids could be limiting for nitrogen use efficiency. Therefore, amino acid concentrations were analyzed in subcellular compartments of leaf mesophyll cells of plants grown under low (0.5 mM NO₃^–) and high (4 mM NO₃^–) nitrogen supply. With high nitrogen supply, young leaves showed an elevated amino acid content, mainly in vacuoles. In old leaves, however, subcellular concentrations were similar under high and low nitrogen conditions, showing that the excess nitrogen had been exported during leaf development. The phloem sap contained up to 650 mM amino acids, more than four times as much than the cytosol of mesophyll cells, indicating a very efficient phloem-loading process. Three amino acid permeases, BnAAP1, BnAAP2, and BnAAP6, were identified and characterized. BnAAP1 and BnAAP6 mediated uptake of neutral and acidic amino acids into Xenopus laevis oocytes at the actual apoplastic substrate concentrations. All three transporters were expressed in leaves and the expression was still detectable during leaf senescence, with BnAAP1 and BnAAP2 mRNA levels increasing from mature to old leaves. We conclude that phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape.     

Genetic diversity of <Emphasis Type="Italic">Pistacia khinjuk</Emphasis> Stocks. using RAPD markers and leaf morphological characters

The aim of this research was to study the genetic diversity of Pistacia khinjuk Stocks. and also to reveal the genetic relationships. Leaf morphological characters of 16 genotypes together with Randomly Amplified Polymorphic DNA (RAPD) marker data were used for this purpose. Leaf morphological characters were used for factor analysis, which determined four main factors. Grouping of genotypes by these factors was performed by Ward’s method. Fifteen primers produced a total of 146 fragments, with an average of 9.73 fragments per primer, of which 129 were polymorphic. The unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard’s similarity coefficient matrix. According to RAPD data, genotypes were separated into three groups. The first contained the genotypes with round leaflets. Genotypes with three lanceolate leaflets were located in the second and third groups, respectively. This study revealed that high diversity exists among genotypes of P. khinjuk, like in P. vera. Also it could be postulated that genotypes of P. khinjuk with oblique and lanceolate leaflets are probably descendents of the genotypes with acuminate and roundish leaflets.     

QTL analysis and the development of closely linked markers for days to flowering in spring oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Days to flowering (DTF) is an important trait impacting cultivar performance in oilseed rape (Brassica napus L.), but the interaction of all loci controlling this trait in spring-type oilseed rape is not fully understood. We identified quantitative trait loci (QTL) for variation in DTF in a doubled haploid (DH) population from the Qinghai–Tibet Plateau that includes 217 lines derived from a cross between spring-type oilseed rape (B. napus L.) line No. 5246 and line No. 4512, the latter of which is responsive to the effective accumulated temperature (EAT). A linkage map was constructed for the DH population, using 202 SSR and 293 AFLP markers. At least 22 DTF QTL were found in multiple environments. Four major QTL were located on linkage groups A7, C2, C8 and C8. Among these QTL, cqDTFA7a and cqDTFC2a were identified in five environments and individually explained 10.4 and 23.0 % of the trait variation, respectively. cqDTFC8, a major QTL observed in spring environments, and a unique winter environment QTL, qDTFC8-3, were identified; these QTL explained 10.0 and 46.5 % of the phenotypic variation, respectively. Minor QTL (for example, cqDTFC2c) and epistatic interactions seemed evident in this population. Two closely linked SSR markers for cqDTFA7a and cqDTFC8 were developed (G1803 and S034). BnAP1, a B. napus gene with homology to Arabidopsis thaliana that was identified as a cqDTFA7a candidate gene, played a major role in this study. The allelic effects of the major and minor QTL on DTF were further validated in the DH population and in 93 breeding genotypes.     

Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) suspension cell cultures

Phosphite (H₂PO₃^–, Phi) prevents the acclimation of plants and yeast to orthophosphate (Pi, HPO₄^2–) deprivation by specifically obstructing the derepression of genes encoding proteins characteristic of their Pi-starvation response. In this study, we report that prolonged (i.e., 3–4 weeks) culture of Brassica napus L. suspension cells in Pi-deficient (–Pi) media leads to programmed cell death (PCD). However, when the B. napus cells were subcultured into –Pi media containing 2 mM Phi, they initiated PCD within 5 days, with 95% cell death observed by day 9. Dying cells exhibited several morphological and biochemical features characteristic of PCD, including protoplast shrinkage, chromatin condensation, and fragmentation of nuclear DNA. Immunoblotting indicated that B. napus cells undergoing PCD upregulated a 30-kDa cysteine endoprotease that is induced during PCD in the inner integument cells of developing B. napus seeds. It is concluded that PCD in B. napus suspension cells is triggered by extended Pi starvation, and that Phi treatment greatly accelerates this process. Our results also infer that the adaptive value of acclimating at the molecular level to Pi-stress is to extend the viability of –Pi B. napus cell cultures by about 3 weeks.Abbreviations APase acid phosphatase (EC 3.1.3.2) - BnCysP B. napus cysteine proteinase - DAPI 4,6-diamidino-2-phenylindole - FDA fluorescein diacetate - PCD programmed cell death - Phi phosphite - +Pi and –Pi Pi-sufficient and -deficient, respectively - PI propidium iodide - PSI Pi-starvation inducible     

Interaction of silicon and cadmium in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

The objective of this study was to determine the effect of silicon (Si) and cadmium (Cd) on root and shoot growth and Cd uptake in two hydroponically cultivated Brassica species (B. juncea (L.) Czern. cv. Vitasso and B. napus L. cv. Atlantic). Both species are potentially usable for phytoextraction. Inhibitory effects of Cd on root elongation were diminished by the impact of Si. Primary roots elongation in the presence of Cd + Si compared with Cd was stronger and the number of lateral roots was lower in B. juncea than in B. napus. Cd content per plant was higher in B. napus roots and shoots compared with B. juncea. Suberin lamellae were formed closer to the root apex in Cd + Si than in Cd treated plants and this effect was stronger in B. napus than in B. juncea. Accelerated maturation of endodermis was associated with reduced Cd uptake. Cd decreased the content of chlorophylls and carotenoids in both species, but Si addition positively influenced the content of photosynthetic pigments which was higher in B. napus than in B. juncea. Si enhanced more substantially translocation of Cd into the shoot of B. napus than of B. juncea. Based on our results B. napus seems to be more suitable for Cd phytoextraction than B. juncea because these plants produce more biomass and accumulate higher amount of Cd. The protective effect of Si on Cd treated Brassica plants could be attributed to more extensive development of suberin lamellae in endodermis.     

Phosphinothricin-resistant <Emphasis Type="Italic">Brassica napus</Emphasis> + <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> somatic hybrids

Hybrid plants resistant to phosphinothricin (PPT) are obtained as a result of experiments with somatic hybridization between Brassica napus L. cv. Kalinins’kyy and Orychophragmus violaceus L. O.E. Shulz. The hybrids inherited PPT resistance from O. violaceus plants that had been previously transformed by a vector containing the maize transposon system Spm/dSPm with bar gene located within the nonautonomous transposon. The morphologically obtained plants occupy an intermediate position between the initial forms, which is in agreement with the results of isoenzyme analyses (analysis of multiple forms of amylase and esterase) and PCR analysis (presence of the genes bar, gus, and SpmTPase). Inheritance of the plastome occurs from oilseed rape, while that of the mitochondrion, from O. violaceus, which is proved by means of PCR-RFLP analysis. The plant hybrids may be utilized for further selection research with oilseed rape following determination of the edible quality of its oil as well as in experiments with chloroplast transformation, a topic which is of critical importance for oilseed rape.     

Cadmium distribution and microlocalization in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) after long-term growth on cadmium-contaminated soil

Brassica napus (L.) was grown from seeds on a reconstituted soil contaminated with 100 mg Cd kg(-1). Compared with roots and stems, leaves accumulated high amounts of Cd. Although the Cd concentration in the leaves remained high throughout plant growth and no appreciable change was noticed in the total, extractable or soluble Cd in the soil adhering to the roots, the symptoms of Cd toxicity (leaf chlorosis, growth retardation) decreased with time. Cd induced a noticeable accumulation of phytochelatins in young plants (aged 22 days), which decreased in parallel to the disappearance of the symptoms of Cd intoxication. The subcellular distribution of Cd in leaves of Cd-acclimated plants was determined using biochemical, microscopic and metal-imaging techniques. Leaf fractionation by differential centrifugations showed that Cd was present predominantly in the 'soluble' fraction corresponding to the vacuoles and the cytoplasm. Transmission electron microscopic analyses revealed that those cell compartments contained electron-dense granules associated with needle-like structures. Cd, and also high amounts of sulfur, was detected in those structures by electron-spectroscopic imaging. This technique also showed Cd binding to cell walls by a mechanism that does not involve sulfur atoms. In contrast, very little Cd was found in chloroplasts, and this is consistent with the preservation of photosynthesis in plants grown on Cd-polluted soil. The microanalytical results presented here confirm that long-term growth of B. napus on Cd-contaminated soil is accompanied by preferential storage of Cd in the vacuoles and the cell walls. This phenomenon diverted Cd ions from metabolically active compartments (cytosol, chloroplasts, mitochondria), resulting in a reduction of Cd toxicity in the leaves.     

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.     

Genetic variation and phylogeographic history of <Emphasis Type="Italic">Picea likiangensis</Emphasis> revealed by RAPD markers

Repeated cycles of retreat and recolonization during the Quaternary ice ages are thought to have greatly influenced current species distributions and their genetic diversity. It remains unclear how this climatic oscillation has affected the distribution of genetic diversity between populations of wind-pollinated conifers in the Qinghai-Tibetan region. In this study, we investigated the within-species genetic diversity and phylogenetic relationships of Picea likiangensis, a dominant forest species in this region using polymorphic DNA (RAPD) markers. Our results suggest that this species has high overall genetic diversity, with 85.42% of loci being polymorphic and an average expected heterozygosity (H _E) of 0.239. However, there were relatively low levels of polymorphism at population levels and the differences between populations were not significant, with percentages of polymorphic bands (PPB) ranging from 46.88 to 69.76%, Nei’s gene diversity (H _E) from 0.179 to 0.289 and Shannon’s indices (Hpop) from 0.267 to 0.421. In accordance with our proposed hypothesis, a high level of genetic differentiation among populations was detected based on Nei’s genetic diversity (G _ST = 0.256) and AMOVA analysis (Phi _st = 0.236). Gene flow between populations was found to be limited (Nm = 1.4532) and far lower than reported for other conifer species with wide distribution ranges from other regions. No clusters corresponding to three morphological varieties found in the south, north and west, respectively, were detected in either UPGMA or PCO analyses. Our results suggest that this species may have had different refugia during the glacial stages in the southern region and that the northern variety may have multiple origins from these different refugia.     

Mapping of the Ogura fertility restorer gene <Emphasis Type="Italic">Rfo</Emphasis> and development of <Emphasis Type="Italic">Rfo</Emphasis> allele-specific markers in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader^® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.     

Chitinase isozymes induced by TYMV and <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> during compatible and incompatible interaction with <Emphasis Type="Italic">Brassica napus</Emphasis>

Accumulation of extracellular chitinases in Brassica napus plants infected with Turnip yellow mosaic virus (TYMV) and fungal pathogen Leptosphaeria maculans was studied in both compatible and incompatible interaction. Analysis of apoplast fluid by means of non-denaturing anodic and cathodic PAGE followed by in-gel detection of chitinase activity revealed a number of chitinase isozymes. TYMV induced 8 acidic and 4 basic isozymes in a systemic way. Except for one acidic and one basic isozyme, all other chitinases were also constitutively present in low amounts in mock inoculated control. In TYMV systemically infected plants, chitinases were detected in leaves expressing symptoms as well as in symptomless ones. Both virulent and avirulent L. maculans isolates induced production of chitinase isozymes in cotyledons in a time dependent manner. Some of them were present in plants constitutively and their content increased after inoculation. Three of five acidic and two of three basic isozymes responded to L. maculans infection. Chitinases started to accumulate before symptom appearance. First two acidic isozymes were detected 24 h after inoculation. The difference between compatible and incompatibe interaction reflected two basic isozymes.     

Effect of a turnip rape <Emphasis Type="Italic">(Brassica rapa)</Emphasis> trap crop on stem-mining pests and their parasitoids in winter oilseed rape <Emphasis Type="Italic">(Brassica napus)</Emphasis>

Concerns about the negative effects of chemical control of oilseed rape (Brassica napus L.) pests on non-target species, human safety, and development of insecticide resistance, require alternative control strategies such as the use of trap crops and biocontrol to be developed. Psylliodes chrysocephala(L.) (Coleoptera: Chrysomelidae) (cabbage stem flea beetle) and Ceutorhynchus pallidactylus (Marsh.) (Coleoptera: Curculionidae) (cabbage stem weevil) are two major stem-mining pests of oilseed rape. This study investigated the phenology of these pests and their main parasitoids in the UK, the potential use of turnip rape (Brassica rapa L.) as a trap crop to reduce oilseed rape infestation, and the effects of insecticide treatment on pest incidence and larval parasitism. Water trap samples, plant dissections and pest larval dissections were done to determine: the incidence of adult pests and their parasitoids, the level of plant infestation by the pests and percentage larval parasitism, respectively. The turnip rape trap crop borders reduced P. chrysocephalabut not C. pallidactylus infestation of oilseed rape plots. Treatment of the trap crop with insecticide had little effect on either pest or parasitoid incidence in the oilseed rape. TersilochusmicrogasterSzép. andT. obscurator Aub. (Hymenoptera: Ichneumonidae) were the main larval parasitoids of P. chrysocephalaand C. pallidactylus, respectively. Tersilochus microgasteris reported for the first time in the UK. The implications for integrated pest management are discussed.     

Genetic analysis of loci associated with partial resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Sclerotinia stem rot is the most devastating disease of rapeseed (Brassica napus L.) in China. Quantitative trait loci (QTLs) involved in resistance to Sclerotinia sclerotiorum were detected in a rapeseed population of 128-F(2:3) families derived from a cross between the male sterility restorer line H5200 and a partial resistant line Ning RS-1. A total of 107 molecular markers including 72 RFLPs, 30 AFLPs, 3 SSRs and 2 RAPDs were employed to construct a genetic linkage map with 23 linkage groups covering 1,625.7 cM with an average space of 15.2 cM. Resistance was assessed empirically at two developmental stages: with a detached leaf inoculation at the seedling stage and in vivo stem inoculation at the mature plant stage. The observed resistance was scored for each plant as leaf resistance at the seedling stage (LRS) and stem resistance at the mature plant stage (SRM). A total of 13 loci were identified by one-way ANOVA and six QTLs were detected with MapMaker-QTL. We found that three of the six QTLs were associated with leaf resistance at the seedling stage and collectively accounted for 40.7% of the total phenotypic variation, each accounting for 23.2%, 16.6% and 13.6% respectively. Three QTLs were found corresponding to the disease resistance at the mature plant stage, explaining 49.0% of the phenotypic variation. Epistasis was observed for the resistance and the additive by additive interactions were the predominant type of epistasis. It was concluded that both single-locus QTLs and epistatic interactions played important roles in Sclerotinia resistance in rapeseed.     

Small RNA changes in synthetic <Emphasis Type="Italic">Brassica napus</Emphasis>

Main conclusion

Small RNAs and microRNAs were found to vary extensively in synthetic Brassica napus and subsequent generations, accompanied by the activation of transposable elements in response to hybridization and polyploidization.

Abstract

Resynthesizing B. napus by hybridization and chromosome doubling provides an approach to create novel polyploids and increases the usable genetic variability in oilseed rape. Although many studies have shown that small RNAs (sRNAs) act as important factor during hybridization and polyploidization in plants, much less is known on how sRNAs change in synthetic B. napus, particularly in subsequent generations after formation. We performed high-throughput sequencing of sRNAs in S₁–S₄ generations of synthetic B. napus and in the homozygous B. oleracea and B. rapa parent lines. We found that the number of small RNAs (sRNAs) and microRNAs (miRNAs) doubled in synthetic B. napus relative to the parents. The proportions of common sRNAs detected varied from the S₁ to S₄ generations, suggesting sRNAs are unstable in synthetic B. napus. The majority of miRNAs (67.2 %) were non-additively expressed in the synthesized Brassica allotetraploid, and 33.3 % of miRNAs were novel in the resynthesized B. napus. The percentage of miRNAs derived from transposable elements (TEs) also increased, indicating transposon activation and increased transposon-associated miRNA production in response to hybridization and polyploidization. The number of target genes for each miRNA in the synthesized Brassica allotetraploid was doubled relative to the parents, enhancing the complexity of gene expression regulation. The potential roles of miRNAs and their targets are discussed. Our data demonstrate generational changes in sRNAs and miRNAs in synthesized B. napus.