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.

     

<Emphasis Type="Italic">BcMF11</Emphasis> and its homologous sequences may form a lncRNA family in <Emphasis Type="Italic">Brassica</Emphasis> diploids

BcMF11 is a long non-coding RNA that has been identified in Brassica rapa and shown to be involved in pollen development. Here, when re-cloned the gene sequence, multiple paralogous copies of BcMF11 were identified in B. rapa (A genome). Multiple paralogous copies of BcMF11 were also found in B. nigra (B genome) and Brassica oleracea (C genome), the other two primary diploids of Brassica U triangle. While in the early diverging Brassicaceae lineage including Arabidopsis thaliana, no BcMF11 homolog was found. Phylogenetic analysis showed that the BcMF11 homologous sequences cloned from A genome or C genome could be clustered into a separate branch, respectively. However, there was no distinct cluster defined for BcMF11 homologous sequences cloned from B genome. The expression of BcMF11 in B. rapa was investigated and revealed a different result in the previous study. In addition, 12 expressed sequence tags from B. napus and B. rapa showing high similarities with BcMF11 were identified in the NCBI database, which further verified that rather than the useless repeat fragments in the genome, the BcMF11 homologous genes could transcribe. It is possible that BcMF11 and its homologous sequences may form a large gene family which might be originated in the recent ancestral lineage of Brassica.     

Molecular and cytological analyses of A and C genomes at meiosis in synthetic allotriploid <Emphasis Type="Italic">Brassica</Emphasis> hybrids (ACC) between <Emphasis Type="Italic">B.napus</Emphasis> (AACC) and <Emphasis Type="Italic">B.oleracea</Emphasis> (CC)

Success of interspecific hybridization relies mostly on the adequate similarity between the implicated genomes to ensure synapsis, pairing and recombination between appropriate chromosomes during meiosis in allopolyploid species. Allotetraploid Brassica napus (AACC) is a model of natural hybridization between Brassica rapa (AA) and Brassica oleracea (CC), which are originally derived from a common ancestor, but genomic constitution of the same chromosomes probably varied among these species through time after establishment, giving rise to cytogenetic difference in the synthetic hybrids. Herein we investigated meiotic behaviors of A and C chromosomes of synthetic allotriploid Brassica hybrids (ACC) at molecular and cytological levels, which result from the interspecific cross between natural B. napus (AACC) and B.oleracea (CC), and the results showed that meiosis course was significantly aberrant in allotriploid Brassica hybrids, and chromosomes aligned chaotically at metaphase I, chromosome bridges and lags were frequently observed from later metaphase I to anaphase II during meiosis. Simultaneously, we also noticed that meiosis-related genes were abruptly down-regulated in allotriploid Brassica hybrids, which likely accounted for irregular scenario of meiosis observed in these synthetic hybrids. Therefore, these results indicated that inter-genomic exchanges of A and C chromosomes could occur frequently in synthetic Brassica hybrids, and provided an efficient approach for genetic changes of homeologous chromosomes during meiosis in polyploid B.napus breeding program.     

Identification of candidate genes of QTLs for seed weight in <Emphasis Type="Italic">Brassica napus</Emphasis> through comparative mapping among <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica</Emphasis>species

Background

Map-based cloning of quantitative trait loci (QTLs) in polyploidy crop species remains a challenge due to the complexity of their genome structures. QTLs for seed weight in B. napus have been identified, but information on candidate genes for identified QTLs of this important trait is still rare.

Results

In this study, a whole genome genetic linkage map for B. napus was constructed using simple sequence repeat (SSR) markers that covered a genetic distance of 2,126.4 cM with an average distance of 5.36 cM between markers. A procedure was developed to establish colinearity of SSR loci on B. napus with its two progenitor diploid species B. rapa and B. oleracea through extensive bioinformatics analysis. With the aid of B. rapa and B. oleracea genome sequences, the 421 homologous colinear loci deduced from the SSR loci of B. napus were shown to correspond to 398 homologous loci in Arabidopsis thaliana. Through comparative mapping of Arabidopsis and the three Brassica species, 227 homologous genes for seed size/weight were mapped on the B. napus genetic map, establishing the genetic bases for the important agronomic trait in this amphidiploid species. Furthermore, 12 candidate genes underlying 8 QTLs for seed weight were identified, and a gene-specific marker for BnAP2 was developed through molecular cloning using the seed weight/size gene distribution map in B. napus.

Conclusions

Our study showed that it is feasible to identify candidate genes of QTLs using a SSR-based B. napus genetic map through comparative mapping among Arabidopsis and B. napus and its two progenitor species B. rapa and B. oleracea. Identification of candidate genes for seed weight in amphidiploid B. napus will accelerate the process of isolating the mapped QTLs for this important trait, and this approach may be useful for QTL identification of other traits of agronomic significance.

     

Genetic mapping of clubroot resistance genes in oilseed rape

Clubroot caused by the obligate biotrophic protist Plasmodiophora brassicae is a major disease of Brassica species. Clubroot resistances introduced from B. oleracea var. ‘Böhmerwaldkohl’ and resistance from B. rapa ECD-04 were genetically mapped in oilseed rape (B. napus L.). A doubled haploid (DH) population of rape seed was developed by crossing a resistant DH-line derived from a resynthesized B. napus with the susceptible cultivar ‘Express’. The DH population was tested in the greenhouse against seven P. brassicae isolates showing low and high virulence toward B. oleracea or/and B. rapa. DH-lines with highest or lowest disease scores were used in a bulked segregant analysis (BSA), and 43 polymorphic AFLPs were identified. A genetic map of the whole genome was constructed using 338 AFLP and 156 anchored SSR markers. Nineteen QTL were detected on chromosomes N02, N03, N08, N13, N15, N16 and N19 giving resistance to seven different isolates. Race-specific effects were observed for all QTL, none of the QTL conferred resistance to all isolates. The phenotypic variance explained by the respective QTL ranged between 10.3 and 67.5%. All QTL could be assigned to both ancestral genomes of B. napus. In contrast to previous reports, a clear differentiation into major QTL from B. rapa and minor QTL from B. oleracea could not be found. Composite interval mapping confirmed the linkage relationships determined by BSA, thus demonstrating that markers for oligogenic traits can be selected by merely testing the distributional extremes of a segregating population.     

<Emphasis Type="Italic">LMI1</Emphasis>-like genes involved in leaf margin development of <Emphasis Type="Italic">Brassica napus</Emphasis>

In rapeseed (Brassica napus L.), leaf margins are variable and can be entire, serrate, or lobed. In our previous study, the lobed-leaf gene (LOBED-LEAF 1, BnLL1) was mapped to a 32.1 kb section of B. napus A10. Two LMI1-like genes, BnaA10g26320D and BnaA10g26330D, were considered the potential genes that controlled the lobed-leaf trait in rapeseed. In the present study, these two genes and another homologous gene (BnaC04g00850D) were transformed into Arabidopsis thaliana (L.) Heynh. plants to identify their functions. All three LMI1-like genes of B. napus produced serrate leaf margins. The expression analysis indicated that the expression level of BnaA10g26320D determined the difference between lobed- and entire-leaved lines in rapeseed. Therefore, it is likely that BnaA10g26320D corresponds to BnLL1.     

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.

     

Expression Profiling of the <Emphasis Type="Italic">CSDP</Emphasis> Transcription Factor Gene Family Points to Roles in Organ Development and Abiotic Stress Response in Tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.)

The cold shock domain proteins (CSDPs) are small group of nucleic acid-binding proteins that act as RNA chaperones in growth regulation, development, and stress adaptation in plants. The functions of CSDPs have been studied in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), wheat (Triticum aestivum), and Chinese cabbage (Brassica rapa). To gain insight into the function of CSDPs in tomato (Solanum lycopersicum), we performed a genome-wide analysis of CSDPs through in silico characterization and expression profiling in different organs and in response to different abiotic stress and phytohormone treatments. We identified five non-redundant SlCSDP genes. The evolutionary analysis and phylogenetic classification indicated that tomato CSDPs are more closely related to potato than those of others. The five SlCSDP genes are distributed on four of the 12 tomato chromosomes and no segmental or tandem duplication events are detected among them. Expression analysis showed broad expression patterns with strong expression in fruit development and ripening. Expression of individual SlCSDP genes was significantly altered by stress and phytohormone treatments. SlCSDP2, SlCSDP3, and SlCSDP4 were highly induced by all four abiotic stresses and by phytohormone treatment in tomato. These findings provide a foundation for future research towards functional biological roles of CSDP gene in particular to develop tomato cultivars with large size, early ripening, and abiotic stress tolerance.     

Glucosinolate profile and oviposition behavior in relation to the susceptibilities of Brassicaceae to the cabbage seedpod weevil

Understanding how host‐plant characteristics affect behavioral and physiological responses of insect herbivores is of considerable importance in the development of resistant crop germplasm. Feeding, oviposition preference, larval development, and oviposition behavior of the cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) (= Ceutorhynchus assimilis Payk.) (Coleoptera: Curculionidae), were investigated on eight Brassicaceae species that differed in their glucosinolate profiles. The least preferred host plants for feeding and oviposition were the Sinapis alba L. lines while the Brassica carinata L. line was most preferred. Larval development occurred most rapidly on Brassica rapa L. and slowest on S. alba. Larval weight was highest on B. napus L. and lowest on S. alba. Total glucosinolate levels did not influence C. obstrictus larval growth or development; however high levels of specific glucosinolates such as p‐hydroxybenzyl and 3‐butenyl glucosinolate were associated with increased developmental time or reduced weight. The time required for oviposition behavioral events was measured on different host‐plant species: B. rapa, B. napus, B. napus×S. alba, B. tournefortii Gouan., B. juncea (L.) Czern, B. carinata, B. nigra (L.) Koch., and S. alba. The early steps in the sequence were completed faster on more susceptible host plants (B. carinata, B. napus, and B. rapa) than on relatively resistant ones (B. tournefortii and B. juncea). Females explored pods of B. nigra and S. alba, but oviposition occurred only rarely on these species. There was no significant difference in the location on the pod on which oviposition occurred among the different plant species. Mean eggs laid per female weevil were highest on the B. napus×S. alba hybrid and lowest on B. nigra and S. alba.     

Physiological Effects of Rapeseed Transformation with the <Emphasis Type="Italic">acb</Emphasis> Gene as Affected by the Genetic Vector Structure

Rapeseed (Brassica napus L.) plants transformed with the acb gene encoding acyl-CoA-binding protein from Arabidopsis thaliana were used to demonstrate the physiological effects of transformation as related to the genetic vector structure. The opportunities and limitations of the genetic engineering technologies in the studies of particular proteins are under discussion.     

Descriptions of eight new species of <Emphasis Type="Italic">Ligophorus</Emphasis> Euzet &; Suriano, 1977 (Monogenea: Ancyrocephalidae) from Red Sea mullets

Eight new species of Ligophorus Euzet &; Suriano, 1977 (Monogenea: Ancyrocephalidae) are described from two species of mullets from the Red Sea. Ligophorus bykhowskyi n. sp. and L. zhangi n. sp. from Crenimugil crenilabris (Forsskål) differ from other species of the genus in the structure of the male copulatory organ, which has a simple accessory piece and a wide copulatory tube that arises from a large, single-chambered, expanded base. Ligophorus simpliciformis n. sp., L. bipartitus n. sp., L. campanulatus n. sp., L. mamaevi n. sp., L. lebedevi n. sp. and L. surianoae n. sp. from Liza carinata (Valenciennes) are differentiated on the basis of the morphometrics of the hard parts of the haptor and male copulatory organ. The eight species represent the first records of species directly attributed to Ligophorus from the Red Sea. Measurements of the haptoral hard-parts and the male copulatory organ of the new species are analysed with the aid of Principal Component Analysis. Three morphological types of male copulatory organ, five types of anchor, and two types of ventral and three types of dorsal bars were distinguished among these species. L. bykhowskyi and L. zhangi from C. crenilabris have the same type of male copulatory organ and anchors. Those species from Liza carinata have only one common morphological character, a thick copulatory tube, but have two types of accessory piece, four types of anchors and three types of bars. All species of Ligophorus found on mullets in the Red Sea have an accessory piece without a distal bifurcation and thus differ from most species of this genus from other regions of the world’s oceans.     

Predictive capability of a leaf optical meter for determining leaf pigment status during senescence

We conducted an experiment to assess the predictive capability of a leaf optical meter for determining leaf pigment status of Acer mono Maxim., A. ginnala Maxim., Quercus mongolica Fisch., and Cornus alba displaying a range of visually different leaf colors during senescence. Concentrations of chlorophyll (Chl) a, Chl b, and total Chl [i.e., Chl (a+b)] decreased while the concentration of carotenoids (Car) remained relatively static for all species as leaf development continued from maturity to senescence. C. alba exhibited the lowest average concentration of Chl (a+b), Chl a, and Car, but the highest relative anthocyanin concentration, while Q. mongolica exhibited the highest Chl (a+b), Chl b, and the lowest relative anthocyanin concentration. A. mono exhibited the highest Chl a and Car concentrations. The relationships between leaf pigments and the values measured by the optical meter generally followed an exponential function. The strongest relationships between leaf pigments and optical measurements were for A. mono, A. ginnala, and Q. mongolica (R ² ranged from 0.64 to 0.95), and the weakest relationships were for C. alba (R ² ranged from 0.13 to 0.67). Moreover, optical measurements were more strongly related to Chl a than to Chl b or Chl (a+b). Optical measurements were not related to Car or relative anthocyanin concentrations. We predicted that weak relationships between leaf pigments and optical measurements would occur under very low Chl concentrations or under very high anthocyanin concentrations; however, these factors could not explain the weak relationship between Chl and optical measurements observed in C. alba. Overall, our results indicated that an optical meter can accurately estimate leaf pigment concentrations during leaf senescence — a time when pigment concentrations are dynamically changing — but that the accuracy of the estimate varies across species. Future research should investigate how species-specific leaf traits may influence the accuracy of pigment estimates derived from optical meters.     

Natural genetic variation in <Emphasis Type="Italic">Brassica</Emphasis> homologs of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> and characterization of its expression domains

FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time.     

Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Competitive allele-specific PCR (KASPar) assay is a user-friendly system that provides flexibility in the numbers of single nucleotide polymorphisms (SNPs) and genotypes. Based on Illumina-GA-IIx genomic data from 10 genotypes with a broad genetic background, 3183 SNPs were selected for KASPar assays development, and 568 were finally converted and selected for Brassica rapa germplasm characterization (17.8%) on the basis of reproducibility, missing data rate, and uniform genetic distribution. High levels of polymorphism of these markers across 231 B. rapa genotypes were verified, illustrating by high polymorphic information content (averaged 0.34), minor allele frequency (0.37), genetic diversity (0.45), and the low observed heterozygosity (0.10). Based on the SNP dataset, structure and principal coordinates analysis, and neighbor-joining phylogenetic methods were used to examine the population structure and gave highly consistent results. The 231 accessions were divided into the four primary subspecies, representing 99 accessions from B. rapa ssp. pekinensis, 85 from B. rapa ssp. chinensis, 30 from B. rapa ssp. rapifera, and 17 from B. rapa ssp. oleifera and were further subdivided into 12 lower-order clusters according to different morphotypes. The genetic variability and pairwise fixation index analysis revealed that the ssp. pekinensis accessions possess the most extensive genetic variation among the four subspecies. The KASPar system is highly useful for validating SNPs and will be valuable for genetics research and breeding applications in B. rapa.