Characterisation of the radish introgression carrying the Rfo restorer gene for the Ogu-INRA cytoplasmic male sterility in rapeseed (Brassica napus L.)

 Bulked segregant analysis and comparative mapping were applied to identify molecular markers linked to the Rfo restorer gene used for the Ogu-INRA cytoplasmic male-sterility system in rapeseed. These markers were then used to localise the radish introgression on the B. napus genetic map constructed from the cross ‘Darmor.bzh’ x ’Yudal’. The introgression mapped on the DY15 linkage group. From the comparison of this latter group to the linkage group constructed on a F₂ progeny segregating for the radish introgression, it was concluded that the introgression had occurred through homoeologous recombination, that it was not distal and that it had replaced a B. napus region of around 50 cM. A QTL involved in aliphatic seed glucosinolate content was located on the DY15 linkage group at a position corresponding to one end of the introgression. The DNA markers identified in this study are being used in map-based cloning of the Rfo gene and in marker-assisted selection. Received: 3 December 1997 / Accepted: 17 December 1997     

Cytoplasmic male sterility in rapeseed (Brassica napus L.)

Summary Restriction patterns of chloroplast (cp) and mitochondrial (mt) DNA in Brassica napus rapeseed reveal the alloplasmic nature of cytoplasmic male sterility in this crop. Both the Shiga and Bronowski systems probably exploit cytoplasmic diversity in B. napus cultivars arising from introgression of cytoplasm from the other rapeseed species, B. campestris. Nuclear genes specific to these systems do not cause sterility in maintainers (Bronowski and Isuzu-natane) because they have a campestris cytoplasm, but give rise to sterility in napus cytoplasms. In the course of hybridization to napus cultivars a line with the triazine resistant cytoplasm (a campestris cytoplasm) has undergone an alteration in the mt genome rendering its restriction pattern more similar than previously to that of napus. The alteration may be an inversion between 7.2 and 3.4 kb in length.     

Large scale purification of rapeseed proteins (Brassica napus L.)

Rapeseed (Brassica napus L.) cruciferin (12S globulin), napin (2S albumin) and lipid transfer proteins (LTP) were purified at a multi-g scale. The procedure developed was simple, rather fast and resolutive; it permitted the recovery of these proteins with a good yield, such as 40% for cruciferin and 18% for napin. Nanofiltration eliminated the major phenolic compounds. The remaining protein fraction was fractionated by cation exchange chromatography (CEC) on a streamline SP-XL column in alkaline conditions. The unbound neutral cruciferin was polished by size exclusion chromatography. The alkaline napin isoforms and LTP, adsorbed on the beads, were eluted as a whole fraction and further separated by an other CEC step at acidic pH. Napins were polished by hydrophobic interaction chromatography (HIC). The fractions were characterized by reverse phase HPLC, electrophoresis, N-terminal sequencing and mass spectrometry. All the fractions contained less than 5% of impurities.     

Fine mapping and candidate gene analysis of the nuclear restorer gene Rfp for pol CMS in rapeseed (Brassica napus L.)

The Polima (pol) system of cytoplasmic male sterility (CMS) in rapeseed is widely used in China for commercial hybrid seed production. Genetic studies have shown that its fertility restorer gene (Rfp) is monogenic dominant. For fine mapping of the Rfp gene, a near isogenic line comprising 3,662 individuals of BC(14)F(1) generation segregating for the Rfp gene was created. Based on the sequences of two SCAR markers, SCAP0612ST and SCAP0612EM2, developed by Zhao et al. (Genes Genom 30(3):191-196, 2008) and the synteny region of Brassica napus and other Brassica species, 13 markers strongly linked with the Rfp gene were identified. By integrating three of these markers to the published linkage map, the Rfp gene was mapped on linkage group N9 of B. napus. Using these markers, the Rfp locus was narrowed down to a 29.2-kb genomic region of Brassica rapa. Seven open reading frames (ORFs) were predicted in the target region, of these, ORF2, encoding a PPR protein, was the most likely candidate gene of Rfp. These results lay a solid foundation for map-based cloning of the Rfp gene and will be helpful for marker-assisted selection of elite CMS restorer lines.     

Linkage between an isozyme marker and a restorer gene in radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Summary Co-segregation studies of isozyme markers and male fertility restoration showed that a restorer gene from radish was introduced into rapeseed along with an isozyme marker (Pgi-2). The radish chromosome segment carrying these genes was introgressed into rapeseed through homoeologous recombination, substituting for some of the rapeseed alleles. By crossing heterozygous restored plants to male-sterile lines and to maintainers, tight linkage was found between the restorer gene and the marker. The recombination fraction was estimated at 0.25 ± 0.02%. Although few restored plants lacked the radish isozyme marker, it was still possible to distinguish male-fertile from male-sterile plants by their PGI-2 patterns. Furthermore, homozygous and heterozygous restored plants could be separated by specific PGI-2 phenotypes. Thus, the Pgi-2 marker is now currently used in restorer breeding programs.     

Embryogenesis following cryopreservation in isolated microspores of rapeseed (Brassica napus L.)

A simple procedure is described for cryopreservation of isolated microspores of rapeseed in liquid nitrogen without loss of embryogenic capacity (i.e. embryogenes is can still be induced following freezing). Microspores frozen in Lichter's (1982) medium with 13% sucrose produced ca. 10% of the embryos yielded by an unfrozen control. Microspores frozen in Lichter's medium with 13% sucrose, and supplemented with 0.5 M glycerol and 0.5 M DMSO produced no embryos. Regeneration of embryos obtained from frozen microspores yielded 88% diploid and 12% haploid plants, while embryos from unfrozen controls produced 7% diploids and 93% haploids. The potential to increase the efficiency of the rapeseed haploidy system using cryopreservation is discussed in light of these results.     

Association mapping of six yield-related traits in rapeseed (Brassica napus L.)

Yield is one of the most important traits for rapeseed (Brassica napus L.) breeding, but its genetic basis remains largely ambiguous. Association mapping has provided a robust approach to understand the genetic basis of complex agronomic traits in crops. In this study, a panel of 192 inbred lines of B. napus from all over the world was genotyped using 451 single-locus microsatellite markers and 740 amplified fragment length polymorphism markers. Six yield-related traits of these inbred lines were investigated in three consecutive years with three replications, and genome-wide association studies were conducted for these six traits. Using the model controlling both population structure and relative kinship (Q + K), a total of 43 associations (P < 0.001) were detected using the means of the six yield-related traits across 3 years, with two to fourteen markers associated with individual traits. Among these, 18 markers were repeatedly detected in at least 2 years, and 12 markers were located within or close to QTLs identified in previous studies. Six markers commonly associated with correlated traits. Conditional association analysis indicated that five of the associations between markers and correlated traits are caused by one QTL with pleiotropic effects, and the remaining association is caused by linked but independent QTLs. The combination of favorable alleles of multiple associated markers significantly enhances trait performance, illustrating a great potential of utilization of the associations in rapeseed breeding programs.     

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.     

Thermal reactivity of rapeseed (Brassica napus L.) under different gas atmospheres

Non-isothermal thermogravimetric method was applied to rapeseed to investigate its thermal reactivity under both individual dynamic atmospheres of nitrogen, steam, carbon dioxide and dry air, and under several mixtures of these gases (nitrogen+steam), (steam+dry air), and (nitrogen+carbon dioxide+steam). In this method, the sample was heated from ambient to 1273 K with a constant heating rate of 20 K/min. The trend of the TGA curves and the derived DTG profiles were interpreted regarding the conversion yields and the reactivity of the samples. Conversion yields of biomass to gaseous products changed in the range of 75-94% on original basis, with respect to the atmosphere under which the experiment was carried out. The maximum rates of the mass losses from the sample were found between 2.6 and 4.3 mg/min. The activation energies were calculated using the method of Coats-Redfern, and varied between 21.3 and 96.0 kJ/mol.     

Fatty acid inheritance in microspore-derived Populations of spring rapeseed (Brassica napus L.)

Summary Hard seededness in soybean [Glycine max (L.) Merr.] is a quantitative trait that affects the germination rate, viability, and quality of stored seeds. We have used 72 restriction fragment length polymorphisms (RFLPs) to identify genomic regions containing quantitative trait loci (QTL) affecting hard seededness in a segregating population from a G. max by a Glycine soja (Sieb. & Zucc.) cross. Five independent RFLP markers were found to be associated with variation in the hard-seeded trait. These markers and the epistatic interactions between them explain 71% of the variation for hard seededness. A genomic region associated with the i locus accounted for 32% of the variation in this segregating population. This study illustrates one approach to physiological genetic studies in plants.Journal Paper No. J-13557 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2763     

Evidence of cross-reactivity between porcine pancreatic and rapeseed (Brassica napus L.) lipases

A cross-reactivity between animal and plant lipases was determined, using immunological techniques. It was shown by ELISA and dot-blotting that these antibodies react with lipases in the rapeseed crude extract and in the different cellular fractions obtained by differential centrifugation. Pre-incubation of the antiserum with the rapeseed crude extract affects the amount of antibodies binding to the porcine pancreatic lipase. Antibodies were able to precipitate lipase activity from 3-day-old rapeseed crude extract. These epitopes seem to be located in the catalytic site, suggesting that a consensus sequence exists in oleaginous lipases and that it will be universal.     

Molecular marker-assisted breeding for improved Ogura cms restorer line (RfoRfo) and mapping of the restorer gene (Rfo) in Brassica juncea

Developing a fully functional hybrid system is a must for hybrid breeding in Brassica juncea. The B. napus Ogura cms hybrid system was transferred into B. juncea by the researchers at INRA, France. The B. juncea restorer (R) line (RfoRfo) exhibited poor vigor, low fertility and was black-seeded due to linkage drag. Our studies indicated that the Rfo gene in B. juncea R line was linked to the 5 C9 markers of B. napus (sN3553F, sS2285, sN3841, sN12905 and At5g58730) and 4 radish markers (At3g27100, At5g25080, At4g13720 and At5g06240) in addition to the 6 radish markers reported before (ScH03, ScA14, OPF3, BolJon, CAB and PGIint). These markers were used to screen for improved restorer plants in the three crosses of B. juncea restorer plant O39-16 (Rforf) × condiment var. Cutlass, O39-16 (Rforf) × canola B. juncea line C668 and O39-16 (Rforf) × resynthesized B. juncea line 15043. One improved homozygous R line VR441 (RfoRfo) with only 1 C9 marker sN12905 and 2 radish markers ScH03 and BolJon was successfully developed via marker-assisted selection in the cross O39-16 × 15043. VR441 had good seed-setting (average: 14.3 seeds/pod), strong growth vigor and was yellow-seeded. Linkage mapping revealed that the Rfo gene was introgressed into chromosome 9 of the A genome in B. juncea. The development of the improved R line VR441 has made the Ogura cms hybrid system fully functional in B. juncea. We are currently using the improved system for developing high yielding hybrid varieties in condiment and canola B. juncea.     

A dynamic and complex network regulates the heterosis of yield-correlated traits in rapeseed (Brassica napus L.)

Although much research has been conducted, the genetic architecture of heterosis remains ambiguous. To unravel the genetic architecture of heterosis, a reconstructed F(2) population was produced by random intercross among 202 lines of a double haploid population in rapeseed (Brassica napus L.). Both populations were planted in three environments and 15 yield-correlated traits were measured, and only seed yield and eight yield-correlated traits showed significant mid-parent heterosis, with the mean ranging from 8.7% (branch number) to 31.4% (seed yield). Hundreds of QTL and epistatic interactions were identified for the 15 yield-correlated traits, involving numerous variable loci with moderate effect, genome-wide distribution and obvious hotspots. All kinds of mode-of-inheritance of QTL (additive, A; partial-dominant, PD; full-dominant, D; over-dominant, OD) and epistatic interactions (additive × additive, AA; additive × dominant/dominant × additive, AD/DA; dominant × dominant, DD) were observed and epistasis, especially AA epistasis, seemed to be the major genetic basis of heterosis in rapeseed. Consistent with the low correlation between marker heterozygosity and mid-parent heterosis/hybrid performance, a considerable proportion of dominant and DD epistatic effects were negative, indicating heterozygosity was not always advantageous for heterosis/hybrid performance. The implications of our results on evolution and crop breeding are discussed.     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

Cryopreservation of isolated micropores of spring rapeseed (Brassica napus L.) for in vitro embryo production

Microspore cryopreservation is a potentially powerful method for long-term storage of germplasm for in vitro embryo production in plant species. In this study, several factors influencing embryo production following the ultra-low temperature (–196 °C in liquid nitrogen) storage of isolated microspores of rapeseed (Brassica napus L.) were investigated. Microspores were prepared in cryogenic vials and subjected to various cooling treatments before immersion in liquid nitrogen for varying periods. Efficiency of microspore cryopreservation was reflected by in vitro embryo production from frozen microspores. Of all the cooling treatments, microspores treated with a cooling rate of 0.25% °C/min and a cooling terminal temperature of –35 °C before immersion in liquid nitrogen produced the highest embryo yields (18% and 40% of unfrozen controls in two genotypes, respectively). Fast thawing in a 35 °C water bath was necessary to recover a high number of embryos from microspore samples being frozen at a higher cooling rate, while thawing speed did not affect samples after freezing at a slower cooling rate. The storage density of cryopreserved microspores affected embryo production. Storage at the normal culture density (8×10⁴ microspores/ml) was less efficient for embryo production than at high densities (4×10⁶ microspores/ml and 1.6×10⁷ microspores/ml), although no significant difference was found between the high densities. Evaluation of plant lines derived from frozen microspores indicated no variation in isozyme pattern and no enhanced cold tolerance of these lines. Isolated microspores of B. napus could be stored for extended period for in vitro embryo production.     

Quantitative trait loci that control the oil content variation of rapeseed (Brassica napus L.)

Key message

This report describes an integrative analysis of seed-oil-content quantitative trait loci (QTL) in Brassica napus , using a high-density genetic map to align QTL among different populations.

Abstract

Rapeseed (Brassica napus) is an important source of edible oil and sustainable energy. Given the challenge involved in using only a few genes to substantially increase the oil content of rapeseed without affecting the fatty acid composition, exploitation of a greater number of genetic loci that regulate the oil content variation among rapeseed germplasm is of fundamental importance. In this study, we investigated variation in the seed-oil content among two related genetic populations of Brassica napus, the TN double-haploid population and its derivative reconstructed-F₂ population. Each population was grown in multiple experiments under different environmental conditions. Mapping of quantitative trait loci (QTL) identified 41 QTL in the TN populations. Furthermore, of the 20 pairs of epistatic interaction loci detected, approximately one-third were located within the QTL intervals. The use of common markers on different genetic maps and the TN genetic map as a reference enabled us to project QTL from an additional three genetic populations onto the TN genetic map. In summary, we used the TN genetic map of the B. napus genome to identify 46 distinct QTL regions that control seed-oil content on 16 of the 19 linkage groups of B. napus. Of these, 18 were each detected in multiple populations. The present results are of value for ongoing efforts to breed rapeseed with high oil content, and alignment of the QTL makes an important contribution to the development of an integrative system for genetic studies of rapeseed.     

Unravelling the complex trait of harvest index in rapeseed (Brassica napus L.) with association mapping

Background

Harvest index (HI), the ratio of grain yield to total biomass, is considered as a measure of biological success in partitioning assimilated photosynthate to the harvestable product. While crop production can be dramatically improved by increasing HI, the underlying molecular genetic mechanism of HI in rapeseed remains to be shown.

Results

In this study, we examined the genetic architecture of HI using 35,791 high-throughput single nucleotide polymorphisms (SNPs) genotyped by the Illumina BrassicaSNP60 Bead Chip in an association panel with 155 accessions. Five traits including plant height (PH), branch number (BN), biomass yield per plant (BY), harvest index (HI) and seed yield per plant (SY), were phenotyped in four environments. HI was found to be strongly positively correlated with SY, but negatively or not strongly correlated with PH. Model comparisons revealed that the A–D test (ADGWAS model) could perfectly balance false positives and statistical power for HI and associated traits. A total of nine SNPs on the C genome were identified to be significantly associated with HI, and five of them were identified to be simultaneously associated with HI and SY. These nine SNPs explained 3.42 % of the phenotypic variance in HI.

Conclusions

Our results showed that HI is a complex polygenic phenomenon that is strongly influenced by both environmental and genotype factors. The implications of these results are that HI can be increased by decreasing PH or reducing inefficient transport from pods to seeds in rapeseed. The results from this association mapping study can contribute to a better understanding of natural variations of HI, and facilitate marker-based breeding for HI.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1607-0) contains supplementary material, which is available to authorized users.     

The genetic basis of flowering time and photoperiod sensitivity in rapeseed (Brassica napus L.)

The objective of this study was to dissect the genetic control of days to flowering (DTF) and photoperiod sensitivity (PS) into the various components including the main-effect quantitative trait loci (QTLs), epistatic QTLs and QTL-by-environment interactions (QEs). Doubled haploid (DH) fines were produced from an F1 between two spring Brassica napus cultivars Hyola 401 and Q2. DTF of the DH lines and parents were investigated in two locations, one location with a short and the other with a long photoperiod regime over two years. PS was calculated by the delay in DTF under long day as compared to that under short day. A genetic linkage map was constructed that comprised 248 marker loci including SSR, SRAP and AFLP markers. Further QTL analysis resolved the genetic components of flowering time and PS into the main-effect QTLs, epistatic QTLs and QEs. A total of 7 main-effect QTLs and 11 digenic interactions involving 21 loci located on 13 out of the 19 linkage groups were detected for the two traits. 3 main-effect QTLs and 4 pairs of epistatic QTLs were involved in QEs conferring DTF. One QTL on linkage group (LG) 18 was revealed to simultaneously affect DTF and PS and explain for the highest percentage of the phenotypic variation. The implications of the results for B. napus breeding have been discussed.