Phenotypic and seed structural comparison in hybrids of different Brassica rapa and B. oleracea

Brassica napus is an important oil species with short history and narrow genetic background. Interspecific hybrids from crosses between B. oleracea and different B. rapa were obtained. We found the hybrids with white petal resembling B. oleracea, the flavonoid and phenolic content decreased in hybrids, agreeing with the expressional changes of flavonoid biosynthesis genes. Seed coat of hybrids resembled diploid parents, or partly resembled to each parent with a clear outline. The palisade layer in hybrids was thicker than parents, with similar pigment accumulation as B. oleracea but more than B. rapa. Differentially sized protein bodies (PBs) were found in hybrids. The radical and inner cotyledon of all hybrids were identified with larger but less PBs than parents. The average size of PBs in outer cotyledon of resynthesized B. napus was also larger than parents, but the number of PBs was not significantly reduced. The phenotypic and seed structural variations after polyploidization of B. napus would be interesting for genetic broadening and breeding of rapeseed.     

Development of molecular markers linked to the Leptosphaeria maculans resistance gene Rlm6 and inheritance of SCAR and CAPS markers in Brassica napus × Brassica juncea interspecific hybrids

Leptosphaeria maculans causes blackleg disease on Brassica napus, an economically important oilseed crop. Brassica juncea has high resistance to blackleg and is a source for the development of resistant B. napus varieties. To transfer the Rlm6 resistance gene from B. juncea into B. napus, an interspecific cross between B. napus “Topas DH16516” and B. juncea “Forge” was produced, followed by the development of F₂ and F₃ generations. Sequence characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers linked to the L. maculans resistance gene Rlm6 were developed. Segregation of SCAR and CAPS markers linked to Rlm6 were confirmed by genotyping of F₂ and F₃ progeny. Segregation of CAPS markers and phenotypes for blackleg disease severity in F₂ plants had a Mendelian ratio of 3:1 in resistant vs. susceptible plants, respectively, supporting the assumption that genetic control of resistance was by a single dominant gene. The molecular markers developed in this study, which show linkage with the L. maculans resistance gene Rlm6, would facilitate marker‐assisted backcross breeding in a variety development programme.     

Transgene introgression from Brassica napus to different varieties of Brassica juncea

Transgene introgression from transgenic rapeseed (Brassica napus) to different varieties of B. juncea was assessed in this study. Crossability between a transgenic rapeseed line Z7B10 (pollen donor) and 80 cultivars of 16 B. juncea varieties (including two wild accessions) was estimated by artificial pollination in a greenhouse. As a result, interspecific crossability between the transgenic Z7B10 line and the 80 B. juncea cultivars varied considerably, with seeds per flower from 0.00–10.67. Seed germination rates of the interspecific F₁ hybrids ranged from 49.0%–89.3%. The estimated frequencies of natural gene flow from the transgenic Z7B10 line to 10 B. juncea cultivars with different uses in the experiment field varied from 0.08% to 0.93%. The natural F₁ hybrids were highly sterile, with seeds per silique ranging from 0.27 to 1.03. In addition, seeds per flower of hybrid descendants varied from 0.02 to 0.22 when F₁ hybrids were self‐pollinated, and those ranged from 0.03 to 0.30 when F₁ hybrids were backcrossed with their corresponding B. juncea parents. Results of this study suggest a low level of transgene introgression from transgenic rapeseed to different B. juncea varieties, which provides a sound scientific basis for the safety management of coexisting transgenic B. napus and B. juncea varieties in China.     

Introgression of Resistance to Shattering in Brassica napus from Brassica juncea through Non-Homologous Recombination

Genetic information conffering non- shattering of siliques has been introgressed in rapeseed (Brassica napus) following; interspecific hybridization between Brassica juncea and B. napus. A reconstituted B. napus plant with complete non-dehiscence of its fruits was isolated in the BC-, generation. This plant had normal meiosis and formed 19 bivalents. Its seed fertility, however, was low (23 percent). It is suggested that the gene(s) for shattering-resistance were incorporated into a B. oleracea chromosome following allosyndetic; chromosome pairing and. segmental exchange between B. nigra and B. oleracea chromosomes in the initial interspecific AABC hybrid.     

Inheritance and mapping of a restorer gene for the rapeseed cytoplasmic male sterile line 681A

A novel cytoplasmic male sterility‐fertility restoration system has been developed in rapeseed (Brassica napus). The cytoplasmic male sterile line 681A was derived from a spontaneous male sterile mutant in a newly released double‐low rapeseed cultivar ‘Xiangyou 13′. The restorer line 714R was identified in the interspecific progeny from a B. napus×B. juncea‐cross. Genetic analysis showed that fertility restoration for 681A cytoplasmic male sterility was controlled by a single dominant nuclear gene which might originate from B. juncea. The RAPD marker S1039_‐520 was found to be linked to the restorer gene in F₂ progeny of 681A × 714R with a recombination frequency of 5.45%.     

Quantitative trait locus analysis of seed germination,seedling vigour and seedling‐regulated hormones in Brassica napus

Good germination and seedling vigour are major breeding targets in winter oilseed rape (Brassica napus), because seedling vigour and prewinter crop establishment are closely associated with postwinter growth and yield. Here, we identified quantitative trait loci (QTL) related to germination, seedling vigour and seedling‐regulated hormones in a doubled haploid (DH) mapping population from a cross between winter oilseed rape parents with high vigour (Express 617) and low vigour (1012‐98). By phenotyping in a climate‐controlled glasshouse, we identified a total of 13 QTL on nine chromosomes for germination and seedling‐related traits at 7 and 14 days after sowing (DAS), explaining up to 11.2% of the phenotypic variation for seedling vigour. Forty‐seven metabolic QTL on 15 chromosomes were identified for auxin, abscisic acid (ABA) and dihydrophaseic acid (DPA) at 5 and 12 DAS, explaining up to 49.4% of phenotypic variation in seedling hormone composition. Multitrait QTL hot spots contribute to our understanding of the genetics and metabolomics of germination and seeding vigour in B. napus, and represent potential targets to breed high‐vigour cultivars.     

Cytoplasmic male sterility with self-incompatibility,a novel approach to utilizing heterosis in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The Polima cytoplasmic male sterility (CMS) system has been successfully used in three/two-line hybrid production in rapeseed (Brassica napus L.). However, the sterility of the Polima (pol) CMS lines is sensitive to temperature fluctuations. Also, traces of pollen can cause self-pollination within the CMS lines, which results in reduced levels of F₁ hybrid seed purity and leads to a significant yield loss. Self-incompatibility (SI) is another important approach for hybrid seed production in rapeseed. Despite having a wide range of restorers and being easily selected in a breeding program, SI system has some drawbacks. In this study, SI genes from a self-incompatible line of Brassica napus were transferred to a pol CMS line and S372A, a novel line of combined cytoplasmic male sterility with self-incompatibility was bred. Due to the SI genes, this line produced very few seeds when it was selfed at low temperature and no seeds at high temperature. This suggested that the line with CMS + SI had combined the advantages and overcome the disadvantages of both the pol CMS and SI systems. Furthermore, our results showed that most of the maintainers and all the restorers of the pol CMS system were also maintainers and restorers of the CMS + SI line, respectively. This indicates that the CMS + SI system can be easily used to establish three-line hybrids of rapeseed, and we believe this novel system could be extended to other species of Brassica.     

Dissection of a major QTL for seed colour and fibre content in Brassica napus reveals colocalization with candidate genes for phenylpropanoid biosynthesis and flavonoid deposition

A major quantitative trait locus (QTL) influencing seed fibre and colour in Brassica napus was dissected by marker saturation in a doubled haploid (DH) population from the black‐seeded oilseed rape line ‘Express 617’ crossed with a yellow‐seeded B. napus line, ‘1012–98’. The marker at the peak of a sub‐QTL with a strong effect on both seed colour and acid detergent lignin content lay only 4 kb away from a Brassica (H+)‐ATPase gene orthologous to the transparent testa gene AHA10. Near the peak of a second sub‐QTL, we mapped a copy of the key phenylpropanoid biosynthesis gene cinnamyl alcohol dehydrogenase, while another key phenylpropanoid biosynthesis gene, cinnamoyl co‐a reductase 1, was found nearby. In a cross between ‘Express 617’ and another dark‐seeded parent, ‘V8’, Bna.CCR1 was localized in silico near the peak of a corresponding seed fibre QTL, whereas in this case Bna.CAD2/CAD3 lay nearby. Re‐sequencing of the two phenylpropanoid genes via next‐generation amplicon sequencing revealed intragenic rearrangements and functionally relevant allelic variation in the three parents.     

Variations in chlorosis and potential usefulness of alloplasmic Brassica rapa with the cytoplasm of male sterile Brassica juncea

To select superior seed parents for vegetable hybrid seed production, we conducted interspecific crosses between male sterile Brassica juncea (2n = 36, AABB) and eight inbred lines of Brassica rapa (2n = 20, AA). Alloplasmic lines of B. rapa with the cytoplasm of B. juncea were developed from B. juncea × B. rapa hybrids by repeated backcrossing using B. rapa as the recurrent male parent until the BC₃ generation. Seed fertility, male sterility and chlorophyll content were investigated in these plants cultivated under four different temperature conditions (5, 10, 12 and 20°C). At 10°C, the alloplasmic lines of B. rapa with the cytoplasm of B. juncea were male sterile with partly chlorotic leaves. The alloplasmic B. rapa had lower chlorophyll a, chlorophyll b and carotenoid contents than those of the original B. rapa. The leaves recovered from chlorosis when the plants were cultivated at 20°C. An alloplasmic line of B. rapa (A6) is available as a seed parent for vegetable hybrid seed production and contributes seed fertility, slight chlorosis and stable male sterility.     

Comparisons of Isozyme Patterns in Resynthesized Amphihaploid Rapeseed (Brassica napus) and Their Parental Species Brassica campestris and Brassica oleracea

Resynthesized rapeseed plants produced by embryo culture and the progeny of their selfed parents from Brassica oleracea and Brassica campestris were analyzed by starch gel electrophoresis staining of enzymes GPI, LAP and SDH. In at least one unequivocal case it could be proved for each enzyme system studied that the alleles of both parents arc independently expressed in the newly synthesized individuals and that in the pattern of the dimeric enzyme GPI interlocus bands of the homoeologous loci arc visible. Comparisons with enzyme patterns in cultivated B. napus leads to the conclusion that in general active enzyme loci of both parental genomes are present. This is of great significance in estimating slide frequencies, degree of heterozygosity and other genetic parameters of B. nupus populations/cultivars.     

Cytological observation of anther structure and genetic investigation of a new type of cytoplasmic male sterile 0A193‐CMS in Brassica rapa L.

Cytoplasmic male sterility (CMS), a maternally transmitted failure in pollen formation, is an effective pollination control system in hybrid rapeseed (Brassica napus) breeding. However, CMS is not widely used in the related oilseed species Brassica rapa. In the past years, several male sterile plants have been isolated from the B. rapa landrace ‘0A193’, collected in Shaanxi, China, in 2011. It is noteworthy that the fertility expression of 0A193‐CMS was affected by temperature. In contrast to pol CMS, fertility tests with 18 B. rapa and 9 B. napus accessions suggest that a different system of maintaining and restoring is responsible for the observed phenotype. Further on, genetic investigation evidenced that fertility of 0A193‐CMS is controlled by both cytoplasmic and one pair of nuclear recessive genes. Interestingly, plants of the 0A193‐CMS type possess a highly specific fragment of the mitochondrial gene orf222, a crucial regulator of male sterility in nap CMS. Our study broadens the CMS resources in B. rapa and provides a highly applicable alternative to pol CMS and ogu CMS for hybrid breeding production.     

Interspecific transfer of Brassica juncea-type high blackleg resistance to Brassica napus

Summary Complete resistance to Leptosphaeria maculans, the cause of blackleg of oilseed rape (Brassica napus), was transferred from B. juncea to B. napus through an interspecific cross. B. juncea-type complete resistance (JR) was recognized first in one F₃ progeny (Onap^JR) by the absence of leaf-lesions on seedlings and canker-free adult plants. The commercially important characters of B. napus were retained in advanced lines of Onap^JR, which combined JR with low erucic acid levels (<0.5%), high seed yield and variable maturity dates.JR appeared to be inherited as a major gene or genes. Segregation for resistance and susceptibility contintied to occur during later generations of selection of Onap^JR. JR was readily transferred from Onap^JR to other suitable B. napus cultivars or lines with partial resistance to blackleg and resulted in highly vigorous carly generation selections adapted to cold, wet situations along with complete resistance to blackleg.     

Production of a desirable Brassica oleracea CMS line using an alloplasmic B. rapa CMS line carrying Diplotaxis erucoides cytoplasm as a bridge plant

In Brassica oleracea, production of F₁ hybrid seeds mainly makes use of the improved Ogura cytoplasmic male sterile (CMS) line. However, reliance on one particular line is a risk, and it would be advantageous to develop other CMS lines. In this study, we transferred Diplotaxis erucoides cytoplasm to B. oleracea cultivars using an alloplasmic B. rapaCMS line as a bridge plant to avoid incompatibility between donor and recipient plants. The new B. oleraceaCMS lines, which were derived by four generations of backcrossing, had small rudimentary anthers with no pollen grain and showed complete male sterility. There was no functional defect in other floral organs, and the ability to receive normal pollen did not appear to be impaired. Moreover, the B. oleraceaCMS lines carrying D. erucoides cytoplasm had larger leaf areas and a normal plastochron. As a consequence, the B. oleraceaCMS lines carrying D. erucoides cytoplasm have the potential to be valuable alternatives for use in commercial B. oleracea hybrid seed production.     

A cytogenetic study of the progenies of hybrids between Brassica napus and Brassica oleracea, Brassica bourgeaui, Brassica cretica and Brassica montana

In this cytogenetic study the progeny of all crosses were investigated in F₁, F₂ and backcross (BC₁) hybrids. Brassica napus and F₁ hybrids between B. napus and B. oleracea, and between B. napus and three wild relatives of B. oleracea (B. bourgeaui, B. cretica and B. montana). Each of the wild relatives has 18 somatic chromosomes. Interspecific F₁ hybrids were obtained through ovary culture mean. These had 28 and 37 chromosomes and their mean pollen fertility was 10.7% and 93.0%, respectively. Many F₂ and BC₁ seeds were harvested from the F₁ hybrids with 37 chromosomes after self‐pollination and open pollination of the F₁ hybrids, and backcrossing with B. napus. Many aneuploids were obtained in the F₂ and BC₁ plants. It is evident from these investigations that the F₁ hybrids may serve as bridge plants to improve B. napus and other Brassica crops.     

Microspore mutagenesis of Brassica species for fatty acid modifications: a preliminary evaluation

A microspore mutagenesis protocol was developed for Brassica rapa, Brassica napus and Brassica juncea for the production of double haploid lines with novel fatty acid profiles in the seed oil. Freshly isolated Brassica microspores were first cultured with ethyl methane sulphonate (EMS) for 1.5 h. The EMS was removed and the microspores were then cultured according to the standard Brassica microspore culture protocol. This protocol was used to generate over 80 000 Brassica haploid/double haploid plants. Field evaluation of B. napus and B. juncea double haploids was conducted between 2000 and 2003. Fatty acid analysis of the B. napus double haploid lines showed that saturated fatty acid proportions ranged from 5.0% to 7.7%. For B. juncea, saturate proportions ranged from 5.4% to 9.5%. Of the 7000 B. rapa lines that were analysed, 197 lines had elevated oleic acid (>55%), 69 lines had reduced α‐linolenic acid (<8%) and 157 lines had low saturated fatty acid proportions (<5%), when compared with the parental lines.     

Genetic analysis of drought stress response in rapeseed (Brassica campestris and B. Napus). III. physiological characters

Summary Leaf proline accumulation, leaf chlorophyll stability, and germination at low osmotic potentials have previously been suggested as possible selection criteria for drought resistance in turnip rape (Brassica campestris) and rapeseed (B. napus) (Richards, 1978a). The feasibility of using these characters in a breeding program is reported. These characters were under significant genetic control and were responsive to selection. Broad sense heritabilities ranged from 40% for proline accumulation in both species to 55% for germination rate in B. campestris and 64% for chlorophyll stability in B. napus, however, narrow sense heritabilities for proline and germination were substantially lower. Their use as selection criteria for yield in a Mediteranean drought would be restricted since in B. campestris these characters were not related to yield while in B. napus only a weak association was observed between yield under drought and proline accumulation.     

Combination of resistance to Verticillium longisporum from zero erucic acid Brassica oleracea and oilseed Brassica rapa genotypes in resynthesized rapeseed (Brassica napus) lines

Resynthesized (RS) forms of rapeseed (Brassica napus L.; genome AACC, 2n = 38) generated from interspecific hybridization between suitable genotypes of its diploid progenitors Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and Brassica oleracea L. (CC, 2n = 18) represent a potentially useful resource to introduce resistance against the fungal pathogen Verticillium longisporum into the gene pool of oilseed rape. Numerous cabbage (B. oleracea) accessions are known with resistance to V. longisporum; however, B. oleracea generally has high levels of erucic acid and glucosinolates in the seed, which reduces the suitability of resulting RS rapeseed lines for oilseed rape breeding. In this study resistance against V. longisporum was identified in the cabbage accession Kashirka 202 (B. oleracea convar. capitata), a zero erucic acid mutant, and RS rapeseed lines were generated by crossing the resistant genotype with two spring turnip rape accessions (B. rapa ssp. olerifera) with zero erucic acid. One of the resulting zero erucic acid RS rapeseed lines was found to have a high level of resistance to V. longisporum compared with both parental accessions and with B. napus controls. A number of other zero erucic acid RS lines showed resistance levels comparable to the parental accessions. In the most resistant RS lines the resistance and zero erucic acid traits were combined with variable seed glucosinolate contents. Erucic acid‐free RS rapeseed with moderate seed glucosinolate content represents an ideal basic material for introgression of quantitative V. longisporum resistance derived from B. oleracea and B. rapa into elite oilseed rape breeding lines.     

Development of self-incompatible Brassica napus: (I) introgression of S-alleles from Brassica oleracea through interspecific hybridization

Cultivars in Brassica napus var. oleifera, a self‐pollinating, self‐compatible species, have traditionally been developed as open‐pollinated lines or populations. Significant yield gains in this species have been realized through the exploitation of heterosis. Commercial hybrid production has been possible as a result of the development of a number of pollination control systems. Self‐incompatibility was transferred from B. oleracea var. italica to B. napus var. oleifera through interspecific hybridization. The response to interspecific pollination, as measured by pod elongation and initial stages of ovule development, was genotype dependent, and two highly responsive B. napus genotypes were identified. Embryo rescue was used to produce the interspecific hybrids. Isoelectric focusing of stigma proteins was used to identify S‐alleles in the interspecific hybrids to facilitate backcrossing. Segregation of the S‐locus through a series of back‐crosses to B. napus was complicated by aneuploidy; however, the S‐locus was found to segregate as a single gene. Usefulness of B. oleracea as a source of S‐alleles for pollination control in B. napus is discussed.     

Pod shatter resistance evaluation in cultivars and breeding lines of Brassica napus, B. juncea and Sinapis alba

Pod shatter susceptibility was investigated in Brassica napus germplasm and shatter resistant species of B. juncea and Sinapis alba. The comparisons were made by measuring seed yield in field plots, detached pod rupture energy (RE) and the half‐life of pod‐opening. Pod shatter resistance was significantly greater in B. napus lines derived from interspecific hybridizations of B. napus with B. rapa, B. carinata and B. juncea, than common B. napus cultivars. While these lines exhibited no significant difference in resistance to pod shatter than B. juncea, an entry of S. alba had no yield loss caused by pod shatter. Resistance to pod shatter was characterized in the field as little or no yield loss after full maturity, delayed shattering in time, and stable yield performance under variable climatic conditions during pod maturity. Yield loss caused by pod shatter ranged from a low of 4% for the B. juncea cv. ‘AC Vulcan’ to a high of 61% for the black seeded B. napus line DH12075 in 2‐year field trials after 1 month maturity. Pod shatter resistance was not significantly associated with specific plant and pod morphological traits, except pod length (P = 0.005) in tested materials. Field visual scores of pod shatter through inspections of average pod shatter per plant within plots were highly correlated with plot yield loss. Indoor quantitative evaluations of pod strength using a pendulum machine to measure pod RE and random impact test to measure half‐life of pod‐opening resistance were highly correlated with field yield loss. Multiple evaluations of pod shatter in method and in time after pod maturity are recommended for reliable evaluation of pod shatter resistance.