Broadening the avenue of intersubgenomic heterosis in oilseed Brassica

Accumulated evidence has shown that each of the three basic Brassica genomes (A, B and C) has undergone profound changes in different species, and has led to the concept of the “subgenome”. Significant intersubgenomic heterosis was observed in hybrids between traditional Brassica napus and first generation lines of new type B. napus. The latter were produced by the partial introgression of subgenomic components from different species into B. napus. To increase the proportion of exotic subgenomic components and thus achieve stronger heterosis, lines of first generation new type B. napus were intercrossed with each other, and subjected to intensive marker-assisted selection to develop the second generation of new type B. napus. The second generation showed better agronomic traits and a higher proportion of introgression of subgenomic components than did the first generation. Compared with the commercial hybrid and the hybrids produced with the first generation new type B. napus, the novel hybrids showed stronger heterosis for seed yield during the 2 years of field trials. The extent of heterosis showed a significant positive correlation with the introgressed subgenomic components in the parental new type B. napus. To increase the content of the exotic subgenomic components further and to allow sustainable breeding of novel lines of new type B. napus, we initiated the development of a gene pool for new type B. napus that contained a substantial amount of genetic variation in the A^r and C^c genome. We discuss new approaches to broaden the avenue of intersubgenomic heterosis in oilseed Brassica.     

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.     

BnaC.Tic40, a plastid inner membrane translocon originating from Brassica oleracea, is essential for tapetal function and microspore development in Brassica napus

Here, we describe the characteristics of a Brassica napus male sterile mutant 7365A with loss of the BnMs3 gene, which exhibits abnormal enlargement of the tapetal cells during meiosis. Later in development, the absence of the BnMs3 gene in the mutant results in a loss of the secretory function of the tapetum, as suggested by abortive callose dissolution and retarded tapetal degradation. The BnaC.Tic40 gene (equivalent to BnMs3) was isolated by a map-based cloning approach and was confirmed by genetic complementation. Sequence analyses suggested that BnaC.Tic40 originated from BolC.Tic40 on the Brassica oleracea linkage group C9, whereas its allele Bnms3 was derived from BraA.Tic40 on the Brassica rapa linkage group A10. The BnaC.Tic40 gene is highly expressed in the tapetum and encodes a putative plastid inner envelope membrane translocon, Tic40, which is localized into the chloroplast. Transmission electron microscopy (TEM) and lipid staining analyses suggested that BnaC.Tic40 is a key factor in controlling lipid accumulation in the tapetal plastids. These data indicate that BnaC.Tic40 participates in specific protein translocation across the inner envelope membrane in the tapetal plastid, which is required for tapetal development and function.     

Fine mapping of the recessive genic male-sterile gene (Bnms1) in Brassica napus L.

A recessive genic male sterility (RGMS) system, S45 AB, has been developed from spontaneous mutation in Brassica napus canola variety Oro, and is being used for hybrid cultivar development in China. The male sterility of S45 was controlled by two duplicated recessive genes, named as Bnms1 and Bnms2. In this study, a NIL (near-isogenic line) population from the sib-mating of S45 AB was developed and used for the fine mapping of the Bnms1 gene, in which the recessive allele was homozygous at the second locus. AFLP technology combined with BSA (bulked segregant analysis) was used. From a survey of 2,560 primer combinations (+3/+3 selective bases), seven AFLP markers linked closely to the target gene were identified, of which four were successfully converted to sequence characterized amplified region (SCAR) markers. For further analysis, a population of 1,974 individuals was used to map the Bnms1 gene. On the fine map, Bnms1 gene was flanked by two SCAR markers, SC1 and SC7, with genetic distance of 0.1 cM and 0.3 cM, respectively. SC1 was subsequently mapped on linkage group N7 using doubled-haploid mapping populations derived from the crosses Tapidor × Ningyou7 and DH 821 × DHBao 604, available at IMSORB, UK, and our laboratory, respectively. Linkage of an SSR marker, Na12A02, with the Bnms1 gene further confirmed its location on linkage group N7. Na12A02, 2.6 cM away from Bnms1, was a co-dominant marker. These molecular markers developed from this research will facilitate the marker-assisted selection of male sterile lines and the fine map lays a solid foundation for map-based cloning of the Bnms1 gene.     

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.     

Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents

Modification of oleic acid (C18:1) and linolenic acid (C18:3) contents in seeds is one of the major goals for quality breeding after removal of erucic acid in oilseed rape (Brassica napus). The fatty acid desaturase genes FAD2 and FAD3 have been shown as the major genes for the control of C18:1 and C18:3 contents. However, the genome structure and locus distributions of the two gene families in amphidiploid B. napus are still not completely understood to date. In the present study, all copies of FAD2 and FAD3 genes in the A- and C-genome of B. napus and its two diploid progenitor species, Brassica rapa and Brassica oleracea, were identified through bioinformatic analysis and extensive molecular cloning. Two FAD2 genes exist in B. rapa and B. oleracea, and four copies of FAD2 genes exist in B. napus. Three and six copies of FAD3 genes were identified in diploid species and amphidiploid species, respectively. The genetic control of high C18:1 and low C18:3 contents in a double haploid population was investigated through mapping of the quantitative trait loci (QTL) for the traits and the molecular cloning of the underlying genes. One major QTL of BnaA.FAD2.a located on A5 chromosome was responsible for the high C18:1 content. A deleted mutation in the BnaA.FAD2.a locus was uncovered, which represented a previously unidentified allele for the high oleic variation in B. napus species. Two major QTLs on A4 and C4 chromosomes were found to be responsible for the low C18:3 content in the DH population as well as in SW Hickory. Furthermore, several single base pair changes in BnaA.FAD3.b and BnaC.FAD3.b were identified to cause the phenotype of low C18:3 content. Based on the results of genetic mapping and identified sequences, allele-specific markers were developed for FAD2 and FAD3 genes. Particularly, single-nucleotide amplified polymorphisms markers for FAD3 alleles were demonstrated to be a reliable type of SNP markers for unambiguous identification of genotypes with different content of C18:3 in amphidiploid B. napus.     

Improvement of the recessive genic male sterile lines with a subgenomic background in Brassica napus by molecular marker-assisted selection

Both the pollination control system and genetic distance are major factors in the utilization of crop heterosis. The recessive genic male sterile line (RGMS) 7-7365A (Bnms3ms3ms4ms4) has been widely applied to hybrid seed production because it can generate a completely male sterile population by crossing with the 7-7365C temporary line (Bnms3ms3rfrf). In this study, the sterile genes of 7-7365A were transferred to the new Brassica napus lines 7-749 and 7-750 with a high content of subgenomes by backcross breeding. We used the amplified fragment length polymorphism (AFLP) technique combined with bulk segregant analysis (BSA) to identify markers linked to the BnMs4 gene. Twelve AFLP markers linked to the BnMs4 gene were identified. Of them, SA06MG09 and P08MG16 were the closest makers, which were on either side of the gene at a distance of 0.9 and 0.8?cM, respectively. Twenty AFLP primer combinations were used to screen the F2, BC1F3, and BC2F4 populations from the breeding program, and the markers linked to the BnMs3 and BnMs4 genes were used to screen the BC2F4 populations. As a result, we obtained two types of improved sterile lines, 7-749A and 7-750A, and their indexes of subgenomic components (ISG) were 44.2?C49.8 and 20.2?C26.6%, respectively. The combining ability analyses of seed yield character were conducted in the crosses from the three sterile lines and ten restorers within a random block design in three environments for two successive years. The general combining ability (GCA) of the two improved sterile lines were significantly higher than the GCA of 7-7365A in every environment tested. The two improved sterile lines had stability in seed yield, and they will be used in the future for hybrid seed production.     

A Large Insertion in bHLH Transcription Factor BrTT8 Resulting in Yellow Seed Coat in Brassica rapa

Yellow seed is a desirable quality trait of the Brassica oilseed species. Previously, several seed coat color genes have been mapped in the Brassica species, but the molecular mechanism is still unknown. In the present investigation, map-based cloning method was used to identify a seed coat color gene, located on A9 in B. rapa. Blast analysis with the Arabidopsis genome showed that there were 22 Arabidopsis genes in this region including at4g09820 to at4g10620. Functional complementation test exhibited a phenotype reversion in the Arabidopsis thaliana tt8-1 mutant and yellow-seeded plant. These results suggested that the candidate gene was a homolog of TRANSPARENT TESTA8 (TT8) locus. BrTT8 regulated the accumulation of proanthocyanidins (PAs) in the seed coat. Sequence analysis of two alleles revealed a large insertion of a new class of transposable elements, Helitron in yellow sarson. In addition, no mRNA expression of BrTT8 was detected in the yellow-seeded line. It indicated that the natural transposon might have caused the loss in function of BrTT8. BrTT8 encodes a basic/helix-loop-helix (bHLH) protein that shares a high degree of similarity with other bHLH proteins in the Brassica. Further expression analysis also revealed that BrTT8 was involved in controlling the late biosynthetic genes (LBGs) of the flavonoid pathway. Our present findings provided with further studies could assist in understanding the molecular mechanism involved in seed coat color formation in Brassica species, which is an important oil yielding quality trait.     

Distribution of <Emphasis Type="Italic">S</Emphasis> haplotypes and its relationship with restorer–maintainers of self-incompatibility in cultivated <Emphasis Type="Italic">Brassica napus</Emphasis>

Brassica napus (AACC, 2n = 38) is a self-compatible amphidiploid plant that arose from the interspecies hybridization of two self-incompatible species, B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18). Self-incompatibility (S) haplotypes in one self-incompatible line and 124 cultivated B. napus lines were detected using S-locus-specific primers, and their relationships with restorer-maintainers were investigated. Two class I (S-I ( SLG ) a and S-I ( SLG ) b) and four class II (S-II ( SLG ) a, S-II ( SLG ) b, S-II ( SP11 ) a and S-II ( SP11 ) b) S haplotypes were observed, of which S-II ( SP11 ) b was newly identified. The nucleotide sequence of SP11 showed little similarity to the reported SP11 alleles. The lines were found to express a total of eleven S genotypes. The self-incompatible line had a specific genotype consisting of S-II ( SP11 ) a, similar to B. rapa S-60, and S-II ( SLG ) a, similar to B. oleracea S-15. Restorers expressed six genotypes: the most common genotype contained S-I ( SLG ) a, similar to B. rapa S-47, and S-II ( SLG ) b, similar to B. oleracea S-15. Maintainers expressed nine genotypes: the predominant genotype was homozygous for two S haplotypes, S-II ( SLG ) a and S-II ( SP11 ) b. One genotype was specific to restorers and four genotypes were specific to maintainers, whereas five genotypes were expressed in both restorers and maintainers. This suggests that there is no definitive correlation between the distribution of S genotypes and restorer-maintainers of self-incompatibility. The finding that restorers and maintainers express unique genotypes, and share some common genotypes, would be valuable for detecting the interaction of S haplotypes in inter- or intra-genomes as well as for developing markers-assisted selection in self-incompatibility hybrid breeding.     

Inheritance and gene mapping of the white flower trait in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Despite being a unique marker trait, white flower inheritance in Brassica juncea remains poorly understood at the gene level. In this study, we investigated a B. juncea landrace with white petal in China. The white petal phenotype possessed defective chromoplasts with less plastoglobuli than the yellow petal phenotype. Genetic analysis confirmed that two independent recessive genes (Bjpc1 and Bjpc2) controlled the white flower trait. We then mapped the BjPC1 gene in a BC₄ population comprising 2295 individuals. We identified seven AFLP (amplified fragment length polymorphism) markers closely linked to the white flower gene. BLAST search revealed the sequence of AFLP fragments were highly homologous with the Scaffold000085 and Scaffold000031 sequences on the A02 chromosome in the Brassica rapa genome. Based on this sequence homology, we developed simple sequence repeat (SSR) primer pairs and identified 13 SSRs linked to the BjPC1 gene, including two that were co-segregated (SSR9 and SSR10). The two closest markers (SSR4 and SSR11) were respectively 0.9 and 0.4 cM on either side of BjPC1. BLAST analysis revealed that these marker sequences corresponded highly to A02 in B. juncea. They were mapped within a 33 kb genomic region on B. rapa A02 (corresponds to a 40 kb genomic region on B. juncea A02) that included three genes. Sequence BjuA008406, homologous to AtPES2 in Arabidopsis thaliana and Bra032956 in B. rapa, was the most likely candidate for BjPC1. These results should accelerate BjPC1 cloning and facilitate our understanding of the molecular mechanisms controlling B. juncea petal color.