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Key message: A candidate gene Bndm1 related to determinate inflorescence was mapped to a 128-kb interval on C02 in Brassica napus. Brassica napus plants with determinate inflorescence exhibit improved traits in field production, such as lower plant height, improved lodging resistance, and consistent maturity. Compared to plants with indeterminate inflorescence, such features are favorable for mechanized harvesting techniques. Here, using a natural mutant 6138 with determinate inflorescence, it is demonstrated that determinate inflorescence reduces plant height significantly without affecting thousand-grain weight and yield per plant. Determinacy was regulated by a single recessive gene, Bndm1. Using a combination of SNP arrays and map-based cloning, we mapped the locus of determinacy to a 128-kb region on C02. Based on sequence comparisons and the reported functions of candidate genes in this region, we predicted BnaC02.knu (a homolog of KNU in Arabidopsis) as a possible candidate gene of Bndm1 for controlling determinate inflorescence. We found a 623-bp deletion in a region upstream of the KNU promoter in the mutant. This deletion led to the significant overexpression of BnaC02.knu in the mutant compared to that in the ZS11 line. The correlation between this deletion and determinate inflorescence was examined in natural populations. The results indicated that the deletion affected the normal transcription of BnaC02.knu in the plants with determinate inflorescence and played an important role in maintaining flower development. This study presents as a new material for optimizing plant architecture and breeding novel canola varieties suitable for mechanized production. Moreover, our findings provide a theoretical basis for analyzing the molecular mechanisms underlying the formation of determinate inflorescence in B. napus. [ABSTRACT FROM AUTHOR]

Key message: The mutations BnA1.CER4 and BnC1.CER4 produce disordered wax crystals types and alter the composition of epidermal wax, causing increased cuticular permeability and sclerotium resistance. The aerial surfaces of land plants are coated with a cuticle, comprised of cutin and wax, which is a hydrophobic barrier for preventing uncontrolled water loss and environmental damage. However, the mechanisms by which cuticle components are formed are still unknown in Brassica napus L. and were therefore assessed here. BnA1.CER4 and BnC1.CER4, encoding fatty acyl-coenzyme A reductases localizing to the endoplasmic reticulum and highly expressed in leaves, were identified and functionally characterized. Expression of BnA1.CER4 and BnC1.CER4 cDNA in yeast (Saccharomyces cerevisiae) induced the accumulation of primary alcohols with chain lengths of 26 carbons. The mutant line Nilla glossy2 exhibited reduced wax crystal types, and wax composition analysis showed that the levels of branched primary alcohols were decreased, whereas those of the other branched components were increased. Further analysis showed that the mutant had reduced water retention but enhanced resistance to Sclerotinia sclerotiorum. Collectively, our study reports that BnA1.CER4 and BnC1.CER4 are fatty acyl-coenzyme A reductase genes in B. napus with a preference for branched substrates that participate in the biosynthesis of anteiso-primary alcohols. [ABSTRACT FROM AUTHOR]

Key message: A candidate gene for male fertility restoration in Brassica juncea, BjRf, was isolated from a 23-kb interval on chromosome A05 using map-based cloning and BSA methods. The cytoplasmic male sterility/fertility restoration (CMS/Rf) system has been extensively used for heterosis in plants. It also provides valuable resources for studying mitochondrial–nuclear coevolution and interaction. The oxa CMS, which is a new CMS type reported in Brassica juncea (B. juncea), has been broadly used in the exploitation and application of heterosis in this species. However, the oxa CMS fertility restorer gene BjRf has not been reported. In this study, a stable restorer line was successfully constructed via continuous testcross and artificial selection. Besides, a new Rf gene was mapped in a 23-kb region on chromosome A05 in B. juncea with a genetic distance of 0.5 cM by the method incorporating bulk segregant analysis (BSA) and conventional map-based cloning. Finally, BjuA017917, a non-PPR Rf gene encoding a guanosine nucleotide diphosphate dissociation inhibitor (GDI), is proposed to be the candidate gene for fertility restoration of the oxa CMS line in B. juncea. Moreover, a functional marker, CRY3, was developed for marker-assisted selection for Brassica juncea breeding. [ABSTRACT FROM AUTHOR]

Key message: A mitochondria-localized pentatricopeptide repeat protein was identified by positional cloning and transferred into the hau CMS line, where it successfully restored fertility Cytoplasmic male sterility (CMS) is a maternally inherited trait that can be controlled by restorer-of-fertility (Rf) genes present in the nucleus. The hau CMS was identified as a new form of CMS associated with the mitochondrial transcript orf288; however, a lack of a restorer gene has limited its utilization in Brassica crops. Here, the combination of Brassica 60 K array with bulk segregant analysis and map-based cloning was used to delimit the Rfh locus to an 82.2-kb region on chromosome A09. A candidate gene encoding a mitochondria-localized pentatricopeptide repeat (PPR) protein was identified and transferred into the hau CMS line, where it successfully restored the fertility of the hau CMS plants. Furthermore, the expression analysis showed that Rfh was highly expressed in the flower buds, and the sequence analysis results implied that functional divergence between RFH and rfh could be due to 59 amino acid residue differences in the deduced protein sequences. In addition, a co-separated molecular marker was developed based on the divergent sequences between the dominant and recessive alleles. These results will help enable the heterosis of Brassica crops in the future. [ABSTRACT FROM AUTHOR]

Purple leaves are rich in health-protecting anthocyanins and food colorants in Brassica juncea. But the causal gene, which is related to leaf color formation, have not been reported in B. juncea. Anthocyanins mainly accumulated throughout the adaxial and abaxial epidermal leaf cells of purple leaves. A genetic analysis indicated that an incompletely dominant gene controls the purple leaf trait in B. juncea. Furthermore, the BjPur gene, which increased anthocyanin accumulation in purple-leaf mustard, was cloned. Blast and phylogenetic analyses revealed that BjPur encodes a new R2R3-MYB transcription factor. Sequence analysis of two alleles revealed a DNA sequence insertion in the first intron of BjPur in green leaves parent line (LY) when compared with the BjPur gene in the purple-leaf parent line (ZY). And this insertion greatly reduced the transcription of BjPur in green leaves. In purple-leaf plants, the transcript level of BjPur was significantly higher in leaves than in roots, stems, siliques, and flower buds. Additionally, molecular markers linked to leaf color were developed to distinguish different genotypes of B. juncea. These results will be helpful for the genetic improvement of the purple leaf color in B. juncea. [ABSTRACT FROM AUTHOR]

Key message: A candidate branching-controlling gene for qDBA09 was identified after delimiting a Brassica napus recessive locus within a 270-kb interval on chromosome A09. Although branching is an important trait associated with the adaptation and yield potential of rapeseed (Brassica napus), the genetic mechanisms underlining branching in this crop remain poorly understood. In this study, we characterized a naturally occurring rapeseed mutant, db1, which showed an ultrahigh branching density phenotype. By combining bulked segregant analysis (BSA) and the Brassica 60K SNP BeadChip Array, we identified two major quantitative trait loci (QTLs), qDBA09 and qDBC06, which were subsequently confirmed using the traditional QTL-mapping approach. Analysis of 208 individuals from a BC1F3 population indicated that the qDBA09 locus is a single Mendelian factor and that the dense branching phenotype is controlled by a single recessive gene. Furthermore, QTL analysis confirmed that qDBA09 explained between 9.5 and 70.5% of the variation in branching-related traits. Using 7785 individuals from the BC1F3 population, we mapped qDBA09 to a DNA fragment of approximately 270 kb in length that contained 27 predicted genes, three of which were identified as potentially involved in the control of the dense branching trait. Based on the reported function of these genes, together with sequence comparisons and co-segregation analysis, we identified a potential candidate gene for the qDBA09 locus. The present findings lay the foundations for further in-depth research on the branching mechanisms of B. napus. [ABSTRACT FROM AUTHOR]

Key message: Using microarray analysis combined with map-based cloning, a major locus positively regulating SL and SW was mapped to a 98.47 kb interval on A09 in rapeseed. In rapeseed, seed yield is closely associated with silique-related traits such as silique length (SL) and seed weight (SW). Previously identified quantitative trait loci (QTLs) revealed that SL and SW are complex traits and many QTLs overlap. However, the genetic characterization of the association between SL and SW is poorly understood. In the present study, a BC3F3 near isogenic line developed from a short silique plant and the long silique cultivar 'ZS11' was analyzed to identify the locus related to SL. Map-based cloning indicated that a major locus acting as a single Mendelian factor was mapped to a 98.47 kb region on chromosome A09. BLAST analysis and DNA sequencing showed SNP variations and a fragment replacement in the upstream region of the candidate gene BnaA09g55530D may alter gene expression and influence SL. The results showed that this SL locus may also positively affect SW as well as in the 186 rapeseed accessions identified by the associated markers. Therefore, selecting plants with appropriate SL and developing functional markers for the associated gene could play important roles in the molecular breeding of high-yield rapeseed varieties. [ABSTRACT FROM AUTHOR]

Key message: Successfully constructing restorer lines for the hau CMS line and molecular mapping of Rfh to a 94 kb candidate region on chromosome A03 in Brassica napus. Cytoplasmic male sterility is a general phenomenon in almost 200 species, and the interaction between chimeric genes in mitochondria and restorer genes in nucleus may be responsible for restoration of male fertility. Orf288 has been identified as a CMS-associated gene in the hau CMS line of Brassica napus and Brassica juncea; however, the restorer lines/genes have not been found yet. We therefore have successfully constructed two restorer lines in B. napus by extensive testcrossing and have mapped a major restorer gene Rfh to a physical distance of 94 kb on chromosome A03 by whole-genome resequencing and molecular markers. We found that the restorer line is indeed restored to male fertility at histological level. Comparative genomics and collinearity analysis between close relatives revealed that rearrangements and recombination may have happened and thus caused the production of Rfh or components of the restoration of fertility complex. Meanwhile, nuclear backgrounds with multiple loci and temperature were related to the variation and instability of restoration of fertility in three different populations. Our study provides new sights into the coevolution between restorer genes and CMS-associated genes as well as the cultivation of superior hybrids via molecular breeding. [ABSTRACT FROM AUTHOR]

Key message: oxa CMS is a new cytoplasmic male sterility type in Brassica juncea. Abstract: oxa CMS is a cytoplasmic male sterility (CMS) line that has been widely used in the production and cultivation of stem mustard in the southwestern China. In this study, different CMS-type specific mitochondrial markers were used to confirm that oxa CMS is distinct from the pol CMS, ogu CMS, nap CMS, hau CMS, tour CMS, Moricandia arvensis CMS, orf220-type CMS, etc., that have been previously reported in Brassica crops. Pollen grains of the oxa CMS line are sterile with a self-fertility rate of almost 0% and the sterility strain rate and sterility degree of oxa CMS is 100% due to a specific flower structure and flowering habit. Scanning electron microscopy revealed that most pollen grains in mature anthers of the oxa CMS line are empty, flat and deflated. Semi-thin section further showed that the abortive stage of anther development in oxa CMS is initiated at the late uninucleate stage. Abnormally vacuolated microspores caused male sterility in the oxa CMS line. This cytological study combined with marker-assisted selection showed that oxa CMS is a novel CMS type in stem mustard ( Brassica juncea). Interestingly, the abortive stage of oxa CMS is later than those in other CMS types reported in Brassica crops, and there is no negative effect on the oxa CMS line growth period. This study demonstrated that this novel oxa CMS has a unique flower structure with sterile pollen grains at the late uninucleate stage. Our results may help to uncover the mechanism of oxa CMS in Brassica juncea. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Yellow-seeded Brassica napus was for the first time developed from interspecific crosses using yellow-seeded B. juncea (AABB), yellow-seeded B. oleracea (CC), and black-seeded artificial B. napus (AACC). Three different mating approaches were undertaken to eliminate B-genome chromosomes after trigenomic hexaploids (AABBCC) were generated. Hybrids (AABCC, ABCC) from crosses AABBCC × AACC, AABBCC × CC and ABCC × AACC were advanced by continuous selfing in approach 1, 2 and 3, respectively. To provide more insight into Brassica genome evolution and the cytological basis for B. napus resynthesis in each approach, B-genome chromosome pairing and segregation were intensively analyzed in AABCC and ABCC plants using genomic in situ hybridization methods. The frequencies at which B-genome chromosomes underwent autosyndesis and allosyndesis were generally higher in ABCC than in AABCC plants. The difference was statistically significant for allosyndesis but not autosyndesis. Abnormal distributions of B-genome chromosomes were encountered at anaphase I, including chromosome lagging and precocious sister centromere separation of univalents. These abnormalities were observed at a significantly higher frequency in AABCC than in ABCC plants, which resulted in more rapid B-genome chromosome elimination in the AABCC derivatives. Yellow or yellow-brown seeds were obtained in all approaches, although true-breeding yellow-seeded B. napus was developed only in approaches 2 and 3. The efficiency of the B. napus construction approaches was in the order 1 > 3 > 2 whereas this order was 3 > 2 > 1 with respect to the construction of yellow-seeded B. napus. The results are discussed in relation to Brassica genome evolution and the development and utilization of the yellow-seeded B. napus obtained here. [ABSTRACT FROM AUTHOR]

A recessive epistatic genic male sterile two-type line, 7365AB ( Bnms3ms3ms4msRrfRf/BnMs3ms3ms4ms4RfRf), combined with the fertile interim-maintainer 7365C ( Bnms3ms3ms4ms4rfrf) is an effective pollination control system in hybrid rapeseed production. We report an effective strategy used to fine map BnMs4 and BnRf. The two genes were both defined to a common microsyntenic region with Arabidopsis chromosome 3 using intron polymorphism (IP) markers developed according to Arabidopsis genome information and published genome organization of the A genome. The near-isogenic lines 7365AC ( Bnms3ms3ms4ms4Rfrf/ Bnms3ms3ms4ms4rfrf) of BnRf and 736512AB ( Bnms3ms3Ms4ms4RfRf/ Bnms3ms3ms4ms4RfRf) of BnMs4 were constructed to screen developed markers and create genetic linkage maps. Nine polymorphic IP markers (P1-P9) were identified. Of these, P2, P3, P4, and P6 were linked to both BnMs4 and BnRf with genetic distances <0.6 cM. Three simple sequence repeat markers, SR2, SR3, and SR5, were also identified by using public information. Subsequently, all markers linked to the two genes were used to compare the micro-collinearity of the regions flanking the two genes with Brassica rapa and Arabidopsis. The flanking regions showed rearrangements and inversion with fragments of different Arabidopsis chromosomes, but a high collinearity with B. rapa. This collinearity provided extremely valuable reference for map-based cloning in polyploid Brassica species. These IP markers could be exploited for comparative genomic studies within and between Brassica species, providing an economically feasible approach for molecular marker-assisted selection breeding, accelerating the process of gene cloning, and providing more direct evidence for the presence of multiple alleles between BnMs4 and BnRf. [ABSTRACT FROM AUTHOR]

In the present study, we have obtained one dwarf mutant ( bnaC.dwf) from the Brassica napus inbred line T6 through chemical mutagen ethyl methanesulfonate (EMS). We have determined the phenotypic effects and genetic characteristics of dwarf mutant ( bnaC.dwf). The dwarf mutant was insensitive to exogenous GA for plant height, suggesting that it is significantly playing a crucial role in the gibberellins response pathway. Genetic analysis revealed that one recessive gene is responsible for controlling the phenotypic expression of dwarf mutant. Amplified Fragment Length Polymorphism (AFLP) technique was applied for selecting markers linked to the BnaC.DWF gene which assisted in screening of dwarf and normal individuals in the BC population. We have screened 1,024 primer combinations and then identified nine AFLP markers linked to the BnaC.DWF gene. Identification and linkage of the markers were carried out by analysing 2,000 individuals from a larger population of the BC. Two markers EA10MC09 and EA12MC02 were located on the flanking region of the BnaC.DWF gene at a distance of 0.2 and 0.05 cM, respectively. Four AFLP markers EA09MG05, EA02MC07, EA01MC01 and EC04MC07 were successfully converted into Sequence Characterised Amplified Region markers namely SCA9G5, SCA2C7, SCA1C1 and SCC4C7. We further integrated BnaC.DWF linked Simple Sequence Repeat markers into two populations (Piquemal et al. Theor Appl Genet 111:1514-1523, ; Cheng et al. Theor Appl Genet 118:1121-1131, ). BnaC.DWF was mapped to the linkage region N18. The molecular markers developed from these investigations will greatly accelerate the selection process for developing dwarf varieties in B. napus by Marker Assisted Selection and genetic engineering. [ABSTRACT FROM AUTHOR]

The inheritance of yield-related traits in rapeseed ( Brassica napus) is poorly understood, and the investigations on mapping of quantitative trait loci (QTL) for such traits are only few. QTL related to six traits were mapped which include plant height (PH), height of lowest primary effective branch (HPB), length of main inflorescence (LMI), silique length (SL), number of primary branches (FB) and silique density (SD). A set of 258 doubled haploid (DH) lines derivatives of a cross between a canola variety Quantum and a resynthesized B. napus line No.2127-17, and a fixed immortalized F2 (designated as IF2) population generated by randomly permutated intermating of these DHs were investigated. A genetic linkage map was constructed using 208 SSR and 189 SRAP markers for the DH population. Phenotypic data were collected from three environments for the two populations. Using composite interval mapping analyses, 30 and 22 significant QTL were repeatedly detected across environments for the six traits in the DH and IF2 populations, respectively. Twenty-nine QTL were common between the two populations. The directions of parental contribution for all common QTL were the same, showing a great potential for marker-assisted selection in improving these traits. Some chromosomal regions harbor QTL for multiple traits, which were consistent with significant phenotypic correlations observed among traits. The results provided a better understanding of the genetic factors controlling yield-related traits in rapeseed. [ABSTRACT FROM AUTHOR]