Dengfeng Hong

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 BC14F1 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.

BnaC9.SMG7b Functions as a Positive Regulator of the Number of Seeds per Silique in Brassica napus by Regulating the Formation of Functional Female Gametophytes.

A major QTL encodes a small protein with an Arabidopsis homolog and promotes the formation of functional ovules, thus yielding more seeds per silique in Brassica napus. Number of seeds per silique (NSS) is an important determinant of seed yield potential in Brassicaceae crops, and it is controlled by naturally occurring quantitative trait loci. We previously mapped a major quantitative trait locus, qSS.C9, on the C9 chromosome that controls NSS in Brassica napus. To gain a better understanding of how qSS.C9 controls NSS in B. napus, we isolated this locus through a map-based cloning strategy. qSS.C9 encodes a predicted small protein with 119 amino acids, designated as BnaC9.SMG7b, that shows homology with the Ever ShorterTelomere1 tertratricopeptide repeats and Ever Shorter Telomere central domains of Arabidopsis (Arabidopsis thaliana) SUPPRESSOR WITH MORPHOGENETIC EFFECTS ON GENITALIA7 (SMG7). BnaC9.SMG7b plays a role in regulating the formation of functional female gametophyte, thus determining the formation of functional megaspores and then mature ovules. Natural loss or artificial knockdown of BnaC9.SMG7b significantly reduces the number of functional ovules per silique and thus, results in decreased seed number, indicating that qSS.C9 is a positive regulator of NSS in B. napus. Sequence and function analyses show that BnaC9.SMG7b experiences a subfunctionalization process that causes loss of function in nonsense-mediated mRNA decay, such as in Arabidopsis SMG7. Haplotype analysis in 84 accessions showed that the favorable BnaC9.SMG7b alleles are prevalent in modern B. napus germplasms, suggesting that this locus has been a major selection target of B. napus improvement. Our results represent the first step toward unraveling the molecular mechanism that controls the natural variation of NSS in B. napus.

Map-based cloning of a recessive genic male sterility locus in Brassica napus L. and development of its functional marker

We previously mapped one male-sterile gene (Bnms3) from an extensively used recessive genic male sterility line (9012AB) in Brassica napus to a 0.14-cM genomic region. In this study, two highly homologous BAC contigs possibly containing the candidate BnMs3 gene were identified using a map-based cloning strategy. A BnMs3-linked SCAR marker (DM1) capable of differentiating the subgenomes between B. rapa and the B. oleracea aided mapping of BnMs3 on the contig derived from the B. napus chromosome C9. One representative BAC clone was sequenced from each of the two contigs and resulted in a larger number of markers according to the sequence difference between the two clones. To isolate BnMs3, these markers were then analyzed in another two BC1 populations with different genetic backgrounds. This assay allowed for a delimitation of the mutated functional region of BnMs3 to a 9.3-kb DNA fragment. Gene prediction suggested that one complete open reading frame (ORF, ORF2) and partial CDS fragments of ORF1 and ORF3 reside in this fragment. Sequence comparison and genetic transformation eventually indicated that ORF1 (designated as BnaC9.Tic40), an analogue of the Arabidopsis gene AT5G16620 which encodes a translocon of the inner envelope of chloroplasts 40 (Tic40), is the only candidate gene of BnMs3. Furthermore, two distinct mutation types in ORF1 both causing the male-sterile phenotype were individually revealed from 9012A and the temporary maintainer line T45. The molecular mechanism of this male sterility as well as the application of BnMs3-associated functional and cosegregated markers in true breeding programs was also discussed.

Abnormal Vacuolization of the Tapetum During the Tetrad Stage is Associated with Male Sterility in the Recessive Genic Male Sterile Brassica napus L. Line 9012A

In the recessive genic male sterile line 9012A of Brassica napus, pollen development is affected during the tetrad stage. According to the light and electron microscopy analysis of tapetal cells and tetrads, the sterile tapetal cells swelled with expanded vacuoles at the early tetrad stage and finally filled the center of the locules where a majority of tetrads encased with the thick callose wall collapsed and degraded. We suggested that an absence of callase, which is a wall-degrading enzyme stored in the vacuoles of tapetal cells before secretion, resulted in the failure of tetrad separation. Moreover, transmission electron microscopy analysis showed that the secretory tapetal cells were not observed in sterile anthers, which indicated that the transition of the tapetum from the parietal type to the secretory type was probably aberrant. In plants, degeneration of the tapetum is thought to be the result of programmed cell death (PCD). PCD of tapetal cells was investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and signals indicative of deoxyribonucleic acid fragmentation were detected much earlier in sterile anther than in fertile anther. This suggests that tapetal breakdown does not occur by the normal procession of PCD and might be following an alternative mechanism of unscheduled apoptosis in line 9012A. This research supports the hypothesis that premature PCD is associated with male sterility in B. napus.

AFLP and SCAR markers linked to the suppressor gene (Rf) of a dominant genetic male sterility in rapeseed (Brassica napus L.)

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene (Ms) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene (Rf). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus. In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual (MsMsrfrf) and a fertile individual (MsMsRfrf) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.

A Mitochondria-Targeted PPR Protein Restores pol Cytoplasmic Male Sterility by Reducing orf224 Transcript Levels in Oilseed Rape.

Document S1. Supplemental Methods, Supplemental Figures 1–4, Supplemental Tables 1–3, and Supplemental ReferencesxDownload (.42 MB ) Document S1. Supplemental Methods, Supplemental Figures 1–4, Supplemental Tables 1–3, and Supplemental References

Molecular analysis and expression of a floral organ-specific polygalacturonase gene isolated from rapeseed (Brassica napus L.)

High throughput screening of stage-specific differentially expressed genes in a Brassica napus two-line Rs1046A/B subtractive library was used to identify the BnQRT3 gene associated with cell wall metabolism. Phylogenetic analysis indicates the protein product of BnQRT3 is polygalacturonase. According to cytological comparisons of Rs1046 sterile and fertile anthers, RT–PCR studies and in situ hybridizations, BnQRT3 is expressed most strongly in floral organs and may play an essential role in pollen maturation. Analysis of the histological staining pattern of BnQRT3 promoter-GUS constructs in transgenic Arabidopsis and Brassica napus revealed that proximal part of 5′-flanking region directed expression in the vascular tissue of filaments, veins in sepal and petals, stigma, branch connective and the floral organ abscission zone during the open flower stage. In the meanwhile, Activity of BnQRT3 was detected in the anthers, which commences at the microsporocyte stage and persists as anther approaches dehiscence. Strong GUS expression also can be observed in the vascular tissue of leaves and stem by compression with forceps or excision, suggesting that the BnQRT3 promoter is responsive to wounding.

A triallelic genetic male sterility locus in Brassica napus: an integrative strategy for its physical mapping and possible local chromosome evolution around it

BACKGROUND AND AIMS Spontaneous male sterility is an advantageous trait for both constructing efficient pollination control systems and for understanding the developmental process of the male reproductive unit in many crops. A triallelic genetic male-sterile locus (BnMs5) has been identified in Brassica napus; however, its complicated genome structure has greatly hampered the isolation of this locus. The aim of this study was to physically map BnMs5 through an integrated map-based cloning strategy and analyse the local chromosomal evolution around BnMs5. METHODS A large F(2) population was used to integrate the existing genetic maps around BnMs5. A map-based cloning strategy in combination with comparative mapping among B. napus, Arabidopsis, Brassica rapa and Brassica oleracea was employed to facilitate the identification of a target bacterial artificial chromosome (BAC) clone covering the BnMs5 locus. The genomic sequences from the Brassica species were analysed to reveal the regional chromosomal evolution around BnMs5. KEY RESULTS BnMs5 was finally delimited to a 0·3-cM genetic fragment from an integrated local genetic map, and was anchored on the B. napus A8 chromosome. Screening of a B. napus BAC clone library and identification of the positive clones validated that JBnB034L06 was the target BAC clone. The closest flanking markers restrict BnMs5 to a 21-kb region on JBnB034L06 containing six predicted functional genes. Good collinearity relationship around BnMs5 between several Brassica species was observed, while violent chromosomal evolutionary events including insertions/deletions, duplications and single nucleotide mutations were also found to have extensively occurred during their divergence. CONCLUSIONS This work represents major progress towards the molecular cloning of BnMs5, as well as presenting a powerful, integrative method to mapping loci in plants with complex genomic architecture, such as the amphidiploid B. napus.

A pentatricopeptide repeat protein restores nap cytoplasmic male sterility in Brassica napus

Map-based cloning provides evidence that the restorer gene Rfn for nap CMS in Brassica napus encodes a pentatricopeptide repeat protein and shares many properties with previously cloned restorer genes.

Exploiting comparative mapping among Brassica species to accelerate the physical delimitation of a genic male-sterile locus ( BnRf ) in Brassica napus

The recessive genic male sterility (RGMS) line 9012AB has been used as an important pollination control system for rapeseed hybrid production in China. Here, we report our study on physical mapping of one male-sterile locus (BnRf) in 9012AB by exploiting the comparative genomics among Brassica species. The genetic maps around BnRf from previous reports were integrated and enriched with markers from the Brassica A7 chromosome. Subsequent collinearity analysis of these markers contributed to the identification of a novel ancestral karyotype block F that possibly encompasses BnRf. Fourteen insertion/deletion markers were further developed from this conserved block and genotyped in three large backcross populations, leading to the construction of high-resolution local genetic maps where the BnRf locus was restricted to a less than 0.1-cM region. Moreover, it was observed that the target region in Brassica napus shares a high collinearity relationship with a region from the Brassica rapa A7 chromosome. A BnRf-cosegregated marker (AT3G23870) was then used to screen a B. napus bacterial artificial chromosome (BAC) library. From the resulting 16 positive BAC clones, one (JBnB089D05) was identified to most possibly contain the BnRfc allele. With the assistance of the genome sequence from the Brassica rapa homolog, the 13.8-kb DNA fragment covering both closest flanking markers from the BAC clone was isolated. Gene annotation based on the comparison of microcollinear regions among Brassica napus, B. rapa and Arabidopsis showed that five potential open reading frames reside in this fragment. These results provide a foundation for the characterization of the BnRf locus and allow a better understanding of the chromosome evolution around BnRf.