Anatomy and transcript profiling of gynoecium development in female sterile Brassica napus mediated by one alien chromosome from Orychophragmus violaceus.

BACKGROUND: The gynoecium is one of the most complex organs of angiosperms specialized for seed production and dispersal, but only several genes important for ovule or embryo sac development were identified by using female sterile mutants. The female sterility in oilseed rape (Brassica napus) was before found to be related with one alien chromosome from another crucifer Orychophragmus violaceus. Herein, the developmental anatomy and comparative transcript profiling (RNA-seq) for the female sterility were performed to reveal the genes and possible metabolic pathways behind the formation of the damaged gynoecium. RESULTS: The ovules in the female sterile Brassica napus with two copies of the alien chromosomes (S1) initiated only one short integument primordium which underwent no further development and the female gametophyte development was blocked after the tetrad stage but before megagametogenesis initiation. Using Brassica_ 95k_ unigene as the reference genome, a total of 28,065 and 27,653 unigenes were identified to be transcribed in S1 and donor B. napus (H3), respectively. Further comparison of the transcript abundance between S1 and H3 revealed that 4540 unigenes showed more than two fold expression differences. Gene ontology and pathway enrichment analysis of the Differentially Expressed Genes (DEGs) showed that a number of important genes and metabolism pathways were involved in the development of gynoecium, embryo sac, ovule, integuments as well as the interactions between pollen and pistil. CONCLUSIONS: DEGs for the ovule development were detected to function in the metabolism pathways regulating brassinosteroid (BR) biosynthesis, adaxial/abaxial axis specification, auxin transport and signaling. A model was proposed to show the possible roles and interactions of these pathways for the sterile gynoecium development. The results provided new information for the molecular mechanisms behind the gynoecium development at early stage in B. napus.

Nucleolar dominance and different genome behaviors in hybrids and allopolyploids.

Many plants are allopolyploids with different nuclear genomes from two or more progenitors, but cytoplasmic genomes typically inherited from the female parent. The importance of this speciation mechanism has stimulated the extensive investigations of genetic consequences of genome mergers in several experimental systems during last 20 years. The dynamic nature of polyploid genomes is recognized, and widespread changes to gene expression are revealed by transcriptomic analysis. These progresses show different stabilities of parental genomes and their unequal contributions to the transcriptome, proteome, and phenotype. We review the results in systems where extensive genetic analyses have been conducted and propose possible mechanisms for biased behavior of parental genomes in allopolyploids, including the role of nucleolar dominance. It is hypothesized that the novel ribosomes with rRNAs from uniparental genome and the ribosomal proteins of biparental origins have some impacts on the biased cellular and genetic behaviors of parental genomes in hybrids and allopolyploids.

Different genome-specific chromosome stabilities in synthetic Brassica allohexaploids revealed by wide crosses with Orychophragmus.

BACKGROUND AND AIMS: In sexual hybrids between cultivated Brassica species and another crucifer, Orychophragmus violaceus (2n = 24), parental genome separation during mitosis and meiosis is under genetic control but this phenomenon varies depending upon the Brassica species. To further investigate the mechanisms involved in parental genome separation, complex hybrids between synthetic Brassica allohexaploids (2n = 54, AABBCC) from three sources and O. violaceus were obtained and characterized. METHODS: Genomic in situ hybridization, amplified fragment length polymorphism (AFLP) and single-strand conformation polymorphism (SSCP) were used to explore chromosomal/genomic components and rRNA gene expression of the complex hybrids and their progenies. KEY RESULTS: Complex hybrids with variable fertility exhibited phenotypes that were different from the female allohexaploids and expressed some traits from O. violaceus. These hybrids were mixoploids (2n = 34-46) and retained partial complements of allohexaploids, including whole chromosomes of the A and B genomes and some of the C genome but no intact O. violaceus chromosomes; AFLP bands specific for O. violaceus, novel for two parents and absent in hexaploids were detected. The complex hybrids produced progenies with chromosomes/genomic complements biased to B. juncea (2n = 36, AABB) and novel B. juncea lines with two genomes of different origins. The expression of rRNA genes from B. nigra was revealed in all allohexaploids and complex hybrids, showing that the hierarchy of nucleolar dominance (B. nigra, BB > B. rapa, AA > B. oleracea, CC) in Brassica allotetraploids was still valid in these plants. CONCLUSIONS: The chromosomes of three genomes in these synthetic Brassica allohexaploids showed different genome-specific stabilities (B > A > C) under induction of alien chromosome elimination in crosses with O. violaceus, which was possibly affected by nucleolar dominance.

Production and cytogenetic characterization of intertribal somatic hybrids between Brassica napus and Isatis indigotica and backcross progenies.

Intertribal somatic hybrids between Brassica napus (2n = 38, AACC) and a dye and medicinal plant Isatis indigotica (2n = 14, II) were obtained by fusions of mesophyll protoplasts. From a total of 237 calli, only one symmetric hybrid (S2) and five asymmetric hybrids (As1, As4, As6, As7 and As12) were established in the field. These hybrids showed some morphological variations and had very low pollen fertility. Hybrids S2 and As1 possessed 2n = 52 (AACCII), the sum of the parental chromosomes, and As12 had 2n = 66 (possibly AACCIIII). Hybrids As4, As6 and As7 were mixoploids (2n = 48-62). Genomic in situ hybridization analysis revealed that pollen mother cells at diakinesis of As1 contained 26 bivalents comprising 19 from B. napus and 7 from I. indigotica and mainly showed the segregation 26:26 at anaphase I (AI) with 7 I. indigotica chromosomes in each polar group. Four BC(1) plants from As1 after pollinated by B. napus resembled mainly B. napus in morphology but also exhibited some characteristics from I. indigotica. These plants produced some seeds on selfing or pollination by B. napus. They had 2n = 45 (AACCI) and underwent pairing among the I. indigotica chromosomes and/or between the chromosomes of two parents at diakinesis. All hybrids mainly had the AFLP banding patterns from the addition of two parents plus some alterations. B. napus contributed chloroplast genomes in majority of the hybrids but some also had from I. indigotica. Production of B. napus-I. indigotica additions would be of considerable importance for genome analysis and breeding.

[Chromosomal behaviors in plant wide hybridizations and their genetic and evolutionary implications].

The wide hybridization and polyploidization play a significant role in the evolution of higher plants. On the contrary, the artificially synthesized allopolyploids are genetically unstable and fail to be used as crops. One reason for this situation may be that the allopolyploids in nature are the products of natural selection and evolution and it is difficult for human to repeat and perform the process in short periods. Another reason is that we know little about the interaction mechanisms between the genomes of different origins. So the genetics and epigenetics after allopolyploidizations are now studied by multidisciplinary approaches. The spatial separation of parental genomes in hybrid cells have been observed in some sexual and somatic hybrids, but the biological meanings remain to clarify. The abnormal chromosome behaviors in plant wide crosses, such as pseudogamy, semigamy, chromosome elimination and the mitotic and meiotic separation of parental genomes, may indicate the incompatibility of two parental species at gametic and chromosomal levels. The systematic studies at different levels on chromosomal behavior and genetics in plant hybridizations are needed to undermine the mechanisms responsible for the formation and evolution of new species.

Production and characterization of intergeneric somatic hybrids between Brassica napus and Orychophragmus violaceus and their backcrossing progenies.

Alien chromosome addition lines have been widely used for identifying gene linkage groups, assigning species-specific characters to a particular chromosome and comparing gene synteny between related species. In plant breeding, their utilization lies in introgressing characters of agronomic value. The present investigation reports the production of intergeneric somatic hybrids Brassica napus (2n = 38) + Orychophragmus violaceus (2n = 24) through asymmetric fusions of mesophyll protoplasts and subsequent development of B. napus-O. violaceous chromosome addition lines. Somatic hybrids showed variations in morphology and fertility and were mixoploids (2n = 51-67) with a range of 19-28 O. violaceus chromosomes identified by genomic in situ hybridization (GISH). After pollinated with B. napus parent and following embryo rescue, 20 BC(1) plants were obtained from one hybrid. These exhibited typical serrated leaves of O. violaceus or B. napus-type leaves. All BC(1) plants were partially male fertile but female sterile because of abnormal ovules. These were mixoploids (2n = 41-54) with 9-16 chromosomes from O. violaceus. BC(2) plants showed segregations for female fertility, leaf shape and still some chromosome variation (2n = 39-43) with 2-5 O. violaceus chromosomes, but mainly containing the whole complement from B. napus. Among the selfed progenies of BC(2) plants, monosomic addition lines (2n = 39, AACC + 1O) with or without the serrated leaves of O. violaceus or female sterility were established. The complete set of additions is expected from this investigation. In addition, O. violaceus plants at diploid and tetraploid levels with some variations in morphology and chromosome numbers were regenerated from the pretreated protoplasts by iodoacetate and UV-irradiation.

Chromosome elimination and fragment introgression and recombination producing intertribal partial hybrids from Brassica napus x Lesquerella fendleri crosses.

The intertribal sexual hybrids between three Brassica napus (2n=38) cultivars and Lesquerella fendleri (2n=12) with the latter as pollen parent were obtained and characterized for their phenotypes and chromosomal and genomic constitutions. F(1) plants and their progenies mainly resembled female B. napus parents, while certain characters of L. fendleri were expressed in some plants, such as longer flowering period, basal clustering stems and particularly the glutinous layer on seed coats related to drought tolerance. Twenty-seven F(1) plants were cytologically classified into five types: type I (16 plants) had 2n=38, type II (2) had 2n=38-42, type III (3) had 2n=31-38, type IV (5) had 2n=25-31, and type V (1) had 2n=19-22. Some hybrids and their progenies were mixoploids in nature with only 1-2 chromosomes or some chromosomal fragments of L. fendleri included in their cells. AFLP (Amplified fragments length polymorphism) analysis revealed that bands absent in B. napus, novel for two parents and specific for L. fendleri appeared in all F(1) plants and their progenies. Some progenies had the modified fatty acid profiles with higher levels of linoleic, linolenic, eicosanoic and erucic acids than those of B. napus parents. The occurrence of these partial hybrids with phenotypes, genomic and fatty acid alterations resulted possibly from the chromosome elimination and doubling accompanied by the introgression of alien DNA segments and genomic reorganization. The progenies with some useful traits from L. fendleri should be new and valuable resource for rapeseed breeding.

Unique chromosome behavior and genetic control in Brassica x Orychophragmus wide hybrids: a review.

Researchers recognized early that chromosome behavior, as other morphological characters, is under genetic control and gave some cytogenetical examples such as the homoeologous chromosome pairing in wheat. In the intergeneric sexual hybrids between cultivated Brassica species and another crucifer Orychophragmus violaceus, the phenomenon of parental genome separation was found under genetic control during mitosis and meiosis. The cytogenetics of these hybrids was species-specific for Brassica parents. The different chromosome behavior of hybrids with three Brassica diploids (B. rapa, B. nigra and B. oleracea) might contribute to the different cytology of hybrids with three tetraploids (B. napus, B. juncea and B. carinata). The finding that genome-specific retention or loss of chromosomes in hybrids of O. violaceus with B. carinata and synthetic Brassica hexaploids (2n=54, AABBCC) is likely related to nucleolar dominance gives new insight into the molecular mechanisms regarding the cytology in these hybrids. It is proposed that the preferential expressions of genes for centromeric proteins from one parent (such as the well presented centromeric histone H3) are related with chromosome stability in wide hybrids and nucleolar dominance is beneficial to the production of centromere-specific proteins of the rRNAs-donor parent and to the stability of its chromosomes.

Intra- and intergenomic homology of B-genome chromosomes in trigenomic combinations of the cultivated Brassica species revealed by GISH analysis.

Intragenomic chromosome homology in the B genome of Brassica nigra and their homoeology with the chromosomes of the A-genome of B. rapa and C-genome of B. oleracea was investigated in triploids (ABC, n = 27) of different origins obtained following hybridizations between natural B. napus (AACC, 2n = 38) x B. nigra (BB, 2n = 16) [AC.B], synthetic B. napus x B. nigra [A.C.B] and B. carinata (BBCC, 2n = 34) x B. rapa (AA, 2n = 20) [BC.A]. A relatively high percentage of pollen mother cells (PMCs) with at least one B-genome chromosome paired allosyndetically with A/C chromosomes was evident in all three combinations. A maximum of three B-genome chromosomes undergoing allosyndesis per cell was observed in AC.B and A.C.B combinations. A maximum of two autosyndetic bivalents within the B genome appeared at diakinesis in all combinations. The accurate analyses of auto- and allo-syndetic pairing for B genome in trigenomic combinations provided further evidence for the hypothesis that the three basic diploid genomes of the cultivated Brassica species evolved from one common ancestral genome with a lower chromosome number. The results showed that Brassica diploids may not be ancient polyploids but may have undergone chromosomal duplications instead of whole-genome duplication. The relevance of these results along with genetic changes of progenitor genomes which occurred during the evolution of Brassica polyploids is discussed.

Extra divisions and nuclei fusions in microspores from Brassica allohexaploid (AABBCC) x Orychophragmus violaceus hybrids.

Abnormal meiosis and microspore development and related defective mutants have often been reported in plants and wide hybrids. Here extra divisions and nuclei fusions were observed to occur in microspore nuclei of partial hybrids between synthetic Brassica hexaploid (2n = 54, AABBCC) and another crucifer Orychophragmus violaceus (2n = 24). Abnormal spindle were formed and chromosomes were separated into several nuclei of variable sizes after bi-, or multi-polar divisions in the four cells of tetrads. As a consequence, more than eight mini-microspores of different sizes were produced by one tetrad. Genomic in situ hybridization results indicated that no chromosome replication occurred during such divisions. In some tetrads, the four nuclei were fused to form one large cell with increased chromosome number. The extra divisions or fusions appeared only in some flower buds of one plant, some anthers in the same buds, or even in individual cells of tetrads. The possible mechanisms behind these cytological phenomena are discussed.