Estimating meiotic chromosome pairing and recombination parameters in telocentric trisomics.

Telocentric trisomics (telotrisomics; one arm of a metacentric chromosome present in addition to two complete genomes) are used in theoretical studies of pairing affinities and chiasma formation in competitive situations and applied in genome analysis, gene localization, gene transfer, and breakage of close linkages. These applications require knowledge of the recombination characteristics of telotrisomics. Appropriate cytological and molecular markers and favorable chromosome morphology are not always available or applicable for quantitative analyses. We developed new mathematical models for extracting the maximum information from simple metaphase I observations. Two types of telotrisomics of the short arm of chromosome 1R of rye (Secale cereale), including several genotypes, were used as test material. In simple telotrisomics, pairing between morphologically identical complete chromosomes was more frequent than pairing between the telocentric and either of the normal chromosomes. In the telocentric substitution, morphologically identical telocentrics paired less frequently with each other than either one with the normal chromosome. Pairing partner switch was significant. Interaction between the two arms was variable. Variation within plants was considerable. Telotrisomics without markers are suitable for analyzing pairing preferences, for gene localization and gene transfer, and for breaking tight linkages, but less so for genome analysis.     

Indistinguishable patterns of recombination resulting from male and female meioses in Brassica napus (oilseed rape).

An F1 individual derived from a cross between two distinct lines of spring oilseed rape (Brassica napus) was used to produce a pair of complementary backcross populations, each consisting of 90 individuals. The F1 donated male gametes to the Male population and female gametes to the Female population. Genetic maps were generated from both populations and aligned using 117 common loci to form an integrated genome map of B. napus with 243 RFLP-defined loci. A comparison of the frequency and distribution of crossovers in the two populations of F1 gametes (assayed in the Male and Female populations) detected no differences. The genetic maps derived from the Male and Female populations each consisted of 19 linkage groups spanning 1544 and 1577 cM, respectively. The maps were aligned with other B. napus maps, and all 19 equivalent linkage groups were unambiguously assigned. The genetic size and general organisation of the new maps were comparable with those of pre-existing B. napus maps in most respects, except that the levels of polymorphism in the constituent A and C genomes were unusually similar in the new cross.     

Homeologous recombination plays a major role in chromosome rearrangements that occur during meiosis of Brassica napus haploids

Chromosomal rearrangements can be triggered by recombination between distinct but related regions. Brassica napus (AACC; 2n = 38) is a recent allopolyploid species whose progenitor genomes are widely replicated. In this article, we analyze the extent to which chromosomal rearrangements originate from homeologous recombination during meiosis of haploid B. napus (n = 19) by genotyping progenies of haploid x euploid B. napus with molecular markers. Our study focuses on three pairs of homeologous regions selected for their differing levels of divergence (N1/N11, N3/N13, and N9/N18). We show that a high number of chromosomal rearrangements occur during meiosis of B. napus haploid and are transmitted by first division restitution (FDR)-like unreduced gametes to their progeny; half of the progeny of Darmor-bzh haploids display duplications and/or losses in the chromosomal regions being studied. We demonstrate that half of these rearrangements are due to recombination between regions of primary homeology, which represents a 10- to 100-fold increase compared to the frequency of homeologous recombination measured in euploid lines. Some of the other rearrangements certainly result from recombination between paralogous regions because we observed an average of one to two autosyndetic A-A and/or C-C bivalents at metaphase I of the B. napus haploid. These results are discussed in the context of genome evolution of B. napus.     

Conserved structure and function of the Arabidopsis flowering time gene CONSTANS in Brassica napus

The Arabidopsis thaliana CONSTANS (CO) gene which promotes flowering in long days was recently isolated by chromosome walking. The mapping of QTLs controlling flowering time in Brassica species has identified genomic regions that contain homologues of the CO gene. Four genes homologous to the Arabidopsis CO gene were isolated from a pair of homoeologous loci in each of two doubled-haploid Brassica napus lines displaying different flowering times, N-o-1 and N-o-9. The four genes, BnCOa1, BnCOa9, BnCOb1 and BnCOb9, are located on linkage groups N10 and N19, and are highly similar to each other and to the Arabidopsis CO gene. Two regions of the proteins are particularly well conserved, a N-terminal region with two putative zinc fingers and a C-terminal region which may contain a nuclear localization signal. All four genes appear to be expressed in B. napus. The BnCOa1 allele was shown to complement the co-2 mutation in Arabidopsis in a dosage-dependent manner causing earlier flowering than in wild type under both long- and short-day conditions.     

Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion

Summary Fertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (11 ratio).     

Genome doubling and chromosome elimination with fragment recombination leading to the formation of Brassica rapa-type plants with genomic alterations in crosses with Orychophragmus violaceus.

In distant hybridization of plants, nonclassical hybrids with unexpected chromosome complements, chromosome elimination, and genetic introgression have been well documented. We obtained intergeneric hybrids between Brassica rapa, B. rapa var. chinensis, and another cruciferous species, Orychophragmus violaceus, following embryo rescue. Hybrids mainly displayed phenotypes of B. rapa, although certain O. violaceus or novel characteristics also appeared. Variable numbers of chromosomes were observed in somatic cells in the roots of plantlets on medium and in ovaries and pollen mother cells (PMCs). However, higher numbers were recorded in the roots. GISH revealed that the majority of ovary cells and PMCs contained 20 chromosomes of B. rapa with or without individual O. violaceus chromosomes or fragments added or introgressed. AFLP analysis showed that fragments deleted from the B. rapa genome were much more frequent than novel and O. violaceus fragments. The mechanisms involved genome doubling and successive elimination of O. violaceus chromosomes accompanied by fragment recombination and introgression, producing B. rapa-type plants with modified genetic constitutions and phenotypes.     

The importance of genetic recombination for fidelity of chromosome pairing in meiosis

In budding yeast, absence of the Hop2 protein leads to extensive synaptonemal complex (SC) formation between nonhomologous chromosomes, suggesting a crucial role for Hop2 in the proper alignment of homologous chromosomes during meiotic prophase. Genetic analysis indicates that Hop2 acts in the same pathway as the Rad51 and Dmc1 proteins, two homologs of E. coli RecA. Thus, the hop2 mutant phenotype demonstrates the importance of the recombination machinery in promoting accurate chromosome pairing. We propose that the Dmc1/Rad51 recombinases require Hop2 to distinguish homologous from nonhomologous sequences during the homology search process. Thus, when Hop2 is absent, interactions between nonhomologous sequences become inappropriately stabilized and can initiate SC formation. Overexpression of RAD51 largely suppresses the meiotic defects of the dmc1 and hop2 mutants. We conclude that Rad51 is capable of carrying out a homology search independently, whereas Dmc1 requires additional factors such as Hop2.     

Homologous chromosome pairing.

Commonly accepted precepts are challenged: (1) that homologous chromosome pairing is normally mediated by nuclear envelope attachment sites; (2) that crossover site establishment awaits synaptic completion; and (3) that it is the function of the synaptonemal complex to hold homologues in register so that equal crossing over can occur, and perhaps to provide machinery for the crossover process. Although these views may eventually be shown to be true, it is felt that currently available evidence does not warrant their full acceptance, and that alternatives should be considered. As examples of alternatives the following ideas, with some supporting evidence, are suggested: (1) homologous chromosome pairing (in non-haplont organisms) may be accomplished by chance meeting of homologue segments (followed by establishment of invisible, elastic connectors) at congression for a mitotic metaphase (in many cases perhaps the premeiotic mitosis); (2) crossover sites may be established before, during, or immediately following initiation of synapsis; and (3) the synaptonemal complex may somehow function in the crossover process at the inception of its formation, but its complete deployment throughout each normal bivalent may serve some other role, such as mediation of the binding of sister chromatids apparently required for chiasma maintenance until anaphase I.     

Mapping PrBn and other quantitative trait loci responsible for the control of homeologous chromosome pairing in oilseed rape (Brassica napus L.) haploids

In allopolyploid species, fair meiosis could be challenged by homeologous chromosome pairing and is usually achieved by the action of homeologous pairing suppressor genes. Oilseed rape (Brassica napus) haploids (AC, n=19) represent an attractive model for studying the mechanisms used by allopolyploids to ensure the diploid-like meiotic pairing pattern. In oilseed rape haploids, homeologous chromosome pairing at metaphase I was found to be genetically based and controlled by a major gene, PrBn, segregating in a background of polygenic variation. In this study, we have mapped PrBn within a 10-cM interval on the C genome linkage group DY15 and shown that PrBn displays incomplete penetrance or variable expressivity. We have identified three to six minor QTL/BTL that have slight additive effects on the amount of pairing at metaphase I but do not interact with PrBn. We have also detected a number of other loci that interact epistatically, notably with PrBn. Our results support the idea that, as in other polyploid species, metaphase I homeologous pairing in oilseed rape haploids is controlled by an integrated system of several genes, which function in a complex manner.     

Characterization of interploid hybrids from crosses between Brassica juncea and B. oleracea and the production of yellow-seeded B. napus

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.     

Indica and japonica crosses resulting in linkage block and recombination suppression on rice chromosome 12

Understanding linkage block size and molecular mechanisms of recombination suppression is important for plant breeding. Previously large linkage blocks ranging from 14 megabases to 27 megabases were observed around the rice blast resistance gene Pi-ta in rice cultivars and backcross progeny involving an indica and japonica cross. In the present study, the same linkage block was further examined in 456 random recombinant individuals of rice involving 5 crosses ranging from F(2) to F(10) generation, with and without Pi-ta containing genomic indica regions with both indica and japonica germplasm. Simple sequence repeat markers spanning the entire chromosome 12 were used to detect recombination break points and to delimit physical size of linkage blocks. Large linkage blocks ranging from 4.1 megabases to 10 megabases were predicted from recombinant individuals involving genomic regions of indica and japonica. However, a significantly reduced block from less than 800 kb to 2.1megabases was identified from crosses of indica with indica rice regardless of the existence of Pi-ta. These findings suggest that crosses of indica and japonica rice have significant recombination suppression near the centromere on chromosome 12.     

Suppressed recombination and a pairing anomaly on the mating-type chromosome of Neurospora tetrasperma

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, Merino et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.     

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.Key words: Synthetic Brassica allohexaploids, Orychophragmus violaceus, intergeneric hybrids, genomic in situ hybridization, amplified fragment length polymorphism, single-strand conformation polymorphism, chromosome elimination, chromosome stability, nucleolar dominance     

PrBn,a major gene controlling homeologous pairing in oilseed rape (Brassica napus) haploids

Precise control of chromosome pairing is vital for conferring meiotic, and hence reproductive, stability in sexually reproducing polyploids. Apart from the Ph1 locus of wheat that suppresses homeologous pairing, little is known about the activity of genes that contribute to the cytological diploidization of allopolyploids. In oilseed rape (Brassica napus) haploids, the amount of chromosome pairing at metaphase I (MI) of meiosis varies depending on the varieties the haploids originate from. In this study, we combined a segregation analysis with a maximum-likelihood approach to demonstrate that this variation is genetically based and controlled mainly by a gene with a major effect. A total of 244 haploids were produced from F(1) hybrids between a high- and a low-pairing variety (at the haploid stage) and their meiotic behavior at MI was characterized. Likelihood-ratio statistics were used to demonstrate that the distribution of the number of univalents among these haploids was consistent with the segregation of a diallelic major gene, presumably in a background of polygenic variation. Our observations suggest that this gene, named PrBn, is different from Ph1 and could thus provide complementary information on the meiotic stabilization of chromosome pairing in allopolyploid species.     

Assessing the level of collinearity between Arabidopsis thaliana and Brassica napus for A. thaliana chromosome 5.

This study describes a comprehensive comparison of chromosome 5 of the model crucifer Arabidopsis with the genome of its amphidiploid crop relative Brassica napus and introduces the use of in silico sequence homology to identify conserved loci between the two species. A region of chromosome 5, spanning 8 Mb, was found in six highly conserved copies in the B. napus genome. A single inversion appeared to be the predominant rearrangement that had separated the two lineages leading to the formation of Arabidopsis chromosome 5 and its homologues in B. napus. The observed results could be explained by the fusion of three ancestral genomes with strong similarities to modern-day Arabidopsis to generate the constituent diploid genomes of B. napus. This supports the hypothesis that the diploid Brassica genomes evolved from a common hexaploid ancestor. Alignment of the genetic linkage map of B. napus with the genomic sequence of Arabidopsis indicated that for specific regions a genetic distance of 1 cM in B. napus was equivalent to 285 Kb of Arabidopsis DNA sequence. This analysis strongly supports the application of Arabidopsis as a tool in marker development, map-based gene cloning, and candidate gene identification for the larger genomes of Brassica crop species.     

Agrobacterium-mediated transformation of Brassica napus and Brassica oleracea

Agrobacterium-mediated transformation is widely used for gene delivery in plants. However, commercial cultivars of crop plants are often recalcitrant to transformation because the protocols established for model varieties are not directly applicable to them. The genus Brassica includes the oil seed crop, canola (B. napus), and vegetable crop varieties of Brassica oleracea, including cauliflower, broccoli and cabbage. Here, we describe an efficient protocol for Agrobacterium-mediated transformation using seedling explants that is applicable to various Brassica varieties; this protocol has been used to genetically engineer commercial cultivars of canola and cauliflower in our laboratory. Young seedling explants are inoculated with Agrobacterium on the day of explant preparation. Explants are grown for 1 week in the absence of a selective agent before being transferred to a selective medium to recover transgenic shoots. Transgenic shoots are subjected to an additional round of selection on medium containing higher levels of the selective agent and a low-carbohydrate source; this helps to eliminate false-positive plants. Use of seedling explants offers flexible experiment planning and a convenient explant source. Using this protocol, transgenic plants can be obtained in 2.5 to 3.5 months.