High efficiency production and genomic<Emphasis Type="Italic">in situ</Emphasis> hybridization analysis of<Emphasis Type="Italic">Brassica</Emphasis> aneuploids and homozygous plants

Interspecific and intergeneric hybridizations have been widely used in plant genetics and breeding to construct stocks for genetic analysis and to introduce into crops the desirable traits and genes from their relatives. The intergeneric crosses between Brassica juncea (L.) Czern. & Coss., B. carinata A. Braun and Orychophragmus violaceus (L.) O. E. Schulz were made and the plants produced were subjected to genomic in situ hybridization analysis. The mixoploids from the cross with B. juncea were divided into three groups. The partially fertile mixoploids in the first group (2n = 36-42) mainly contained the somatic cells and pollen mother cells (PMCs) with the 36 chromosomes of B. juncea and additional chromosomes of O. violaceus. The mixoploids (2n = 30-36) in the second and third groups were morphologically quite similar to the mother plants B. juncea and showed nearly normal fertility. The plants in the second group produced the majority of PMCs (2n = 36) with their chromosomes paired and segregated normally, but 1-4 pairs of the O. violaceus chromosomes were included in some PMCs. The plants in the third group produced only PMCs with the 36 B. juncea chromosomes, which were paired and segregated normally. The mixoploids (2n = 29-34) from the cross with B. carinata produced the majority of PMCs (2n = 34) with normal chromosome pairing and segregation, but some plants had some PMCs with 1-3 pairs of chromosomes from O. violaceus and other plants had only PMCs with the B. carinata chromosomes. The Brassica homozygous plants and aneuploids with complete or partial chromosome complements of Brassica parents and various numbers of O. violaceus chromosomes were derived from these progeny plants. The results in this study provided the molecular cytogenetic evidence for the separation of parental genomes which was previously proposed to occur in the hybridizations of these two genera.     

Parental genome separation and elimination of cells and chromosomes revealed by AFLP and GISH analyses in a Brassica carinata x Orychophragmus violaceus cross

BACKGROUND AND AIMS: The phenomenon of parental genome separation during the mitotic divisions of hybrid cells was proposed to occur under genetic control in intergeneric hybrids between cultivated Brassica species and Orychophragmus violaceus (2n = 24). To elucidate further the cytological and molecular mechanisms behind parental genome separation, Brassica carinata (2n = 34) x O. violaceus hybrids were resynthesized and their chromosome/genomic complements analysed. METHODS: F(1) hybrids of the cross were obtained following embryo rescue, and were investigated for their cytological behaviour and subjected to genomic in situ hybridization (GISH) and amplified fragment length polymorphism (AFLP) to determine the contribution of parental genomes. KEY RESULTS: All the F(1) plants with high fertility closely resembled B. carinata in morphological attributes. These were mixoploids with 2n chromosome numbers ranging from 17 to 35; however, 34, the same number as in B. carinata, was the most frequent number of chromosomes in ovary and pollen mother cells (PMCs). GISH clearly identified 16 chromosomes of B. nigra in ovary cells and PMCs with 2n = 34 and 35. However, no O. violaceus chromosome was detected, indicating the presence of the intact B. carinata genome and elimination of the entire O. violaceus genome. However, some AFLP bands specific for O. violaceus and novel for the two parents were detected in the leaves. Cells with fewer than 34 chromosomes had lost some B. oleracea chromosomes. F(2) plants were predominantly like B. carinata, but some contained O. violaceus characters. CONCLUSIONS: The cytological mechanism for the results involves complete and partial genome separation at mitosis in embryos of F(1) plants followed by chromosome doubling, elimination of cells with O. violaceus chromosomes and some introgression of O. violaceus genetic information.     

甘蓝型油菜与诸葛菜属间杂种无性系的变异研究

从形态及细胞学两方面对甘蓝型油菜与诸葛菜属间杂种无性系的变异进行了研究。经过长期继代培养后,杂种在形态上越来越偏向母本甘蓝型油菜,而表现出较少的父本诸葛菜性状,但表现出对等双分枝的新性状。经细胞学观察表明,杂种体内杂种细胞比例下降,而甘蓝型油菜细胞比例大幅度上升并远高于核质杂种细胞（具有油菜细胞质与诸葛菜细胞核）的比例,以至较多的甘蓝型油菜染色体组被遗传下去。在该杂种继代培养中还观察到杂种细胞内的染色体消除及体细胞配对现象。     

Production and cytogenetics of intergeneric hybrids between the three cultivated Brassica diploids and Orychophragmusviolaceus

It has been proposed that both complete and partial separation of the parental genomes during mitosis and meiosis occurs in the intergeneric hybrids between Orychophragmus violaceus (2n=24) and the three cultivated Brassica tetraploids (B. napus, B. carinata and B. juncea). The hypothesis has been that this and the variations in chromosome numbers of these hybrids and their progenies result from the different roles of the A, B and C genomes originating from Brassica. To test this hypothesis, we produced hybrids between O. violaceus and the cultivated Brassica diploids. The hybrids with B. oleracea (2n=18, CC) had an intermediate morphology, but their petals were purple like those of O. violaceus. They were sterile and had the expected chromosome number (2n=21) in their mitotic and meiotic cells. The hybrid with B. campestris (2n=20, AA) was morphologically intermediate, except for its partial fertility and its yellow petals, which were similar to those of B. campestris. It was mixoploid (2n=23–42), and cells with 2n=34 were most frequent. Partial separation of parental genomes during mitosis, leading to the addition of O. violaceus chromosomes to the B. campestris complement, was proposed to explain the findings in the mitotic and meiotic cells of the hybrid and its progeny. In crosses with B. nigra (2n=16, BB), the majority of the F₁ plants were of the maternal type (2n=16), a small fraction had B. nigra morphology but were mixoploids (2n=16–18), predominantly with 2n=16 cells and three plants, each with a specific morphology, were mixoploids consisting of cells with varying ranges of chromosome numbers (2n=17–26, 11–17 and 14–17). The origin of these different types of plants was inferred to be a result of the complete and partial separation of parental genomes and the loss of O. violaceus chromosomes. Our findings in the three crosses suggest that the A genome was more influential than the C genome with respect to complete genome separation during mitosis and meiosis of the hybrids with B. napus. Possible complete and partial genome separation during mitotic divisions of the hybrids with B. carinata was mainly attributed to the role of the B genome. The combined roles of the A and B genomes would thus contribute to the most variable chromosome numbers of mitotic and meiotic cells in the hybrids with B. juncea and their progenies. The possible cytological mechanisms pertaining to these hybrids and the potential of genome separation in the production of Brassica aneuploids and homozygous plants are discussed. Received: 8 February 1998 / Accepted: 12 March 1999     

甘蓝型油菜与诸葛菜属间杂种后代非整倍体类型及细胞遗传学研究

在甘蓝型油菜与诸葛菜属间五倍体杂种后代中,鉴定出多种非整倍体类型。在P3群体中,细胞学观察发现诸葛菜染色体替代甘蓝型油菜的1对染色体异代换植株,体细胞由36~38条染色体的3种类型组成,但具38条的体细胞与花粉母细胞（PMC）占绝对优势,育性正常。在P4群体后代中鉴定出3株具有37条染色体的单体类型,形态与结实率均不相同。其中1株生长势很强,其体细胞与PMC均由多种类型构成,但具37条的体细胞与花粉母细胞占主要比例,未配对的染色体形态较小,对生长无明显影响。在染色体数为44与41的两种超倍体（P3群体）后代,鉴定出具有29~32条染色体的4种非整倍体,除染色体数为29的植株外,其余均为混倍体,它们的减数分裂异常,花粉育性低。本文对这些非整倍体的来源和应用的前景进行讨论。     

Genomic in situ hybridization in Brassica amphidiploids and interspecific hybrids

Genomic in situ hybridization (GISH) methods were used to detect different genome components within Brassica amphidiploid species and to identify donor chromatin in hybrids between Brassica napus and Raphanus sativus. In Brassica juncea and Brassica carinata the respective diploid donor genomes could be reliably distinguished by GISH, as could all R-genome chromosomes in the intergeneric hybrids. The A- and C-genome components in B. napus could not be clearly distinguished from one another using GISH, confirming the considerable homoeology between these genomes. GISH methods will be extremely beneficial for monitoring chromatin transfer and introgression in interspecific Brassica hybrids. Received: 20 May 1997 / Accepted: 28 July 1997     

埃塞俄比亚芥与诸葛菜属间杂种的基因组原位杂交分析

用幼胚培养方法重新获得的埃塞俄比亚芥(Brassica carinata A.Braun,2n=34)与诸葛菜(Orychophragmus violaceus (L.)O.E.Schulz,2n=24)的属间杂种仍为混倍体(2n=14～34),2n=34细胞的频率最高;绝大多数花粉母细胞(PMCs)表现正常的17个二价体配对和17：17的分离。基因组原位杂交分析结果表明,在所有体细胞和PMCs中不含有整条的诸葛菜染色体,2n=34的体细胞和PMCs中包含了来自黑芥(B．nigra (L．)Koch,2n=16)的16条染色体。这些具有完全或部分埃塞俄比亚芥染色体组成的细胞,可能来源于以前提出的杂种细胞在有丝分裂中完全或部分亲本染色体组分开和染色体复制,并伴随诸葛菜染色体的消除。     

植物逆转座子及其在基因功能和基因组分析中的应用

植物逆转座子及其在基因功能和基因组分析中的应用路铁刚孙敬三（中国科学院植物研究所,北京１０００９３）ＰｌａｎｔＲｅｔｒｏｔｒａｎｓｐｏｓｏｎｓａｎｄｔｈｅＰｅｒｓｐｅｃｔｉｖｅｏｆＴｈｅｉｒＡｐｐｌｉｃａｔｉｏｎｉｎＧｅｎｅＦｕｎｃｔｉｏｎａｌａｎｄ...     

RFLP analysis and genomic in situ hybridization (GISH) in somatic hybrids and their progeny between Lycopersicon esculentum and Solanum lycopersicoides

 RFLP (restriction fragment length polymorphism) and GISH (genomic in situ hybridization) analyses were employed to identify the chloroplast and nuclear genomes of the somatic hybrids and progeny between tomato ‘Ohgata zuiko’ and Solanum lycopersicoides (‘LA 2386’). A random distribution of the chloroplast genotype was determined using a cloned 19.6-kb BamHI fragment (Ba1) of tobacco chloroplast DNA. Eight selected hybrids were analyzed for their chromosomal compositions; 4 were tetraploids (2n=48) with an equal number of chromosomes derived from each parent as accurately determined by GISH, and the other 4 were hexaploids, containing an average of two sets of tomato chromosomes and one set from the wild parent. RFLP analysis with six tomato nuclear probes of known chromosomal locations revealed no major variation among the 44 hybrid plants surveyed. However, it also showed the presence of both parent-specific alleles and the loss of some and the presence of a few non-parental alleles, indicating rearrangement and/or recombination of the nuclear DNA. The relevance of the molecular and cytological methods and the potential use of somatic hybrids for plant breeding are demonstrated. Received: 20 July 1997 / Accepted: 6 October 1997     

Ogura细胞质雄性不育紫菜苔×萝卜属间杂种F_1的获得及细胞遗传学研究

以Ogura细胞质雄性不育紫菜苔(AA, 2n＝20)为母本,以不同萝卜品种(RR,2n＝18)为父本进行杂交,获得了大量的属间杂种.杂种F1幼苗在低温下子叶及真叶均不缺绿.以红萝卜为父本获得的杂种F1植株叶柄、叶脉呈紫红色;以白萝卜为父本获得的杂种F1植株叶柄、叶脉不呈紫红色.所有的杂种F1植株都开白花,蜜腺正常,雄配子高度不育,但是雌配子具有部分育性.杂种F1花粉母细胞的染色体数目为预期的2n＝19,染色体平均配对构型为15.53+1.34+0.25+0.01,多数染色体以单价体的形式存在,但也有一些二价体、三价体甚至四价体,最多达到6+3+1,参加配对的染色体数达到13条,表明A染色体组和R染色体组具有一定的同源性.对该杂种的遗传及育种意义进行了讨论.     

Genetic and genomic aspects of hybridization in ferns

The morphological and ecological intermediacy of hybrid taxa has long interested and challenged fern biologists, resulting in numerous systematic contributions focused on disentangling relationships within reticulate species complexes. From a genetic perspective, hybrid ferns are especially interesting because they represent the union of divergent parental genomes in unique evolutionary entities. This review summarizes advances in our knowledge of the genetic and genomic aspects of hybridization in ferns from the mid-20th century to the present. The different organismal products of hybridization, evolutionary aspects of additive and non-additive gene expression in allopolyploids, genetic and genomic mechanisms leading to gene silencing and loss, the roles of multiple origins and introgression for imparting genetic variation to hybrid fern taxa and their progenitors, and the utility of allopolyploid ferns to investigate mechanisms of genome evolution in the homosporous ferns are discussed. Comparisons are made to other plant lineages and important future research directions are highlighted, with the goal of stimulating additional research on hybrid ferns.     

Regeneration of plants from protoplasts of Capsella bursa-pastoris and somatic hybridization with rapid cycling Brassica oleracea

Fertile rooted plantlets were recovered from leaf mesophyll protoplasts of Capsella bursa-pastoris. Protoplasts cultured over a feeder layer of Brassica napus cells produced 221 colonies, 7 of which regenerated multiple plantlets. The nuclear DNA content of most regenerates varied from 0.89 to 1.0 pg/nucleus, close to the value for seed-grown C. bursa-pastoris (0.94±0.03 pg/nucleus). Two regenerants had a tetraploid DNA content (1.8– 2.0 pg). Plants with a DNA content close to Capsella produced seeds, both in vitro and in soil. Intertribal somatic hybrids were obtained by polyethylene glycol-mediated fusion of untreated C. bursa-pastoris protoplasts with iodoacetate-treated protoplasts of rapid-cycling B. oleracea. Plants were confirmed as somatic hybrids by isozyme and RAPD analysis. The nuclear DNA content of the hybrids ranged from 3.2 to 6.4 pg, higher than the sum of the parental genomes. One of two hybrids tested was resistant to Alternaria brassicicola, like the Capsella fusion partner. Hybrids rooted easily and produced sterile flowers when transplanted to soil. Received: 13 April 1998 / Revision received: 25 August 1998 / Accepted: 31 August 1998     

Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus

The intergeneric hybrid between Brassica napus and Orychophragmus violaceus was obtained by means of embryo culture technique with the latter as the pollen parent. The hybrid was morphologically intermediate between its parents, but could produce a lot of seeds when selfed. Somatic separation of the genomes from the two parental species was observed during the mitotic divisions of some of the hybrid cells. Thus, the hybrid became the mixoploid in nature, consisting of haploid and diploid cells of B. napus, and a nuclear — cytoplasmic hybrid, with the cytoplasm of B. napus and the nuclei of O. violaceus, and the hybrid cells. Pollen mother cells with 19, 12 and 6 bivalents, respectively, were produced by the hybrid. From the selfed progeny of the hybrid, mainly two kinds of plants, B. napus and the hybrid, were found. The hybrid plants of the selfed progeny again produced two kinds of plants, B. napus and the hybrid.     

Comparative genomic in situ hybridization (cGISH) analysis of the genomic relationships among Sinapis arvensis, Brassica rapa and Brassica nigra

To further understand the relationships between the SS genome of Sinapis arvensis and the AA, BB genomes in Brassica, genomic DNA of Sinapis arvensis was hybridized to the metaphase chromosomes of Brassica nigra (BB genome), and the metaphase chromosomes and interphase nucleus of Brassica rapa (AA genome) by comparative genomic in situ hybridization (cGISH). As a result, every chromosome of B. nigra had signals along the whole chromosomal length. However, only half of the condensed heterochromatic areas in the interphase nucleus and the chromosomes showed rich signals in Brassica rapa. Interphase nucleus and the metaphase chromosomes of S. arvensis were simultaneously hybridized with digoxigenin-labeled genomic DNA of B. nigra and biotin-labeled genomic DNA of B. rapa. Signals of genomic DNA of B. nigra hybridized throughout the length of all chromosomes and all the condensed heterochromatic areas in the interphase nucleus, except chromosome 4, of which signals were weak in centromeric regions. Signals of the genomic DNA of B. rapa patterned the most areas of ten chromosomes and ten condensed heterochromatic areas, others had less signals. The results showed that the SS genome had homology with AA and BB genomes, but the homology between SS genome and AA genome was clearly lower than that between the SS genome and BB genome.     

A simple method for enhancing hybridization efficiency in chromosome and array comparative genomic hybridization

Abstract

The accuracy of comparative genomic hybridization (CGH) analysis is affected by hybridization efficiency. We describe here a simple method for enhancing hybridization efficiency. The hybridization procedure is essentially the same as that of conventional methods. Hybridization solution containing denatured DNA probe mixture was applied to a metaphase chromosome slide or DNA chip slide and covered with a coverslip. In the new method, however, the slide was inverted by turning the coverslip downward prior to hybridization. We termed this method the inverted slide method. To estimate the efficiency of the new method, metaphase chromosome slides and DNA chip slides were treated by both the conventional and inverted slide methods and incubated in a moist chamber at 37°C for 12, 24, 48, and 72 h. Hybridization signals were approximately 1.5 to 2 times brighter on the slides using the inverted slide method than those using the conventional method after 48 and 72 h of incubation. Furthermore, topographical differences in fluorescence intensity were smaller in slides using the inverted-slide method than in those prepared by the conventional method. The inverted slide method is methodologically very simple and improves the resolution of CGH.     

Genome-wide comparative analysis of NBS-encoding genes between Brassica species and Arabidopsis thaliana

Background

Plant disease resistance (R) genes with the nucleotide binding site (NBS) play an important role in offering resistance to pathogens. The availability of complete genome sequences of Brassica oleracea and Brassica rapa provides an important opportunity for researchers to identify and characterize NBS-encoding R genes in Brassica species and to compare with analogues in Arabidopsis thaliana based on a comparative genomics approach. However, little is known about the evolutionary fate of NBS-encoding genes in the Brassica lineage after split from A. thaliana.

Results

Here we present genome-wide analysis of NBS-encoding genes in B. oleracea, B. rapa and A. thaliana. Through the employment of HMM search and manual curation, we identified 157, 206 and 167 NBS-encoding genes in B. oleracea, B. rapa and A. thaliana genomes, respectively. Phylogenetic analysis among 3 species classified NBS-encoding genes into 6 subgroups. Tandem duplication and whole genome triplication (WGT) analyses revealed that after WGT of the Brassica ancestor, NBS-encoding homologous gene pairs on triplicated regions in Brassica ancestor were deleted or lost quickly, but NBS-encoding genes in Brassica species experienced species-specific gene amplification by tandem duplication after divergence of B. rapa and B. oleracea. Expression profiling of NBS-encoding orthologous gene pairs indicated the differential expression pattern of retained orthologous gene copies in B. oleracea and B. rapa. Furthermore, evolutionary analysis of CNL type NBS-encoding orthologous gene pairs among 3 species suggested that orthologous genes in B. rapa species have undergone stronger negative selection than those in B .oleracea species. But for TNL type, there are no significant differences in the orthologous gene pairs between the two species.

Conclusion

This study is first identification and characterization of NBS-encoding genes in B. rapa and B. oleracea based on whole genome sequences. Through tandem duplication and whole genome triplication analysis in B. oleracea, B. rapa and A. thaliana genomes, our study provides insight into the evolutionary history of NBS-encoding genes after divergence of A. thaliana and the Brassica lineage. These results together with expression pattern analysis of NBS-encoding orthologous genes provide useful resource for functional characterization of these genes and genetic improvement of relevant crops.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-3) contains supplementary material, which is available to authorized users.     

芸薹属多倍体植物基因组进化的RAPD分析

多倍化是促进高等植物发生进化的重要力量。为了更清楚地了解多倍体在形成之后其基因组是如何进化的,利用38个随机引物对芸薹属Brassica L.禹氏三角（U’Triangle）中的多倍体物种及其祖先二倍体物种进行了研究。根据扩增出的273条带计算了遗传距离,并用UPGMA法进行了聚类分析。结果发现,二倍体物种B.campestris（AA）与B.oleracea（CC）的亲缘关系比与B.nigra（BB）的要近;异源多倍体B.napus（AACC）比起其二倍体祖先之一B.campestris（AA）与另一个     

芸薹属A，B和C基因组之间关系研究进展

芸薹属A,B和C基因组之间的亲缘关系近年来取得了很大进展,大量细胞遗传学和分子生物学的研究结果表明A和C基因组之间的亲缘关系较A和B基因组以及B和C基因组之间更为接近。A,B和C基因组之间的比较基因组结果表明,这3个基因组是由更加原始物种进化而来的。在芸薹属基因组演化过程中发生了大量的染色体变异,如重复、缺失、重排等,从而造成了现在不同基因组之间的差别。最后,文章对芸薹属不同基因组和拟南芥基因组之间的亲缘关系进行了综述。     

Somatic hybrids with substitution type genomic configuration TCBB for the transfer of nuclear and organelle genes from Brassica tournefortii TT to allotetraploid oilseed crop B. carinata BBCC

Oilseed crop Brassica carinata BBCC is a natural allotetraploid of diploid species B. nigra BB and B. oleracea CC. To transfer the nuclear and organelle genes in a concerted manner from an alien species, B. tournefortii TT, to B. carinata, we produced somatic hybrids with genomic configuration TCBB using B. nigra and B. oleracea stocks that carried selectable marker genes. B. tournefortii TT was sexually crossed with hygromycin-resistant B. oleracea CC. Protoplasts isolated from shoot cultures of hygromycin-resistant F₁ hybrids of B. tournefortiixB. oleracea TC were fused with protoplasts of kanamycin-resistant B. nigra BB. In two different fusion experiments 80 colonies were obtained through selection on media containing both hygromycin and kanamycin. Of these, 39 colonies regenerated into plants. Analysis of 15 regenerants by random amplified polymorphic DNA (RAPD) markers showed the presence of all three genomes, thereby confirming these to be true hybrids. Restriction fragment length polymorphism (RFLP) analysis of organelle genomes with heterologous chloroplast (cp)and mitochondrial (mt) DNA probes showed that the chloroplast genome was inherited from either of the two parents while mitochondrial genomes predominantly showed novel configurations due to either rearrangements or intergenomic recombinations. We anticipate that the TCBB genomic configuration will provide a more conducive situation for recombination between the T and C genomes during meiosis than the TTCCBB or TCCBB type configurations that are usually produced for alien gene transfer. The agronomic aim of producing TCBB hybrids is to transfer mitochondrial genes conferring cytoplasmic male sterility and nuclear genes for fertility restoration from B. tournefortii to B. carinata.     

Genome constitution of Elymus tangutorum (Poaceae: Triticeae) inferred from meiotic pairing behavior and genomic in situ hybridization

Elymus tangutorum (Nevski) Hand.-Mazz (2n = 6x = 42) is a perennial species in the tribe Triticeae, which distributes in Nepal and north and northwest China. However, the genome constitution of E. tangutorum is controversial and its taxonomic status is not clear. Hybridizations of E. tangutorum were carried out with E. wawawaiensis J. R. Carlson & Barkworth (StH), Roegneria grandis Keng (StY), and E. dahuricus Turcz. ex Griseb. (StYH). Meiotic pairing in the hybrids E. tangutorum × E. wawawaiensis (StH), E. tangutorum × R. grandis (StY), and E. tangutorum × E. dahuricus (StYH) averaged 10.48, 11.12, and 20.92 bivalents per cell, respectively. The results suggested that E. tangutorum is an allohexaploid and contains the StYH genomes. Results of genomic in situ hybridization analysis strongly supported the chromosome pairing data. Therefore, E. tangutorum should be treated as Campeiostachys dahurica var. tangutorum (Nevski) B. R. Baum, J. L. Yang & C. Yen. Intergenomic rearrangements of E. tangutorum may be affected by environmental factors.