Genomic in situ hybridization (GISH) analysis of intergeneric hybrids in Colchicaceae

Some intergeneric hybrids produced by the crosses among several colchicaceous ornamentals, Gloriosa superba ??Lutea?? (2n = 2x = 22), G. ??Marron Gold?? (2n = 4x = 44), G. ??Verschild?? (2n = 7x = 77), Littonia modesta Hook. (2n = 2x = 22), and Sandersonia aurantiaca Hook. (2n = 2x = 24), were subjected to genomic in situ hybridization (GISH) analysis in order to clarify their genome constitutions. Chromosome preparation was made from root tip cells of L. modesta × G. superba ??Lutea??, L. modesta × S. aurantiaca, S. aurantiaca × G. superba ??Lutea??, L. modesta × G. ??Marron Gold??, S. aurantiaca × G. ??Marron Gold?? and G. ??Verschild?? × S. aurantiaca. Total DNA of one parent was labeled with digoxigenin or biotin and used as probe, and chromosomes were counterstained with 4??-6-diamidono-2-phenylindole (DAPI). For all the nine intergeneric hybrids, chromosomes from each parent could be clearly distinguished by GISH analysis. Thus GISH analysis is a powerful tool for identifying the genome constitution of intergeneric hybrids in colchicaceous ornamentals. The results obtained by GISH analysis study may be important for further progress in breeding of colchicaceous ornamentals.     

Intra- and intergenomic relationships in interspecific hybrids between Brassica (B. rapa, B. napus) and a wild species B. maurorum as revealed by genomic in situ hybridization (GISH)

Interspecific hybridization plays a crucial role in plant genetics and breeding. The efficiency of interspecific crosses to a considerable extent depends on the genetic relatedness of genomes from parental species. Interspecific hybrids involving Brassica maurorum (2n = 16, MM) and two Brassica crop species, viz B. rapa (2n = 20, AA) and B. napus (2n = 38, AACC), were produced and analyzed for their meiotic chromosome pairings in pollen mother cells (PMCs) by using genomic in situ hybridization (GISH) with the labeled DNA of B. maurorum (MM) as probe. In hybrids B. maurorum × B. rapa (2n = 18, MA), all chromosomes remained unpaired in 28% PMCs, and the maximum of autosyndetic bivalents was two and one among the chromosomes of A and M genomes, with the average per cell being 0.27 and 0.12, respectively. Up to two allosyndetic bivalents between A and M genomes appeared, averagely 0.48 per cell. In hybrids B. maurorum × B. napus (2n = 27, MAC), the maximum of autosyndetic bivalents in M genome was two and the average was 0.11, while the maximum of allosyndetic bivalents between M and A/C genomes was two and the average was 0.78. The 2–7 bivalents formed by A/C-genome chromosomes showed their high homology. The results were compared and discussed with the chromosome pairings in the hybrids of B. maurorum with B. juncea and B. carinata with respect to the genome relationships and the potential for chromosome recombination.     

Detection of wheat-barley translocations by genomic in situ hybridization in derivatives of hybrids multiplied in vitro

Wheat-barley translocations were identified by genomicin situ hybridization (GISH) in backcross progenies originating from in vitro regenerated wheat (Triticum aestivum L. cv. Chinese Spring) × barley (Hordeum vulgare L. cv. Betzes) hybrids. The regenerated hybrids were pollinated with the wheat line Martonvásári 9 kr1. Five translocated wheat-barley chromosomes were recovered among 51 BC₂F₂ progeny from the in vitro regenerated wheat × barley hybrids. All were single breakpoint translocations with the relative positions of the breakpoints ranging from the centromere to about 0.8 of the relative arm length. Of the four translocations with intercalary breakpoints, three were transfers of terminal barley segments to wheat chromosomes; one was a transfer of a terminal wheat segment to a barley chromosome. Because of the absence of diagnostic N-bands, the identity of three barley segments could not be determined; in one translocation the barley chromosome involved had a NOR so it must have been 5H or 6H, and the centric translocation was 4HS.2BL. Following selfing, homozygotes of four translocations were selected. The experiment suggests that in vitro culture conditions are conducive for major genome rearrangements in wheat-barley hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production and meiotic pairing of intergeneric hybrids of Triticum × Dasypyrum species

Summary Crossing experiments between Dasypyrum villosum (2 x) and 14 taxa of Triticum sensu lato (seven diploids, five tetraploids and two hexoploids) were performed. Adult hybrids were obtained in all but three of the combinations with diploid species. A haploid plant was obtained from the combination T. aestivum x D. villosum. Cytogenetic data on the meiotic pairing in the intergeneric hybrids revealed that, in general, very little pairing occurred between the V genome of Dasypyrum and the different genomes in Triticum. There may be comparatively large differences in pairing behaviour in hybrids including different parental accessions, which shows that the Triticum as well as the Dasypyrum genotypes may influence the pairing. The combination T. aestivum x D. hordeaceum (4 x) was also produced and from the meiotic pairing in the hybrid it is evident that D. hordeaceum is an autoploid.     

Genome differentiation between Lycopersicon esculentum and L. pennellii as revealed by genomic in situ hybridization

The interaction between the Se1 and the Ef1 loci, which chiefly control the photoperiod sensitivity (PS) and the basic vegetative growth (BVG) period of rice (Oryza sativa L.) respectively, was investigated using four tester lines different in genotype for the two heading time loci from each other. The four tester lines were grown under 10, 13, 14, 15, and16h day lengths to estimate their BVG period and PS. The Taiwanese cultivar Taichung 65(T65), one of the tester lines, has an extremely long BVG period that has been considered to be conferred by a late heading-time allele ef1 at the Ef1 locus. Experimental results, however, showed that the extremely long BVG of T65was conferred not by a single effect ofef1 but by a complementary effect ofef1 and Se1-e, a photoperiod insensitivity allele, at theSe1 locus. It was also found that a complementary effect of a PS allele Se1-n at the Se1 locus and ef1stimulates the PS of rice. Gene analysis for heading time under an optimum daylength (10 h) as well as under natural day length confirmed the presence of the complementary effect of the two nonallelic genes on BVG, which was found only with homozygosity of both the genes. Based on these results and earlier reports on the Se1 locus, the roles of the Se1 andEf1 loci on the durations of pre-flowering developmental phases in rice were discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Alteration of the genomic composition of Solanum nigrum (+) potato backcross derivatives by somatic hybridization: selection of fusion hybrids by DNA measurements and GISH

Fusion experiments were performed with a first (BC₁‐6738) and a second (BC₂‐9017) generation backcross hybrid of 6x Solarium nigrum (+) 2x potato somatic hybrids with potato cultivars. Because no progeny was obtained from the BC₂ genotypes, alternative approaches were sought to overcome the sexual crossing barrier. Five potato genotypes, one of which contains the hygromycin resistance gene, were used in the fusion experiments. All vigorous regenerants were used for the estimation of nuclear DNA content using flow cytometry. Plants with a DNA content higher than that of the BC₁‐6738 or BC₂ genotypes were considered potential somatic hybrids. Forty‐nine potential somatic hybrids resulted from fusion experiments with BC₁‐6738, from which 20 grew vigorously in the greenhouse and flowered. After pollination with several 4x potato cultivars, eight genotypes produced seeded berries and five genotypes gave seedless berries. In addition, 11 of these 13 somatic hybrids were selected for genomic in situ hybridization (GISH) analysis to determine their genomic composition. Nine had exactly or approximately the expected number of 36 S. nigrum and 60 potato chromosomes. In one genotype, only 22 instead of 36 S. nigrum chromosomes were found and one potato chromosome was possibly missing. Only five potential somatic hybrids were detected among the 79 regenerants from BC₂‐9017 (+) 2x potato fusion experiments that were analysed by flow cytometry. Two of these hybrids were rather vigorous and did flower, but pollinations with potato have not yet set any berries.     

Characterization of Triticum aestivum/Psathyrostachys juncea derivatives by genomic in situ hybridization

Using the genomic in situ hybridization (GISH) technique, one translocation line, seven translocation-addition lines, five translocation plus translocation addition lines and two ditelosomic addition lines were identified in backcross progenies of Triticum aestivum L. -Psathyrostachys juncea (Fisch.) Nevski intergeneric hybrids. No complete P. juncea chromosomes were detected in the 25 lines studied. The results suggest that intact P. juncea chromosomes may be difficult to isolate in a wheat background. This revised version was published online in July 2006 with corrections to the Cover Date.     

Meiotic behavior in Brachiaria humidicola (Poaceae) hybrids

In the genus Brachiaria, genetic variation can be exploited directly from germplasm collections or released using sexual reproduction in normally apomictic polyploids. The discovery of a natural sexual polyploid accession H031 of Brachiaria humidicola collected in Africa, opened new opportunities to exploit the genetic variation in this species. This accession was crossed with an apomictic cultivar BRS Tupi with the same chromosome number (2n = 36) and 361 F₁ hybrids were obtained. Following visual selection for leafiness, vigor, growth habit, and the mode of reproduction, 50 hybrids were selected for further agronomic evaluation. The parents and 45 of the 50 selected hybrids were evaluated for the regularity of meiosis. In the female parent (H031), meiosis was somewhat irregular, with 16.3% of abnormal tetrads, whereas the male (cv. BRS Tupi) meiosis was very regular, with only 3.1% of abnormal tetrads. Among hybrids (sexual and apomictic), the percentage of abnormal tetrads ranged from 15.8 to 98.3%. The abnormalities included irregular chromosome segregation, chromosome stickiness and the absence of cytokinesis. Considering that apomixis in the genus Brachiaria is pseudogamic, and that meiotic aberrations can compromise pollen viability, the results of this study present another parameter to aid selection for more stable microsporogenesis. Apomictic derivatives with stable meiosis are candidates for new cultivars whereas sexual hybrids can be retained in breeding for another round of recombination.     

Meiotic behavior in interspecific hybrids between Brachiaria ruziziensis and Brachiaria brizantha (Poaceae)

The meiotic behavior of two half-sib interspecific tetraploid (2n = 4x = 36) promising hybrids, a sexual and an apomictic one, from crosses B. ruziziensis and B. brizantha, was evaluated. Although chromosome paired predominantly as bivalents, a few tri- and quadrivalents were recorded. Results suggest that B. brizantha and B. ruziziensis are closely related and genetic recombination is expected in hybrids. Introgression of specific target genes from B. ruziziensis into B. brizantha and vice-versa may be foreseen. However, abnormalities such as irregular chromosome segregation, chromosome stickiness and abnormal cytokinesis reported in these hybrids affect pollen fertility. More than 65% of pollen grains are sterile. Since the distinctive cytological feature of these hybrids is abnormal cytokinesis, this fact suggests that both parental genomes are unable to coordinate their activities with regard to this cytological phenomenon. Deployment of such hybrids in the process of developing varieties is discussed.     

Cytoplasmic male sterility in ryegrasses (Lolium SPP.) detected after intergeneric hybridization

Summary Among diploid derivatives of a tetraploid intergeneric hybrid originating from a cross between a diploid F₄ hybrid of Lolium perenne x L. multiflorum and an autotetraploid plant of Festuca pratensis as female and male parent, respectively, a great number of male-sterile Lolium plants have been found. The male sterility appears to be based on an interaction of one or two recessive nuclear genes and sterilizing cytoplasm. The data available indicate a high frequency of B types (O types) in perennial ryegrass.     

The origin of Darwin hybrid tulips analyzed by flow cytometry, karyotype analyses and genomic in situ hybridization

Chromosome morphology was studied in diploid cultivars of Tulipa fosteriana and T. gesneriana (2n = 2x = 24) and triploid Darwin hybrids (2n = 3x = 36) developed from interspecific crosses of T. gesneriana and T. fosteriana. Chromosomes were arranged in the karyotype according to decreasing total length. Based on our karyotypic analysis, we propose that median chromosomes may serve as markers for diploid genotypes. Discriminant analysis with respect to total chromosome length and short arm length showed a significant difference between the size of the larger median chromosomes of T. gesneriana and T. fosteriana Comparison of median chromosome length in Darwin hybrid tulips showed that two larger chromosomes and one smaller chromosome were derived from T. gesneriana and T. fosteriana, respectively. This finding was clearly and unambiguously confirmed by simultaneous hybridization of differentially labeled genomic probes of T. fosteriana and T. gesneriana to metaphase chromosomes of the triploid cultivar ‘Yellow Dover’, thereby enabling us to distinguish between the 24 chromosomes derived from T. gesneriana and 12 chromosomes derived from T. fosteriana. Thus, genomic in situ hybridization and median chromosome analyses can be useful to identify the genome constitution of triploid Darwin hybrid tulips. In addition, their hybridity was readily verified by flow cytometry using vegetative tissue of Darwin hybrid tulips. Our results clarify the process of Tulipa cultivar formation and will be useful for interspecific hybridization breeding.The first and second author have contributed equally to this paper     

Effect of the ph1b mutant on chromosome pairing in hybrids between Dasypyrum villosum and Triticum aestivum

Chromosome pairing was analysed in F₁ hybrids of the wheat cultivar ‘Chinese Spring’ (CS) and its ph1b mutant (CSphlb) with Dasypyrum villosum. On average, 1.61 chromosomes per cell paired in the hybrid CS ×D. villosum, but 14.43 in the hybrid CS ph1b×D. villosum. Genomic fluorescence in situ hybridization (GISH) revealed three types of homoeologous association between wheat (W) and D. villosum (D) chromosomes (W‐D, D‐W‐W and D‐W‐D) in pollen mother cells of the CS ph1b×D. villosum hybrid, and only one type (W‐W), in the CS ×D. villosum hybrid. Both F₁ hybrids were self‐sterile. The seed set of the backcross of CS ×D. villosum with CS was 6.67% and that of CS ph1b×D. villosum with CS or CS ph1b was only 0.45%. The chromosome number of BC₁ plants varied from 48 to 72. Translocations of chromosome segments or entire arms between wheat and D. villosum chromosomes were detected by GISH in the BC₁ plants from the backcross of CS ph1b×D. villosum to CS ph1b.     

Cytogenetic analysis of F1, F2 and BC1 plants from intergeneric sexual hybridization between Sinapis alba and Brassica oleracea by genomic in situ hybridization

By intergeneric sexual hybridization between Sinapis alba and Brassica oleracea , F₁, F₂ and BC₁ progeny plants were produced. S. alba plants (genome SS, 2n = 24) were pollinated with B. oleracea (genome CC, 2n = 18), and the fertile F₁ plants were pollinated with B. oleracea to obtain BC₁ plants. GISH analysis showed that 10 out of 12 F₁ plants had 12 S. alba chromosomes (one full S chromosome set) and nine B. oleracea chromosomes (one C chromosome sets), representing the expected hybrids. However, two F₁ plants had 12 S chromosomes and 18 C chromosomes (two C chromosome sets), indicating unexpected hybrids. A maximum of three trivalents between C and S chromosomes were identified at metaphase I of semi-fertile F₁ pollen mother cells (PMCs), which indicates homology and chromosome pairing between these two genomes. The C genome had obviously been doubled in two F₂ plants from selfed semi-fertile F₁ plants. BC₁ plants consisted of 18 C chromosomes and different numbers of one, five and six additional S chromosomes, respectively. Monosomic alien addition lines developed in the present study can be used for B. oleracea breeding and Sinapis alba gene mapping.     

Maternal inheritance of cytoplasmic organelles in intergeneric hybrids of Carica papaya L. and Vasconcellea spp. (Caricaceae Dumort., Brassicales)

The mode of inheritance of chloroplast and mitochondrial DNA has been determined in intergeneric hybrids between C. papaya and four different Vasconcellea species by employing a restriction fragment length polymorphism analysis of a PCR-amplified chloroplast and mitochondrial DNA region. Artificial F₁ hybrids were produced between a female specimen of C. papaya and male specimens of either V. parviflora, V. goudotiana, V. cundinamarcensis or V. quercifolia. The hybridization patterns of all hybrids correspond in all cases with that of the C. papaya mother, and are different from that of the paternal Vasconcellea species, thus indicating the maternal inheritance of cpDNA and mtDNA in intergeneric hybrids between C. papaya and wild relatives of the Vasconcellea genus.     

Progenies of allotriploids of Oriental × Asiatic lilies (Lilium) examined by GISH analysis

With the aim of utilizing allotriploid (2n = 3x = 36) lily hybrids (Lilium) in introgression breeding, different types of crosses were made. First, using diploid Asiatic lilies (2n = 2x = 24), reciprocal crosses (3x − 2x and 2x − 3x) were made with allotriploid hybrids (AOA) obtained by backcrosses of F₁ Oriental × Asiatic hybrids (OA) to Asiatic cultivars (A). Secondly, the AOA allotriploids were crossed with allotetraploid (OAOA, 2n = 4x = 48), in 3x − 4x combination. Finally, the AOA allotriploids where crossed to 2n gamete producer F₁ OA hybrids (3x − 2x (2n)). Two types of triploids were used as parents in the different types of crosses, derived from: (a) mitotic polyploidization and (b) sexual polyploidization. Ploidy level of the progeny was determined by estimating the DNA values through flowcytometry as well as chromosome counting. The aneuploid progeny plants from 3x − 2x and reciprocal crosses had approximate diploid levels and in 3x − 4x crosses and 3x − 2x (2n) the progeny had approximate tetraploid levels. Balanced euploid gametes (x, 2x and 3x) were formed in the AOA genotypes. Recombinant chromosomes were found in the progenies of all crosses, except in the case of 2x − 3x crosses through genomic in situ hybridization (GISH) analyses. Recombinant chromosomes occurred in the F₁ OA hybrid when the triploid AOA hybrid was derived through sexual polyploidization, but not through mitotic polyploidization with two exceptions. Those recombinant chromosomes were transmitted to the progenies in variable frequencies.     

Enhanced grain yield in rice hybrids through complementation of fertility restoration by Rf3 and Rf4 genes as revealed by multilocation evaluation of tropical japonica derived rice (Oryza sativa) hybrids

Thirty-one tropical japonica derived Rf gene carrying rice hybrids were classified into three classes as Rf3, Rf4 and Rf3 + Rf4 hybrids. These hybrids were tested under three different mega-environments. Between Rf3 class and both the classes of hybrids possessing Rf4 genes, significant variation for spikelet and pollen fertility and grain yield was found. The pollen fertility was five times higher among Rf4 hybrids than that of hybrids carrying Rf3 alone. Likewise, spikelet fertility among Rf4 hybrids was two times higher than that of Rf3 hybrids. Parallel grain yield increase in Rf4 hybrids was 2.4 times than in Rf3 hybrids. However, Rf3 gene was found complementing Rf4 to truncate the range of pollen and spikelet fertility. Hybrids possessing Rf3 alone exhibited partial pollen and spikelet sterility, and significant negative standard heterosis for grain yield. The high yielding hybrid, “HYB36” carrying both the Rf genes was found to be widely adapted. The present study established that Rf4 gene is essential either alone or in combination with Rf3 for fertility restoration to achieve enhanced grain yield in WA-CMS based hybrids.     

水仙荧光原位杂交体系的建立

摘要：建立以水仙染色体为靶、rDNA为探针的荧光原位杂交实验技术,为进一步利用荧光原位杂交技术分析水仙的亲缘和进化关系奠定了基础。水仙的染色体制片以去壁低渗-火焰干燥法较好,容易获得大量清晰、分散的有丝分裂中期相;切刻平移法地高辛标记探针、染色体和探针共变性90℃ 5 min能有效的进行水仙rDNA的染色体荧光原位杂交定位。     

Somatic chromosome elimination and meiotic chromosome pairing in the triple hybrid 6x-(Solanum acaule × S. bulbocastanum) × 2×-S. phureja

Summary Two selected hexaploid F₁ clones from the cross Solanum acaule x S. bulbocastanum were intercrossed and the resulting hybrid plants pollinated with the diploid species S. phureja, in order to obtain tetraploid triple hybrids with the same ploidy level as S. tuberosum cultivars (2n=4x=48).Apart from three trihaploids a large population of triple hybrids was obtained, showing chromosome mosaicism in root tip cells (euploid + hypoploid chromosome numbers) and a uniform, mostly hypoploid chromosome number in the pollen mother cells. It is demonstrated that somatic chromosome elimination in the early stages of development is the most probable cause.From detailed pachytene observations as well as from the chromosome associations observed at metaphase I it is evident, that there is normal pairing between the four genomes in the triple hybrids. Although S. bulbocastanum is a quite distinct species with a rigid crossability barrier with S. phureja, the chromosomes of these two species appear to have a high degree of homology. Especially the formation of quadrivalents involving all twelve groups of four homeologous chromosomes, indicated that the four parental genomes (two from S. acaule, one from S. bulbocastanum and one from S. phureja) are not differentiated to the extent of affecting normal pairing and chiasma formation. These results support the view, that the transfer of valuable characters from S. bulbocastanum to S. tuberosum cultivars is feasible even when these characters are polygenically controlled.