RFLP Analysis of Progeny from Hybrid of Oryza alta×O.sativa

Oryza alta Swallen is a species of allotetraploid wild rice (CCDD, 2n = 48) with high biomass production and resistant to several insects and diseases. The high polymorphism (RFLP) between O. alta and O. sativa L. (average 90%) reveals the long phylogenical distance between these two species which may impede the crossing. The chromosome constitutions of several progeny from O. alta × O. sativa were analyzed with 22 mapped rice RFLP markers. The results showed that almost all triploid sterile plants which had 36 chromosomes were, as expected, composed of the chromosomes from two parents. But the two partial fertile regenerated plants from 02428 (O. sativa) × WHS601-2 (O. alta) which had only 24 and 25 chromosomes respectively, in most cases, had RFLP patterns similiar to their O. sativa parent. Therefore the chromosomes constituted their genomes were mainly from O. sativa or have high homology with the chromosomes of O. sativa. It seems evident that chromosome elimination had occurred during the wide hybridization as O. alta RFLP patterns could be found in them. Furthermore, that the introgression of O. alta chromosome fragment into that of O. sativa in an F2 plant was indicative that some kind of recombination also occurred. Regarding the rare chromosome pairing in PMCs, the probability of the existence of a nonconventional mechanism for the wild rice chromosomes (chromosome fragments ) to introgressing into cultivated rice genomes in a relatively short time was discussed.     

RFLP analysis of the progeny from Oryza alta Swallen x Oryza sativa L.

RFLP analyses were carried out in the progeny from a cross of two phylogenetically distant rice species, wild rice Oryza alta Swallen (CCDD, 2n = 48) and cultivated rice O. sativa L. (AA, 2n = 24). The sterile plants gave heterozygous RFLP patterns at most of the loci detected. They looked more like their wild rice parent, with 36 chromosomes in their root-tip cells and pollen mother cells. In two partially fertile plants, however, most of the markers that were used showed RFLP patterns similar to the cultivated parent, O. sativa. By cytological study, it was found that nearly one-third of the chromosomes had been eliminated in the partially fertile plants. Their seeds have short awns, which is a characteristic of their wild parent, O. alta. An introgression occurred in one of the partially fertile plants, which led to the discussion about a nonconventional mechanism in wide hybridization for transference of wild rice chromosome segments to cultivated rice chromosomes.     

Characterization of six wheat x Thinopyrum intermedium derivatives by GISH, RFLP, and multicolor GISH.

Restriction fragment length polymorphism (RFLP) analysis and multicolor genomic in situ hybridization (GISH) are useful tools to precisely characterize genetic stocks derived from crosses of wheat (Triticum aestivum) with Thinopyrum intermedium and Thinopyrum elongatum. The wheat x Th. intermedium derived stocks designated Z1, Z2, Z3, Z4, Z5, and Z6 were initially screened by multicolor GISH using Aegilops speltoides genomic DNA for blocking and various combinations of genomic DNA from Th. intermedium, Triticum urartu, and Aegilops tauschii for probes. The probing (GISH) results indicated that lines Z1 and Z3 were alien disomic addition lines with chromosome numbers of 2n = 44. Z2 was a substitution line in which chromosome 2D was substituted by a pair of Th. intermedium chromosomes; this was confirmed by RFLP and muticolour GISH. Z4 (2n = 44) contained two pairs of wheat--Th. intermedium translocated chromosomes; one pair involved A-genome chromosomes, the other involved D- and A- genome chromosomes. Z5 (2n = 44) contained one pair of wheat--Th. intermedium translocated chromosomes involving the D- and A-genome chromosomes of wheat. Z6 (2n = 44) contained one pair of chromosomes derived from Th. intermedium plus another pair of translocated chromosomes involving B-genome chromosomes of wheat Line Z2 was of special interest because it has some resistance to infection by Fusarium graminearum.     

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.     

RFLP analysis of rice (Oryza sativa L.) introgression lines

Summary Fifty-two introgression lines (BC₂F₈) from crosses between two Oryza sativa parents and five accessions of O. officinalis were analyzed for the introgression of O. officinalis chromosome segments. DNA from the parents and introgression lines was analyzed with 177 RFLP markers located at approximately 10-cM intervals over the rice chromosomes. Most probe/enzyme combinations detected RFLPs between the parents. Of the 174 informative markers, 28 identified putative O. officinalis introgressed chromosome segments in 1 or more of the introgression lines. Introgressed segments were found on 11 of the 12 rice chromosomes. In most cases of introgression, O. sativa RFLP alleles were replaced by O. officinalis alleles. Introgressed segments were very small in size and similar in plants derived from early and later generations. Some nonconventional recombination mechanism may be involved in the transfer of such small chromosomal segments from O. officinalis chromosomes to those of O. sativa. Some of the introgressed segments show association with genes for brown planthopper (BPH) resistance in some introgressed lines, but not in others. Thus, none of the RFLP markers could be unambiguously associated with BPH resistance.     

Origin of an alien disomic addition with an aberrant homologue of chromosome-10 of tomato and its meiotic behaviour in a potato background revealed through GISH

 While characterising potato (Solanum tuberosum, 2n=4x=48) clones with alien tomato (Lycopersicon esculentum) chromosome additions, a single addition for chromosome-10 of tomato was identified through restriction fragment length polymorphism (RFLP) analysis. This plant, 2101–1, was a BC₂ derivative from a cross between a potato (+) tomato fusion hybrid backcrossed to potato. Cytological analysis of its somatic chromosomes through genomic in situ hybridisation (GISH) indicated the presence of four genomes of potato with two alien tomato chromosomes, of which one was much smaller than the other. Analysis of chromosome pairing at the pachytene and metaphase-I stages of microsporogenesis indicated that the large and small chromosomes were homologues. Thus, it was a disomic addition for chromosome-10 of tomato. The size difference was found to be due to a deletion. Fluorescent in situ hybridisation (FISH) experiments, using the telomeric repeat pAtT4 from Arabidopsis thaliana and the sub-telomeric repeat TGRI, showed intact telomeres and sub-telomeres for both alien chromosomes. Thus, the deletion that the smaller of the homologues suffered was interstitial and most probably occurred in the centromeric heterochromatic region of the long arm. The pattern of distribution of large and small chromosomes to telophase-II nuclei during microsporogenesis indicated that the deletion did not affect the meiotic behaviour of the smaller chromosome. In contrast, the frequencies of transmission of the large and the small chromosomes through the female parent, estimated in 96 BC₃ progeny of plants by RFLP and GISH analyses, appeared to be very different, 69.2% and 3.8% respectively. This study also provides evidence that two different chromatids of a pair of homologues, rather than two chromatids of a single chromosome, are most likely to be involved in the origin of a disomic. The aberrant chromosome can be used for the physical mapping of chromosome-10. Received: 9 June 1998 / Accepted: 28 October 1998     

小偃麦附加系Z1和Z2中外源染色体2Ai-2的结构组成

小偃麦附加系Z1和Z2中外源染色体2Ai-2的结构组成@张增燕$中国农业科学院作物育种栽培研究所!北京100081@辛志勇$中国农业科学院作物育种栽培研究所!北京100081@陈孝$中国农业科学院作物育种栽培研究所!北京100081小偃麦;;附加系;;染色体     

Mapping RFLP Loci in Maize Using B-a Translocations

Plants hypoploid for specific segments of each of the maize (Zea mays L.) chromosomes were generated using 24 different B-A translocations. Plants carrying each of the B-A translocations were crossed as male parents to inbreds, and sibling progeny hypoploid or not hypoploid for specific chromosomal segments were recovered. Genomic DNAs from the parents, hypoploid progeny, and nonhypoploid (euploid or hyperploid) progeny for each of these B-A translocations were digested with restriction enzymes, electrophoresed in agarose gels, blotted onto reusable nylon membranes, and probed with nick-translated, cloned DNA fragments which had been mapped previously by restriction fragment length polymorphism (RFLP) analysis to the chromosome involved in the B-A translocation. The chromosomal segment on our RFLP map which was uncovered by each of the B-A translocations was determined. This work unequivocally identified the short and long arms of each chromosome on this map, and it also identified the region on each chromosome which contains the centromere. Because the breakpoints of the B-a translocations were previously known on the cytological and the conventional genetic maps, this study also allowed this RFLP map to be more highly correlated with these maps.     

Construction and molecular and cytogenetic analyses of euploid (2n = 42) and telocentric addition (2n = 42 + 2t) alloplasmic lines (Hordeum marinum subsp gussoneanum)-Triticum aestivum

Individual plants from the BC1F5 and BC1F6 backcross progenies of barley--wheat (= H. geniculatum All.) (2n = 28) x T. aestivum L. (2n = 42)] and the BC1F6 progeny of their amphiploids were used to obtain alloplasmic euploid (2n = 42) lines L-28, L-29, and L-49 and alloplasmic telocentric addition (2n = 42 + 2t) lines L-37, L-38, and L-50. The lines were examined by genomic in situ hybridization (GISH), microsatellite analysis, chromosome C-banding, and PCR analysis of the mitochondrial 18S/5S repeat. Lines L-29 and L-49 were characterized by substitution of wild barley chromosome 7H1 for common wheat chromosome 7D. In line L-49, common wheat chromosomes 1B, 5D, and 7D were substituted with homeologous barley chromosomes. Lines L-37, L-38, and L-50 each contained a pair of telocentric chromosomes, which corresponded to barley chromosome arm 7H'L. All lines displayed heteroplasmy for the mitochondrial 18S/5S locus; i.e., both barley and wheat sequences were found.     

Molecular cytogenetic analysis of wheat-barley hybrids using genomic in situ hybridization and barley microsatellite markers.

In the present investigation, genomic in situ hybridization (GISH) and barley microsatellite markers were used to analyse the genome constitution of wheat-barley hybrids from two backcross generations (BC1 and BC2). Two BC1 plants carried 3 and 6 barley chromosomes, respectively, according to GISH data. Additional chromosomal fragments were detected using microsatellites. Five BC2 plants possessed complete barley chromosomes or chromosome segments and six BC2 plants did not preserve barley genetic material. Molecular markers revealed segments of the barley genome with the size of one marker only, which probably resulted from recombination between wheat and barley chromosomes. The screening of backcrossed populations from intergeneric hybrids could be effectively conducted using both genomic in situ hybridization and molecular microsatellite markers. GISH images presented a general overview of the genome constitution of the hybrid plants, while microsatellite analysis revealed the genetic identity of the alien chromosomes and chromosomal segments introgressed. These methods were complementary and provided comprehensive information about the genomic constitution of the plants produced.     

Genetic Mapping of Rice Using RFLP Markers and a Double Haploid Population of a Cross Between indica and japonica Varieties

A genetic linkage map of rice was constructed using a double haploid (DH) population from "Gui 630” (Oryza sativa subsp, indica)/"02428" (O. sativa subsp, japonica, wide compatibility variety) and RFLP markers. It consists of 233 loci and covers rice genomes about 2070 cM (centimorgan), and compares well with the other published rice maps. 25 RFLP markers, 2 telomeres and sh-2 (shattering ability) gene were first located on the molecular map of rice. RFLPs between "Gui 630' and "02428' mainly came from base substitution and a few DNA construction variance, not distributed evenly among chromosomes and on chromosome. This was probably resulted from the difference genetic stability among chromosomes and regions, in exchanging recombination ability in different segments of chromosome.     

Molecular tagging of genes for brown planthopper resistance and earliness introgressed from Oryza australiensis into cultivated rice, O. sativa.

Restriction fragment length polymorphism analysis was carried out to tag the alien genes for brown planthopper (BPH) resistance and earliness introgressed from wild species Oryza australiensis into cultivated rice, O. sativa L. One introgression line (IR65482-4-136-2-2), resistant to biotypes 1, 2, and 3 of BPH and early in flowering, was selected from BC2F4 of the cross between O. sativa (IR31917-45-3-2) and O. australiensis (accession 100882). Recurrent parent, O. australiensis, and introgression line were surveyed for RFLP using probes of chromosomes 10 and 12. Two probes, RG457 and CDO98, detected introgression from O. australiensis. Cosegregation between introgressed characters and molecular markers was studied in F2 derived from the cross between the introgression line and recurrent parent. The gene for BPH resistance is linked with RG457 of chromosome 12 at a distance of 3.68 +/- 1.29 cM, and the gene for earliness is linked with CDO98 of chromosome 10 at a distance of 9.96 +/- 3.28 cM. Such close linkage is useful in marker-based selection while transferring BPH resistance from introgression line into other elite breeding lines. Introgression at the molecular level indicates that the mechanism of alien gene transfer is probably genetic recombination through crossing over rather than substitution of whole or large segment of chromosomes of wild species.     

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

The genomic structures of Oryza sativa （A genome） and O. meyeriana （G genome） were comparatively studied using bicolor genomic in situ hybridization （GISH）. GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa （A-A）, O. sativa-O, meyeriana （A-G） and O. meyeriana-O, meyeriana （G-G）, were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.     

Molecular characterization of a Thinopyrum intermedium group 2 chromosome (2Ai-2) conferring resistance to barley yellow dwarf virus.

The wheat--Thinopyrum intermedium addition lines Z1 and Z2 carry 21 pairs of wheat chromosomes and one pair of Th. intermedium chromosomes (2Ai-2) conferring resistance to barley yellow dwarf virus (BYDV). GISH results using the genomic DNA of Pseudoroegneria strigosa (S genome) as the probe indicated that the 2Ai-2 chromosome in Z1 and Z2 is an S-J intercalary translocation. Most of the 2Ai-2 chromosome belongs to the S genome, except for about one third in the middle region of the long arm that belongs to the J genome. The results of detailed RFLP analyses confirmed that the 2Ai-2 chromosome is extensively homoeologous to wheat group 2 chromosomes. Some new RFLP markers specific to the 2Ai-2 chromosome were identified. A RAPD marker, OP-R16(340), specific to the 2Ai-2 chromosome, was screened. We converted the RAPD marker into a sequence-characterized amplified region (SCAR) marker (designated SC-R16). The study establishes the basis for selecting translocation lines with small segments of the 2Ai-2 chromosome and localizing the BYDV resistance gene when introgressed into a wheat background.     

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     

Characterization of interspecific hybrids and backcross progenies from a cross between Oryza minuta and Oryza sativa

Oryza minuta, a tetraploid wild relative of cultivated rice, is an important source for the genetic improvement. Interspecific hybrids were obtained from the cross of O. sativa L. (IR24) and O. minuta (Acc. No. 101133) with 5.58% crossability, which ranged from 0.11% to 1.62% in the backcross generations. The chromosome numbers of the backcross progenies were 24 to 48. Seven yield-related traits of the parents, hybrid F₁, and backcross progenies were evaluated. Simple sequence repeat markers analysis showed that the polymorphism ratio of SSR bands between IR24 and Acc. No. 101133 was 93.2%. The average donor segment number, length, donor genome size, and percentage of donor genome of 92 BC₃F₁ plants (2n=24) were 24.1, 17.8 cM, 438.4 cM and 26.2%, respectively. They were complex variation and uneven among the chromosomes. These introgression lines could be used to identify the favorable genes of O. minuta and provide a new platform for the genetic improvement of cultivated rice.     

Genomic in situ hybridization differentiates between A/D- and C-genome chromatin and detects intergenomic translocations in polyploid oat species (genus Avena).

The genomic in situ hybridization (GISH) technique was used to discriminate between chromosomes of the C genome and those of the A and A/D genomes in allopolyploid oat species (genus Avena). Total biotinylated DNA from A. strigosa (2n = 2x = 14, AsAs genome) was mixed with sheared, unlabelled total DNA from A. eriantha (2n = 2x = 14, CpCp) at a ratio of 1:200 (labelled to unlabelled). The resulting hybridization pattern consisted of 28 mostly labelled and 14 mostly unlabelled chromosomes in the hexaploids. Attempts to discriminate between chromosomes of the A and D genomes in A. sativa (2n = 6x = 42, AACCDD) were unsuccessful using GISH. At least eight intergenomic translocation segments were detected in A. sativa 'Ogle', several of which were not observed in A. byzantina 'Kanota' (2n = 6x = 42, AACCDD) or in A. sterilis CW 439-2 (2n = 6x = 42, AACCDD). At least five intergenomic translocation segments were observed in A. maroccana CI 8330 'Magna' (2n = 4x = 28, AACC). In both 'Ogle' and 'Magna', positions of most of these translocations matched with C-banding patterns.     

萝卜与甘蓝属间杂种基因组原位杂交分析

用基因组原位杂交方法（Genomic in situ hybridization, 简称GISH）研究了萝卜( Raphanus sativus,2n=18,RR)和甘蓝（Brassica oleracea , 2n=18, CC）属间杂种F1减数分裂过程。结果表明杂种体细胞染色体组成为RC,2n=18,但花粉母细胞有三种不同类型：1. RC,2n=18, 终变期染色体平均配对构型为14.87Ⅰ+1.20Ⅱ+0.04Ⅲ+0.06Ⅳ, 染色体配对主要发生在萝卜和甘蓝染色体之间, 后期Ⅰ9条萝卜染色体主要以5/4和6/3的分离比移向两极, 所形成配子的染色体数目和组成均不平衡,配子败育; 2. RRCC,4n=36, 终变期染色体形成18个二价体,后期Ⅰ染色体均衡分离,形成RC不减数配子;3. RRCC缺体,4n=30－34, 少数萝卜染色体丢失,形成的配子具有全套的甘蓝染色体和部分萝卜染色体。     

Molecular and cytogenetic characterization of an Oryza officinalis–O. sativa chromosome 4 addition line and its progenies

The wild species Oryza officinalis Wall. ex Watt (2n = 24, CC) is a valuable genetic resource for rice (O. sativa L., 2n = 24, AA) breeding and genomics research. Genomic in situ hybridization (GISH) and molecular approaches were used to determine the nature and composition of the additional chromosome in a monosomic alien addition line (MAAL) of O. officinalis and its backcross progenies. The extra wild species chromosome in the MAAL (2n = 2x = 25) was a mosaic one, comprising of the long arm of chromosome 4 from O. officinalis and the short arm from O. sativa. Comparative analysis showed that O. sativa and O. officinalis shared high synteny of restriction fragment length polymorphism (RFLP) markers and low synteny of simple sequence repeat (SSR) markers. A DNA methylation alteration was revealed at C619 in the MAAL and progenies. Analysis of progenies of the MAAL indicated that introgression segments were small in size and introgression was not evenly distributed along the long arm. One recombination hot spot between C513 and RG177 was identified, which is in a gene-rich region.     

Transfer of a male-sterility-inducing cytoplasm from onion to leek ( Allium ampeloprasum)

Two interspecific triploid (AAC) hybrids (84/1-94 and 99/1-94) from crosses between onion [ Allium cepa (2 n=2 x=16, CC)] and leek [ A. ampeloprasum (2 n=4 x=32, AAAA)] were backcrossed to leek in order to transfer a male-sterility-inducing cytoplasm from onion that would enable the production of hybrid leek. GISH evaluations of meiosis in the interspecific hybrids revealed irregularities due to univalent onion chromosomes producing micronuclei from onion chromatin, whereas the pairing of the two sets of leek chromosomes was nearly normal. Attempts to use colchicine to double the chromosome number of the hybrids failed. Backcrosses of 84/1-94 to leek as the pollen parent were not successful. The first backcross of 99/1-94 to tetraploid leek produced 11 BC(1) plants with chromosome numbers between 38 and 41. Identification of parental chromosomes by GISH showed that all eight onion chromosomes and 30-33 leek chromosomes were transmitted to the backcross progenies due to unreduced egg cells. Onion chromosomes were eliminated during the second backcross. Southern hybridization confirmed the transfer of the T-cytoplasm like source of CMS from onion to the BC(2) progenies. After the third backcross to leek, 158 plants were obtained with varying numbers of onion chromosomes and some intergenomic recombinant chromosomes. Alloplasmic leek plants without onion chromatin were selected for further characterization of male sterility and quality traits.