Inheritance and Sequence Homology Analysis of the Maize DNA Introgressed into the Wheat Doubled Haploid Plant Through Wheat×Maize Cross

A maize (Zea mays L.) genome-specific repeated DNA sequence (clone MR64) has been transferred into one DH line of wheat through wheat (Triticum persicum Vav. ex Zhuk.) and maize cross. In the present study by RFLP analysis the authors proved that this DNA sequence could stably transmit into DH3 plants, the next generation derived from DH2 self-crossing. A similarity search in all DNA databases using BLASTN program showed that the DNA sequence of MR64 had as high as 93% identity to PREM-2 and 79% to Opie-2 in nucleotides. Both PREM-2 and Opie-2 are known as retrotransposons in maize genome, suggesting that MR64 likely is the partial sequence of a maize retrotransposon. Therefore, the results indicate that some retrotransposon might involve the DNA introgression from maize to wheat genome through wide fertilization. Stable inheritance of this maize genome-specific retrotransposon-like DNA in the wheat genome opens up the possibility of using retrotransposon as a new tool for gene tagging, function analysis, and insertional mutagenesis in wheat genome.     

导入小麦加倍单倍体植株的玉米DNA在后代中的遗传及序列同源性分析

作者曾报道了一个玉米（Ｚｅａ ｍａｙｓ Ｌ．）基因组特异的重复序列ＤＮＡ（克隆ＭＲ６４）通过小麦（Ｔｒｉｔｉｃｕｍ ｐｅｒｓｉｃｕｍ Ｖａｖ．ｅｘ Ｚｈｕｋ．）与玉米杂交导入一个小麦加倍单倍体（ＤＨ）植株中。利用分析主宰该重复ＤＮＡ序列可以稳定地传递到小麦ＤＨ３代植株。通过Ｉｎｔｅｒｍｅｔ在ＤＮＡ数据库中进行序列相似性搜寻和同源性比较,结果显示,ＭＲ６４的ＤＮＡ序列和玉米的最近报道的两个逆转座子Ｐ     

RFLP Variations of Common Wheat Doubled Haploid Progenies from Wheat×Maize Crosses

Wheat ( Triticum aestivum L. ) and maize ( Zea mays L. ) crosses (the chromosome elimination system) can be used to produce frequently a large number of doubled haploid (DH) wheat lines by embryo rescue and doubling treatment. The resulting DH lines are genetically homogeneous. Significant RFLP variations were detected in common wheat DH progenies from wheat and maize crosses by using wheat rDNA clone pta71 and two maize DNA clones (MR13 and MRSO) homologous to wheat genome as probes. The results revealed that the copy number and restriction fragment length of rDNA in some wheat DH progenies was changed, and also that deletion was detected in several DH plants when probed with MR13 and MR5O. In particular, the RFLP pattern of DH line No. 18 was greatly changed using MR13 as a probe. In this line, three new bands, 40.0 kb, 2.5 kb and 2.0 kb emerged while a 4.3 kb intense band from the parental common wheat genome disappeared. This change may be related to a quite large DNA rearrangement within the wheat genomic DNA or an insertion by alien maize DNA fragment.     

由小麦×玉米获得的普通小麦加倍单倍体后代的RFLP变异

用小麦（Ｔｒｉｔｉｃｕｍ　ａｅｓｔｉｖｕｍＬ．）的ｒＤＮＡ克隆ｐＴａ７１和与小麦基因组有部分同源性的玉米（ＺｅａｍａｙｓＬ．）ＤＮＡ克隆作探针,对由小麦ｘ玉米获得的普通小麦加倍单倍体（ＤＨ）后代群体进行ＲＦＬＰ分析。结果发现,不但用ｐＴａ７１在这些ＤＨ后代中检测到ｒＤＮＡ所发生的明显的减少和扩增及非转录间隔区的限制性片段长度的变化,而且用与小麦基因组部分同源的玉米克隆ＭＲ１３和ＭＲ５０在一些ＤＨ后代中检测到缺失变异,特别是用ＭＲ１３在普通小麦ＤＨ系的１８号株的基因组中检测到大幅度的限制性片段长度的变化,即原来的４．３ｋｂ的强信号带消失,取而代之的是４０．０ｋｂ、２．５ｋｂ和２．０ｋｂ三条杂交带。这可能与小麦基因组ＤＮＡ较大的重排事件有关,也可能是由外源的玉米ＤＮＡ插入造成的。     

禾木科植物染色体消除型远缘杂交的研究进展

植物远缘杂交是作物育种中广泛应用的技术。除了核型稳定的种间杂交可以获得杂种以外,还可以利用核型不稳定的种间杂交后父本染色体消除的现象,通过胚培养和染色体加倍处理获得加倍单倍体(DH)植株。然而从小麦×玉米杂交获得的DH后代与其理论上应完全同质的遗传表现却不相符,总有2～5％的DH植株发生了形态学变异。最近的研究证明。通过小麦×玉米的受精作用,一些玉米特异DNA可以被转移到小麦DH后代的基因组中。     

Synthesizing double haploid hexaploid wheat populations based on a spontaneous alloploidization process

Doubled haploid (DH) populations are useful to scientists and breeders in both crop improvement and basic research. Current methods of producing DHs usually need in vitro culture for extracting haploids and chemical treatment for chromosome doubling. This report describes a simple method for synthesizing DHs (SynDH) especially for allopolyploid species by utilizing meiotic restitution genes. The method involves three steps: hybridization to induce recombination, interspecific hybridization to extract haploids, and spontaneous chromosome doubling by selfing the interspecific F1s. DHs produced in this way contain recombinant chromosomes in the genome(s) of interest in a homogeneous background. No special equipment or treatments are involved in the DH production and it can be easily applied in any breeding and/or genetic program. Triticum turgidum L. and Aegilops tauschii Coss, the two ancestral species of common wheat (Triticum aestivum L.) and molecular markers were used to demonstrate the SynDH method.     

The agronomic performance of wheat doubled haploid lines derived from wheat x maize crosses

Summary The agronomic performance of 9 doubled haploid (DH) lines of Chinese Spring, 6 DH lines of Hope, 14 DH lines of the single chromosome substitution line Chinese Spring (Hope 5 A) and their respective parents was analyzed under field conditions. Seventeen Chinese Spring DH lines derived from wheat x Hordeum bulbosum crosses were also included for comparison. No significant variation was detected in either population of Chinese Spring DH lines and neither DH population differed from its parent. The Hope DH lines differed significantly for tiller biomass, spikelet number per ear, ear grain weight and 50-grain weight. However, all the variation could be attributed to the poor performance of only one line. Chinese Spring (Hope 5 A) DH lines showed significant variation for ear emergence time, but this was probably due to genetic heterogeneity in the parental stock. Overall, the results suggest that most DH lines produced by the wheat x maize method resemble their wheat parent, and that the variation induced in DH production is likely to be similar to that found in DHs from wheat x Hordeum bulbosum crosses.     

Improvements in the production of doubled haploids in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) through isolated microspore culture

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C₁₇ induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F₁ crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named ‘Lanuza’, has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.     

Inheritance and genetic mapping of self-incompatibility in Coffea canephora Pierre

Cross-compatibility behaviour of doubled haploid (DH) and hybrid genotypes of Coffea camphora was established using both phenotypic bioassay and in situ seed-set examination. The availability of DHs provided the opportunity of working with genetically homogenous pollen and female parents. The aniline blue fluorescence (ABF) method was applied to detect callose accumulation in pollen and pistil. Clear cross-compatibility/incompatibility situations were observed and confirmed by in situ seed-set analysis. Cross-compatibility analysis of hybrid combinations involving different DHs corroborated the crossing behaviour observed at the DH level. Expression of the self-incompatibility system did not appear to be affected by the low vigour of the DH. The crossing-behaviour distribution observed within DHs derived from clone IF200 confirmed that self-incompatibility in C. canephora is a gametophytic self-incompatibility system controlled by a single locus (S-locus). Reduced seed-set developments following incompatible crosses may indicate the occurrence of pseudo-incompatibility. Molecular marker linkage analysis showed that the S-locus is associated with an RFLP marker on linkage group 9. The availability of a linked DNA marker should facilitate the genetic analysis of self-incompatibility in relation to coffee breeding programmes.     

Nitrogen addition has a stronger effect on stoichiometries of non-structural carbohydrates,nitrogen and phosphorus in <Emphasis Type="Italic">Bothriochloa ischaemum</Emphasis> than elevated CO<Subscript>2</Subscript>

The current study attempted to obtain candidate doubled haploid (DH) wheat lines by serially combining two approaches: conventional chemical mutagenesis and anther culture. Additionally, the salt tolerance levels were examined between stress-treated (100 mM NaCl) and non-treated DH groups. For the molecular analysis, IRAP markers were used to characterize retrotransposon insertion polymorphisms induced by haploidization, chromosome doubling, and/or mutagenesis in the DH lines. Various sodium azide (NaN₃) concentrations (from 0 to 5 mM) were applied to seeds of the Pehlivan wheat cultivar to obtain an M₁ generation mutant population. Anther culture was set up from the M₁ mutant population. Green plant regeneration, the frequency of selected candidate mutants within the DH form and the levels of salt tolerance between samples were screened. A total of eight thousand anthers were cultured, and sixteen candidate salt-tolerant DH mutant lines, twenty-seven candidate DH mutant lines with different characteristics and one hundred and two candidate DH lines with morphologically normal appearances were obtained from the NaN₃-mutagenized population. The IRAP patterns were quite similar between the control DH lines, and the genetic differences between the controls and DHs originating from possible mutants showed close relatedness. According to previous studies, chemical mutagenesis and anther culture were combined for the first time to detect candidate salt tolerant genotypes at the DH stage. This approach might also be useful for determining the threshold dose and efficiency of wheat mutagens.     

Synthesizing double haploid hexaploid wheat populations based on a spontaneous alloploidization process

Doubled haploid(DH) populations are useful to scientists and breeders in both crop improvement and basic research.Current methods of producing DHs usually need in vitro culture for extracting haploids and chemical treatment for chromosome doubling.This report describes a simple method for synthesizing DHs(SynDH) especially for allopolyploid species by utilizing meiotic restitution genes.The method involves three steps:hybridization to induce recombination,interspecific hybridization to extract haploids,a...     

Maize DNA enrichment by representational difference analysis in a maize chromosome addition line of oat

 The recent recovery of maize (Zea mays L.) single-chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses has provided novel source materials for the potential isolation of maize chromosome-specific sequences for use in genetic mapping and gene cloning. We report here the application of a technique, known as representational difference analysis (RDA), to selectively isolate maize sequences from a maize chromosome-3 addition line of oat. DNA fragments from the addition line and from the oat parent were prepared using BamHI digestion and primer ligation followed by PCR amplification. A subtractive hybridization technique using an excess of the oat parental DNA was employed to reduce the availability for amplification of DNA fragments from the addition lines that were in common with the ones from the oat parental line. After three rounds of hybridization and amplification, the resulting DNA fragments were cloned into a plasmid vector. A DNA library containing 400 clones was constructed by this method. In a test of 18 clones selected at random from this library, four (22%) detected maize-specific repetitive DNA sequences and nine (50%) showed strong hybridization to genomic DNA of maize but weak hybridization to genomic DNA of oat. Among these latter nine clones, three detected low-copy DNA sequences and two of them detected DNA sequences specific to chromosome 3 of maize, the chromosome retained in the source maize addition line of oat. The other eight out of the 13 clones that had strong hybridization to maize DNA detected repetitive DNA sequences or high-copy number sequences present on most or all maize chromosomes. We estimate that the maize DNA sequences were enriched from about 1.8% to over 72% of the total DNA by this procedure. Most of the isolated DNA fragments detected multiple or repeated DNA sequences in maize, indicating that the major part of the maize genome consists of repetitive DNA sequences that do not cross-hybridize to oat genomic sequences. Received: 18 November 1997 / Accepted: 12 March 1998     

Cytochrome Oxidase Subunit II Gene from Carrot Contains an Intron

Introns in the cytochrome oxidase subunit II (COXII) gene of plant mitochondrial DNA (mtDNA) have been observed only in monocots. The COXII genes in dicots investigated to date do not contain introns. This is the first report of an intron in the COXII gene of a dicot. The presence of an intron in the carrot COXII intron was verified by restriction mapping and hybridization using specific maize and wheat COXII probes. Regions of the carrot COXII intron are homologous to the maize COXII intron and homologous to the wheat COXII intron-insert as demonstrated by hybridization. Homology of these regions was confirmed by sequencing portions of the gene. A comparison of the restriction map of the carrot COXII gene with the restriction maps of the COXII genes from pea, Oenothera, maize, wheat, and rice revealed that the carrot map coincides with the rice restriction map.     

Mechanism of conjugation and recombination in bacteria. XVIII. Polarity of donor DNA strands transferred to the recipient as determined by DNA-RNA hybridization.

Polarity of donor DNA strand transferred into recipient during conjugation in Escherichia coli K-12 was determined by DNA-3H-RNA hybridization. Lambda prophage was used as a marker. The defective lysogen Hfr H (lambdat11) as a donor and thermosensitive F- CR34 dnaB strain as recipient were used. Two sets of hybridization experiments, with 1-strand specific lambda mRNA and lambda mRNA specific for both phage strands but with large excess of r-strand specific mRNA, were carried out. Strand 1 of lambda DNA was detected preferentially in recipient cells mated at restrictive temperature, when Hfr transferred its genophore in the order gal-lambda-bio. Thus the genophore is transferred with 5'OH at its origin.     

Identification and chromosomal location of a new tandemly repeated DNA in maize.

Two clones of a new family of tandemly repeated DNA sequences have been isolated from a maize random genomic DNA library. MR68 is 410 bp, representing a monomeric unit and MR77 is 1222 bp, containing three units. The copy number was estimated to be about 3000 per 1C maize genome. Its methylation pattern was also determined. Fluorescent in situ hybridization (FISH) indicates that the sequence is located on the subtelomeric region of the long arm of chromosomes 3 and 6, as well as on the satellite of chromosome 6.     

Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants

Intergeneric somatic hybridization was performed between albino maize (Zea mays L.) protoplasts and mesophyll protoplasts of wheat (Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.     

Identification of RAPD markers linked to genetic factors controlling the milling energy requirement of barley

Doubled haploid (DH) populations of barley have been used in combination with PCR-based polymorphic-assay procedures to identify molecular markers linked to genes controlling the milling energy requirement of the grain. Milling energy (ME) is a quantitative trait and locating individual quantitative trait loci (QTLs) involved the construction of bulks by combining DNA from DH families representing the extreme members of the distribution for ME. In addition, the individuals had alternative alleles at theRrn2 locus that has previously been shown to be linked to an ME QTL. The DNA bulks were screened with Randomly Amplified Polymorphic DNA (RAPD) markers and polymorphic amplification products tested for linkage to genes influencing the expression of ME in a DH population. Several markers were identified which are linked to a QTL controlling ME and the recombination fraction determined by maximum likelihood procedures. The results indicate that DHs in combination with RAPDs and bulked segregant analysis provide an efficient method for locating QTLs in barely. Furthermore, this approach is applicable to mapping other QTLs in a range of organisms from which DH or recombinant inbred lines can be extracted.     

Electroporation of immature maize zygotic embryos and regeneration of transgenic plants

Using the pulse-discharging electroporation system HPES-3, we have transferred the neomycin phosphotransferase II (nptII) gene and -glucuronidase (gus) gene into mechanically-woulded immature zygotic embryo cells of an elite local maize cultivar Huanong Supersweet No. 42 and have produced transgenic maize plants. DNA hybridization and NPTII dot assay showed that the foreign genes were integrated into the genomes and expressed stably in the cells of the transgenic calluses and plants.     

脱水应答转录因子CBF1的克隆与转基因小麦的分子检测

根据已发表的小麦(T.aestivum)转录因子CBF1基因序列(GenBank Accession No.AF376136),设计引物从小麦品种‘京花1号’叶片中克隆出该基因,用拟南芥RD29B基因为启动子构建含CBF1基因的逆境诱导表达载体pBAC127F(6 967 bp),以‘99-92’、‘5-98’、‘104’和‘轮选987’等冬小麦品种(系)的幼穗和幼胚为材料,基因枪转化该表达载体。经筛选与植株再生,共获得14株转基因植株及其后代株系。这14个株系经PCR分析和点杂交检测,最终确认了5-98-40、5-98-41这2个株系为转基因株系,结果表明拟南芥RD29B启动子调控下的转录因子CBF1基因已稳定整合到转基因植株中。