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...     

甜瓜属植物种间杂交研究进展

在甜瓜属野生种Cucumis hystrix Chakr. 和栽培黄瓜之间的种间杂交取得成功的基础上,简要回顾了甜瓜属植物种质资源和系统学的研究情况,然后从甜瓜属植物种间杂交的概况、种间杂交的障碍及其解决途径等三个方面,回顾和讨论了甜瓜属植物的种间杂交的研究进展,并对进一步通过种间杂交研究利用甜瓜属野生植物资源的前景进行了展望。     

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.     

Genetic map of Triticum turgidum based on a hexaploid wheat population without genetic recombination for D genome

ABSTRACT: BACKGROUND: A synthetic doubled-haploid hexaploid wheat population, SynDH1, derived from the spontaneous chromosome doubling of triploid F1 hybrid plants obtained from the cross of hybrids Triticum turgidum ssp. durum line Langdon (LDN) and ssp. turgidum line AS313, with Aegilops tauschii ssp. tauschii accession AS60, was previously constructed. SynDH1 is a tetraploidization-hexaploid doubled haploid (DH) population because it contains recombinant A and B chromosomes from two different T. turgidum genotypes, while all the D chromosomes from Ae. tauschii are homogenous across the whole population. This paper reports the construction of a genetic map using this population. RESULTS: Of the 606 markers used to assemble the genetic map, 588 (97%) were assigned to linkage groups. These included 513 Diversity Arrays Technology (DArT) markers, 72 simple sequence repeat (SSR), one insertion site-based polymorphism (ISBP), and two high-molecular-weight glutenin subunit (HMW-GS) markers. These markers were assigned to the 14 chromosomes, covering 2048.79 cM, with a mean distance of 3.48 cM between adjacent markers. This map showed good coverage of the A and B genome chromosomes, apart from 3A, 5A, 6A, and 4B. Compared with previously reported maps, most shared markers showed highly consistent orders. This map was successfully used to identify five quantitative trait loci (QTL), including two for spikelet number on chromosomes 7A and 5B, two for spike length on 7A and 3B, and one for 1000-grain weight on 4B. However, differences in crossability QTL between the two T. turgidum parents may explain the segregation distortion regions on chromosomes 1A, 3B, and 6B. CONCLUSIONS: A genetic map of T. turgidum including 588 markers was constructed using a synthetic doubled haploid (SynDH) hexaploid wheat population. Five QTLs for three agronomic traits were identified from this population. However, more markers are needed to increase the density and resolution of this map in the future study.     

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。     

Nuclear DNA amount and genome downsizing in natural and synthetic allopolyploids of the genera Aegilops and Triticum

Recent molecular studies in the genera Aegilops and Triticum showed that allopolyploidization (interspecific or intergeneric hybridization followed by chromosome doubling) generated rapid elimination of low-copy or high-copy, non-coding and coding DNA sequences. The aims of this work were to determine the amount of nuclear DNA in allopolyploid species of the group and to see to what extent elimination of DNA sequences affected genome size. Nuclear DNA amount was determined by the flow cytometry method in 27 natural allopolyploid species (most of which were represented by several lines and each line by several plants) as well as 14 newly synthesized allopolyploids (each represented by several plants) and their parental plants. Very small intraspecific variation in DNA amount was found between lines of allopolyploid species collected from different habitats or between wild and domesticated forms of allopolyploid wheat. In contrast to the constancy in nuclear DNA amount at the intraspecific level, there are significant differences in genome size between the various allopolyploid species, at both the tetraploid and hexaploid levels. In most allopolyploids nuclear DNA amount was significantly less than the sum of DNA amounts of the parental species. Newly synthesized allopolyploids exhibited a similar decrease in nuclear DNA amount in the first generation, indicating that genome downsizing occurs during and (or) immediately after the formation of the allopolyploids and that there are no further changes in genome size during the life of the allopolyploids. Phylogenetic considerations of the origin of the B genome of allopolyploid wheat, based on nuclear DNA amount, are discussed.     

Cytogenetics of Hordeum chilense: current status and considerations with reference to breeding

Hordeum chilense Roem. et Schult. has a number of characteristics interesting for breeding: high crossability with other Triticeae, resistance to biotic and abiotic stresses and high variability for quality traits such as endosperm storage proteins or carotenoid content. xTritordeum, the amphiploids between H. chilense and different Triticum spp, are bridge species which facilitate the transfer of traits from H. chilense to wheat or triticale. The chromosome pairing between H. chilense and wheat chromosomes is very low (if existing) even in the absence of the action of the Ph1 gene. Nevertheless, translocation between H. chilense and wheat chromosomes has been observed frequently in genomic combinations where univalents of both species are present and therefore a method is available for using H. chilense in wheat or triticale breeding. Hybrids and amphiploids with other crop species of the Triticeae, such as rye or barley, have also been obtained, although to date the production of stable introgression stocks has not been completed. The technique of chromosome painting, using both high- and low-repeated DNA sequences in combination with genomic in situ hybridization have been used as effective methods for basic cytogenetic research in H. chilense, allowing analysis of genome evolution, and monitoring H. chilense chromosomes in interspecific hybridization breeding programs.     

应用基因组原位杂交鉴定蓝粒小麦及其诱变后代

应用基因组原位杂交技术（GISH）对普通小麦（Triticum aestivumL.)和长穗偃麦草[Agropyron elongatum(Host)Beauv,2n=10x=70]杂交后选育出的蓝粒小麦蓝-58及其诱变后代的染色体组成进行了鉴定。结果表明,GISH可方便地检测到小麦遗传背景中的长穗偃麦草染色体或易位的片段。如前人报道,蓝-58（2n=42)是一个具有2条长穗偃麦草4E染色体的异代换系（4E/4D）。LW004可能是一个具有两对相互易位染色体的纯合系,其田间表现磷高效特性,LW43-3-4为41条染色体的蓝单体（40W 1’4E）,种子颜色为浅蓝色,通过此法还检测出一些染色体结构发生很大变异的材料如4E的单端体（40W 1‘4E),种子颜色为浅蓝色,通过此法还检测出一些染色结构发生很大变异的材料如4E的单端体（40W 1‘t4E)以及组型为39W 1‘4E 1‘t4E的个体,此项研究结果更为直观地表明控制蓝粒体状的基因的确在来自长穗偃麦草的染色体上。同时说明有效的突变方法与灵活方便的检测手段的有机结合在染色体工程材料的创制和染色体工程育种中起着至关重要的作用。     

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.     

Timing and rate of genome variation in triticale following allopolyploidization.

The timing and rate of genomic variation induced by allopolyploidization in the intergeneric wheat-rye (Triticum spp. - Secale cereale L.) hybrid triticale (x Triticosecale Wittmack) was studied using amplified fragment length polymorphism (AFLP) analyses with 2 sets of primers, EcoRI-MseI (E-M) and PstI-MseI (P-M), which primarily amplify repetitive and low-copy sequences, respectively. The results showed that allopolyploidization induced genome sequence variation in triticale and that a great degree of the genome variation occurred immediately following wide hybridization. Specifically, about 46.3% and 36.2% of the wheat parental band loss and 74.5% and 68.4% of the rye parental band loss occurred in the F1 hybrids (before chromosome doubling) for E-M and P-M primers, respectively. The sequence variation events that followed chromosome doubling consisted of continuous modifications that occurred at a very small rate compared with the rate of variation before chromosome doubling. However, the rate of sequence variation involving the rye parental genome was much higher in the first 5 generations following chromosome doubling than in any subsequent generation. Surprisingly, the highest rate of rye genomic variation occurring after chromosome doubling was in C3 or later, but not in C1. The data suggested that the cytoplasm and the degree of the relationship between the parental genomes were the key factors in determining the direction, amount, timing, and rate of genomic sequence variation occurring during intergeneric allopolyploidization.     

Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in <Emphasis Type="Italic">Phalaenopsis</Emphasis> species

Endopolyploidy was observed in the protocorms of diploid Phalaenopsis aphrodite subsp. formosana with ploidy doubling achieved by in vitro regeneration of excised protocorms, or protocorm-like bodies (PLBs). Thirty-four per cent of the PLBs regenerated from the first cycle of sectioned protocorms were found to be polyploids with ploidy doubled once or twice as determined by flow-cytometry. The frequency of ploidy doubling increased as the sectioning cycles increased and was highest in diploid followed by the triploid and tetraploid. Regeneration of the endopolyploid cells in the tissue of the protocorms or PLBs is proposed as the source of the development of ploidy doubled plantlets. The frequency of ploidy doubling was similar in seven other Phalaenopsis species, although the rate of increase within cycles was genotype specific. In two species, a comparison of five parameters between 5-month-old diploid and tetraploid potted plants showed only the stomata density differed significantly. The flowers of the tetraploid plant were larger and heavier than those of the diploids. This ploidy doubling method is a simple and effective means to produce large number of polyploid Phalaenopsis species plants as well as their hybrids. The method will be beneficial to orchid breeding programs especially for the interspecific hybridization between varieties having different chromosome sizes and ploidy levels.     

Polyploid formation pathways have an impact on genetic rearrangements in resynthesized Brassica napus

? Polyploids can be produced by the union of unreduced gametes or through somatic doubling of F(1) interspecific hybrids. The first route is suspected to produce allopolyploid species under natural conditions, whereas experimental data have only been thoroughly gathered for the latter. ? We analyzed the meiotic behavior of an F(1) interspecific hybrid (by crossing Brassica oleracea and B.rapa, progenitors of B.napus) and the extent to which recombined homoeologous chromosomes were transmitted to its progeny. These results were then compared with results obtained for a plant generated by somatic doubling of this F? hybrid (CD.S?) and an amphidiploid (UG.S?) formed via a pathway involving unreduced gametes; we studied the impact of this method of polyploid formation on subsequent generations. ? This study revealed that meiosis of the F? interspecific hybrid generated more gametes with recombined chromosomes than did meiosis of the plant produced by somatic doubling, although the size of these translocations was smaller. In the progeny of the UG.S? plant, there was an unexpected increase in the frequency at which the C1 chromosome was replaced by the A1 chromosome. ? We conclude that polyploid formation pathways differ in their genetic outcome. Our study opens up perspectives for the understanding of polyploid origins.     

Mapping of QTL for tiller number at different stages of growth in wheat using double haploid and immortalized F<Subscript>2</Subscript> populations

Effective tiller number is one of the most important traits for wheat (Triticum aestivum L.) yield, but the inheritance of tillering is poorly understood. A set of 168 doubled haploid (DH) lines derivatives of a cross between two winter wheat cultivars (Huapei 3 and Yumai 57), and an immortalized F₂ (IF₂) population generated by randomly permutated intermating of these DHs were investigated, and QTLs of tillering related to the maximum tillering of pre-winter (MTW), maximum tillering in spring (MTS), and effective tillering in harvest (ETH) were mapped. Phenotypic data were collected for the two populations from two different environments. Using inclusive composite interval mapping (ICIM), a total of 9 and 18 significant QTL were detected across environments for tillering in the DH and IF₂ populations, respectively. Four QTLs were common between two populations. A major QTL located on the 5D chromosome with the allele originating from Yumai 57 was detected and increased 1.92 and 3.55 tillers in MTW and MTS, respectively. QTLs (QMts6D, QEth6D) having a neighbouring marker interval at Xswes679.1 and Xcfa2129 on chromosome 6D was detected in MTS and ETH. These results provide a better understanding of the genetic factors for selectively expressing the control of tiller number in different growth stages and facilitate marker-assisted selection strategy in breeding.     

Flower senescence in daylily (Hemerocallis)

This paper reviews recent developments in the use of molecular probes for analyzing the genetic makeup of somatic hybrids. Successful application of somatic hybridization to the interspecific transfer of traits encoded in the nucleus is still having limited success. A major difficulty is hybrid infertility, particularly in hybrids between sexually incompatible species. The formation of asymmetric hybrids is being explored as an approach for improving hybrid fertility. Evaluation of the degree of chromosome elimination and chromosome stability and instability in asymmetric hybrids is difficult when the traditional approaches of chromosome counting and isozyme analysis are used. Two new approaches are resolving this difficulty. The use of species-specific repetitive DNA probes in dot blotting and in situ hybridization to chromosomes is providing quantitative data on chromosome elimination and allows detection of translocations. Use of restriction fragment length polymorphism (RFLP) probes for analysis of hybrids between genetically mapped species makes it possible to account for the presence or absence of individual chromosomes and chromosomes arms. Wider use of such molecular probes should greatly improve our understanding of the genetics of both symmetric and asymmetric somatic hybrids and may lead to new strategies for the effective interspecific transfer of nucleus-encoded traits by protoplast fusion.     

Meiotic restitution in wheat polyhaploids (amphihaploids): a potent evolutionary force

Polyploidy is well recognized as a major force in plant speciation. Among the polyploids in nature, allopolyploids are preponderant and include important crop plants like bread wheat, Triticum aestivum L. (2n = 6x = 42; AABBDD genomes). Allopolyploidy must result through concomitant or sequential events that entail interspecific or intergeneric hybridization and chromosome doubling in the resultant hybrids. To gain insight into the mechanism of evolution of wheat, we extracted polyhaploids of 2 cultivars, Chinese Spring (CS) and Fukuhokomugi (Fuko), of bread wheat by crossing them with maize, Zea mays L. ssp. mays. The derived Ph1-polyhaploids (2n = 3x = 21; ABD) showed during meiosis mostly univalents, which produced first-division restitution (FDR) nuclei that in turn gave rise to unreduced (2n) male gametes with 21 chromosomes. The haploids on maturity set some viable seed. The mean number of seeds per spike was 1.45 +/- 0.161 in CS and 2.3 +/- 0.170 in Fuko. Mitotic chromosome preparations from root tips of the derived plantlets revealed 2n = 42 chromosomes, that is, twice that of the parental polyhaploid, which indicated that they arose by fusion of unreduced male and female gametes formed by the polyhaploid. The Ph1-induced univalency must have produced 2n gametes and hence bilateral sexual polyploidization and reconstitution of disomic bread wheat. These findings highlight the quantum jump by which bread wheat evolved from durum wheat in nature. Thus, bread wheat offers an excellent example of rapid evolution by allopolyploidy. In the induced polyhaploids (ABD) that are equivalent of amphihaploids, meiotic phenomena such as FDR led to regeneration of parental bread wheat, perhaps a simulation of the evolutionary steps that occurred in nature at the time of the origin of hexaploid wheat.     

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5–1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1–7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1–3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.     

Novel technologies in doubled haploid line development

haploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter‐ and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere‐mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis.     

Molecular evidence on maize specific DNA fragment transferred into wheat through sexual hybridization

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Painting of parental chromatin in Beta hybrids by multi-colour fluorescent in situ hybridization

Sugar beet (Beta vulgaris L.) is a relatively young crop and has a narrow gene pool. In order to introduce genetic variability into the crop, interspecific hybrids, selected from crosses with wild beets of the sections Corollinae and Procumbentes, have been generated. The introgressed B. procumbens chromatin carries resistance genes to beet cyst nematode Heterodera schachtii Schm. These lines are important for breeding of nematode-resistant sugar beet, while Corollinae species are potential donors of tolerance to biotic and abiotic stresses such as drought or saline soils. We have used in situ hybridization of genomic DNA to discriminate the parental chromosomes in these interspecific hybrids. Suppression of cross-hybridization by blocking DNA was not necessary indicating that the investigated Beta genomes contain sufficient species-specific DNA enabling the unequivocal determination of the genomic composition of the hybrids. Interspecific hybrid lines with an additional chromosome (2n = 18 + 1), chromosome fragment (2n = 18 + fragment) or translocation of B. procumbens (2n = 18) were analysed by genomic in situ hybridization (GISH) at mitosis and meiosis. Species-specific satellites and ribosomal genes used in combination with genomic DNA or in rehybridization experiments served as landmark probes for chromosome identification in hybrid genomes. The detection of a B. procumbens translocation of approx. I Mbp demonstrated the sensitivity and resolution of GISH and showed that this approach is a powerful method in genome analysis projects of the genus Beta.