Ancestry of American polyploid Hordeum species with the I genome inferred from 5S and 18S-25S rDNA

* BACKGROUND AND AIMS: The genus Hordeum exists at three ploidy levels (2x, 4x and 6x) and presents excellent material for investigating the patterns of polyploid evolution in plants. Here the aim was to clarify the ancestry of American polyploid species with the I genome. * METHODS: Chromosomal locations of 5S and 18S-25S ribosomal RNA genes were determined by fluorescence in situ hybridization (FISH). In both polyploid and diploid species, variation in 18S-25S rDNA repeated sequences was analysed by the RFLP technique. * KEY RESULTS: Six American tetraploid species were divided into two types that differed in the number of rDNA sites and RFLP profiles. Four hexaploid species were similar in number and location of both types of rDNA sites, but the RFLP profiles of 18S-25S rDNA revealed one species, H. arizonicum, with a different ancestry. * CONCLUSIONS: Five American perennial tetraploid species appear to be alloploids having the genomes of an Asian diploid H. roshevitzii and an American diploid species. The North American annual tetraploid H. depressum is probably a segmental alloploid combining the two closely related genomes of American diploid species. A hexaploid species, H. arizonicum, involves a diploid species, H. pusillum, in its ancestry; both species share the annual growth habit and are distributed in North America. Polymorphisms of rDNA sites detected by FISH and RFLP analyses provide useful information to infer the phylogenetic relationships of I-genome Hordeum species because of their highly conserved nature during polyploid evolution.     

Origin and evolution of North American polyploid Silene (Caryophyllaceae)

Nuclear DNA sequences from introns of the low-copy nuclear gene family encoding the second largest subunit of RNA polymerases and the ribosomal internal transcribed spacer (ITS) regions, combined with the psbE-petL spacer and the rps16 intron from the chloroplast genome were used to infer origins and phylogenetic relationships of North American polyploid Silene species and their closest relatives. Although the vast majority of North American Silene species are polyploid, which contrasts to the diploid condition dominating in other parts of the world, the phylogenetic analyses rejected a single origin of the North American polyploids. One lineage consists of tetraploid Silene menziesii and its diploid allies. A second lineage, Physolychnis s.l., consists of Arctic, European, Asian, and South American taxa in addition to the majority of the North American polyploids. The hexaploid S. hookeri is derived from an allopolyploidization between these two lineages. The tetraploid S. nivea does not belong to any of these lineages, but is closely related to the European diploid S. baccifera. The poor resolution within Physolychnis s.l. may be attributed to rapid radiation, recombination among homoeologues, homoplasy, or any combination of these factors. No extant diploid donors could be identified in Physolychnis s.l.     

植物多倍体研究的回顾与展望

多倍化是促进植物进化的重要力量。多倍体主要是通过未减数配子融合,体细胞染色体加倍以及多精受精三种方式起源的。其中,不减数配子是多倍体形成的主要机制。三倍体可能在四倍体的进化中起了重要作用。过去认为多倍体只能是进化的死胡同,现在发现很多多倍体类群都是多元起源的而不是单元起源的。当多倍体形成后,基因组中的重复基因大部分保持原有的功能,也有相当比例的基因发生基因沉默。多倍体通常表现出不存在于二倍体祖先的表型,并且超出了其祖先的分布范围,因为在多倍体中发生了很多基因表达的变化。主要从多倍体的起源、影响多倍体发生的因素及多倍体基因组的进化等方面回顾并展望多倍体的研究。     

Recurrent deletions of puroindoline genes at the grain hardness locus in four independent lineages of polyploid wheat

Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the A(m) genome of hexaploid Triticum zhukovskyi (A(m)AG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed.     

Other tetraploid species and conspecific diploids as sources of genetic variation for an autotetraploid

? Premise of the study: Most plants are polyploid and have more than two copies of the genome. The evolutionary success of polyploids is often attributed to their potential to harbor increased genetic variation, but it is poorly understood how polyploids can attain such variation. Because of their formation bottleneck, newly formed tetraploids start out with little variation. Tetraploids may attain genetic variation through a combination of new mutations, recurrent formation, and gene exchange with diploid ancestors or related tetraploid species. We explore the role of gene exchange and introgression in autotetraploid Rorippa amphibia, a species that harbors more genetic variation than its diploid ancestors. ? Methods: We crossed autotetraploid R. amphibia to diploid conspecifics and tetraploid R. sylvestris and backcrossed resulting F(1) hybrids. We used flow cytometry to determine the ploidy of all progeny. ? Key results: Tetraploids of R. amphibia and R. sylvestris were interfertile; F(1) hybrids were fertile and could backcross. Crosses between diploids and tetraploids yielded a small number of viable, often tetraploid progeny. This indicates that unreduced gametes can facilitate gene flow from diploids to tetraploids. We detected a frequency of unreduced gametes of around 2.7 per 1000, which was comparable between diploids and tetraploids. ? Conclusions: Introgression from tetraploid R. sylvestris provides a realistic source of variation in autotetraploid R. amphibia. Only in a scenario where other compatible partners are absent, for example immediately after tetraploidization, gene flow through unreduced gametes from diploids could be an important source of genetic variation for tetraploids.     

Sequence evidence for sporadic intergeneric DNA introgression from wheat into a wild Aegilops species

Introgressive hybridization has played a crucial role in the evolution of many plant species, especially polyploids. The duplicated genetic material and wide geographical distribution facilitate hybridization and introgression among polyploid species having either homologous or homoeologous genomes. Such introgression may lead to the production of recombinant genomes that are more difficult to form at the diploid level. Crop genes that have introgressed into wild relatives can increase the capability of the wild relatives to adapt to agricultural environments and compete with crops or to compete with other wild species. Although the transfer of genes from crops into their conspecific immediate wild progenitors has been reported, little is known about spontaneous gene movement from crops to more distantly related species. We describe recent spontaneous DNA introgression from domesticated polyploid wheat into distantly related, wild tetraploid Aegilops peregrina (syn. Aegilops variabilis) and the stabilization of this sequence in wild populations despite not having homologous chromosomes. Our results show that DNA can spontaneously introgress between homoeologous genomes of species of the tribe Triticeae and, in the case of crop-wild relatives, possibly enrich the wild population. These results also emphasize the need for fail-safe mechanisms in transgenic crops to prevent gene flow where there may be ecological risks.     

Allopolyploid origin of Cardamine asarifolia (Brassicaceae): incongruence between plastid and nuclear ribosomal DNA sequences solved by a single-copy nuclear gene

Interspecific hybridization and polyploidization have played central roles in plant diversification. However, technical difficulties in the analyses of low-copy genes have limited the study of the origins of hybrid and polyploid plants. Here, we present a phylogenetic analysis of the hexaploid Cardamine asarifolia, distributed in the southern European Alps and northern Apennines. Our study included all relevant taxa of the genus found in Europe. A marked discrepancy was revealed between the trnL-trnF region of cpDNA and internal transcribed spacer (nrDNA ITS) sequences. To solve the incongruence, we sequenced a single-copy nuclear CHS gene (chalcone synthase) using a novel method to design homoeologue-specific PCR primers to bypass artefacts caused by artificial recombination of homoeologues during PCR and/or cloning. Three homoeologues were isolated from C. asarifolia, providing evidence for its allopolyploid origin. One homoeologue, showing the same phylogenetic position as the ITS sequences, most likely originated from an extinct parent. Furthermore, we documented recurrent polytopic hybridizations between C. asarifolia and diploid C. amara. The allohexaploidization and the following hybridization with a diploid species exemplify the ongoing dynamic processes of speciation in the genus Cardamine.     

High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.)

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs.     

Towards an understanding of the molecular mechanisms regulating gene expression during diploidization in phylogenetically polyploid lower vertebrates

Polyploidization and regional gene duplication have occurred frequently during vertebrate evolution, providing the genetic material necessary for creating evolutionary novelties. Mammals, including man, can be regarded as diploid species with a polyploid history of evolution. Polyploidization steps during the phylogeny of mammals probably took place in the genomes of amphibian- or fish-like mammalian ancestors. The polyploid status has subsequently been shaped by the process of diploidization, leading to genomes that are polyploid with respect to the amount of genetic material and the number of gene copies, and diploid with respect to the level of gene expression and chromosomal characteristics. Phylogenetically tetraploid amphibian and teleost species together with their diploid close relatives can be used as a model system to study the effect of polyploidization and the mechanisms of diploidization of a parallel event during early mammalian evolution. Experimental evidence permits the assumption that the diploidization of gene expression in tetraploid cyprinid fish may be functionally correlated with structural modifications of the ribosomal components, RNA and protein. These findings are discussed in the light of reduced protein synthesis in diploidized tetraploid species and a mechanism to explain diploidization during mammalian evolution.     

A phylogenetic analysis of indel dynamics in the cotton genus

Genome size evolution is a dynamic process involving counterbalancing mechanisms whose actions vary across lineages and over time. Whereas the primary mechanism of expansion, transposable element (TE) amplification, has been widely documented, the evolutionary dynamics of genome contraction have been less thoroughly explored. To evaluate the relative impact and evolutionary stability of the mechanisms that affect genome size, we conducted a phylogenetic analysis of indel rates for 2 genomic regions in 4 Gossypium genomes: the 2 coresident genomes (A(T) and D(T)) of tetraploid cotton and its model diploid progenitors, Gossypium arboreum (A) and Gossypium raimondii (D). We determined the rates of sequence gain or loss along each branch, partitioned by mechanism, and how these changed during species divergence. In general, there has been a propensity toward growth of the diploid genomes and contraction in the polyploid. Most of the size difference between the diploid species occurred prior to polyploid divergence and was largely attributable to TE amplification in the A/A(T) genome. After separating from the true parents of the polyploid genomes, both diploid genomes experienced slower sequence gain than in the ancestor, due to fewer TE insertions in the A genome and a combination of increased deletions and decreased TE insertions in the D genome. Both genomes of the polyploid displayed increased rates of deletion and decreased rates of insertion, leading to a rate of near stasis in D(T) and overall contraction in A(T) resulting in polyploid genome contraction. As expected, TE insertions contributed significantly to the genome size differences; however, intrastrand homologous recombination, although rare, had the most significant impact on the rate of deletion. Small indel data for the diploids suggest the possibility of a bias as the smaller genomes add less or delete more sequence through small indels than do the larger genomes, whereas data for the polyploid suggest increased sequence turnover in general (both as small deletions and small insertions). Illegitimate recombination, although not demonstrated to be a dominant mechanism of genome size change, was biased in the polyploid toward deletions, which may provide a partial explanation of polyploid genomic downsizing.     

Rapid Elimination of Low-Copy DNA Sequences in Polyploid Wheat: A Possible Mechanism for Differentiation of Homoeologous Chromosomes

To study genome evolution in allopolyploid plants, we analyzed polyploid wheats and their diploid progenitors for the occurrence of 16 low-copy chromosome- or genome-specific sequences isolated from hexaploid wheat. Based on their occurrence in the diploid species, we classified the sequences into two groups: group I, found in only one of the three diploid progenitors of hexaploid wheat, and group II, found in all three diploid progenitors. The absence of group II sequences from one genome of tetraploid wheat and from two genomes of hexaploid wheat indicates their specific elimination from these genomes at the polyploid level. Analysis of a newly synthesized amphiploid, having a genomic constitution analogous to that of hexaploid wheat, revealed a pattern of sequence elimination similar to the one found in hexaploid wheat. Apparently, speciation through allopolyploidy is accompanied by a rapid, nonrandom elimination of specific, low-copy, probably noncoding DNA sequences at the early stages of allopolyploidization, resulting in further divergence of homoeologous chromosomes (partially homologous chromosomes of different genomes carrying the same order of gene loci). We suggest that such genomic changes may provide the physical basis for the diploid-like meiotic behavior of polyploid wheat.     

Genomic in situ hybridization (GISH) discriminates between the A and the B genomes in diploid and tetraploid Setaria species.

Genomic in situ hybridization (GISH) was used to investigate genomic relationships between different Setaria species of the foxtail millet gene pool (S. italica) and one interspecific F1 hybrid. The GISH patterns obtained on the two diploid species S. viridis (genome A) and S. adhaerans (genome B), and on their F1 hybrid showed clear differentiation between these two genomes except at the nucleolar organizing regions. Similar GISH patterns allowed differentiation of S. italica from S. adhaerans. However, GISH patterns did not distinguish between the genomes of S. italica and its putative wild ancestor S. viridis. GISH was also applied to polyploid Setaria species and enabled confirmation of the assumed allotetraploid nature of S. faberii and demonstration that both S. verticillata and S. verticillata var. ambigua were also allotetraploids. All these tetraploid species contained two sets of 18 chromosomes each, one from genome A and the other from genome B. Only one polyploid species, S. pumila, was shown to bear an unknown genomic composition that is not closely related either to genome A or to genome B.     

Molecular evidence for Triticum speltoides as a B-genome progenitor of wheat (Triticum aestivum).

In polyploid wheat, the origin of the B-genome donor has remained relatively unknown in spite of a number of investigations attempting to identify the parental species. A project was designed to isolate and clone a genome-specific DNA sequence from Triticum speltoides L. to determine if that species could be the B-genome donor. A cloning scheme involving the prescreening of 1-kb fragments followed by colony, dot blot, and Southern blot hybridization screenings was used to isolate a speltoides-specific sequence (pSp89.XI). The methods used allowed for rapid isolation of a genome-specific sequence when screened against total DNA from closely related species. Subsequent analyses showed that the sequence was barely detected in any of the other genomes of the annual Sitopsis section. The results of dot blot and Southern blot analyses established that (i) the sequence pSP89.XI, specific to T. speltoides relative to the other species of the Sitopsis section, was present in the genomes of tetraploid and hexaploid wheat, (ii) the relative abundance of pSp89.XI seemed to decrease from the diploid to the polyploid wheats, and (iii) the existence of a related, but modified B genome in polyploid wheat compared with that in modern T. speltoides was probable. Key words : genome-specific, DNA.     

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.     

种间杂种染色体配对所揭示的披碱草属植物StY基因组分化及其进化意义

在同倍体(homoploid)植物杂交-分化的物种形成(speciation)过程中,杂交后代与亲本之间的有效生殖隔离是新物种形成的关键。杂种与亲本在时间、空间、生态环境和基因水平上的隔离,保证了杂种后代的分化和稳定,并逐渐形成新物种。为了研究披碱草属(Elymus)含StY基因组四倍体物种的系统演化关系,本文对来自亚洲不同地理分布区的26种披碱草属植物进行了大规模的种间杂交和杂种F1减数分裂染色体配对行为的分析。结果表明各物种之间有不同程度的杂交亲合力,杂交结实率在各杂交组合之间有较大的变异(在4．8％-100％之间);但各物种之间的杂种F1完全不育。证明各物种之间形成了明显的生殖隔离。种间杂种F1减数分裂中期-I染色体配对分析的结果进一步表明,StY基因组随各披碱草属物种地理分布的不同而有不同程度的分化。来自同一分布区(如东亚或西亚分布区之内)物种的StY基因组分化程度较低,但来自不同分布区(如东亚和西亚)物种之间相同的StY基因组具有显著的分化。表明地理隔离对含StY基因组物种的分化起到了十分重要的作用。通过对种间和种内杂种F1的减数分裂异常现象和染色体配对频率变化规律的分析,作者认为细胞学水平的变化,如基因组同源性的分化和染色体结构的变异等都在杂种后代与亲本之间产生生殖隔离并逐渐形成新物种的进化过程中起到了积极的作用。     

The origin of polyploid genomes of bluegrasses Poa L. and gene flow between Northern Pacific and sub-Antarctic islands

The involvement of present-day diploid bluegrass species in the formation of polyploid genomes was investigated using comparison of sequences of internal transcribed spacers ITS1 and ITS2, and the 5.8S rDNA sequence. It was demonstrated that highly polyploid New Zealand bluegrasses, P. cita (2n = 84; ca. 96 to 100), P. chathamica (2n = 112), and P. litorosa (2n = 263 to 266) formed separate highly supported clade together with tetraploids (2n = 28) P. intrusa, P. anceps, and P. trioides (Austrofestuca littoralis). Among the diploid species (2n = 14), the closest relatives of these species, as well as of the polyploid species of section Poa, are the genomes of Eurasian species P. remota, P. chaixcii (sect. Homalopoa), P densa (Bolbophorum), and P. sibirica (sect. Macropoa). Nuclear genomes of polyploid Stenopoa, Tichopoa, Oreinos, and Secundae are definitely related to the genome of Arctic species P. pseudabbreviata (sect. Abbreviatae). On the contrary, judging by the genes for nuclear 45S rRNA, genomes of diploid P. trivialis (sect. Pandemos), P. annua, and P. supina (sect. Ochlopoa both) are only remotely related to the genomes of highly polyploid species (distances p between them and other bluegrass species from different sections of subgenus Poa constitute 6-10% and 11-15%, respectively). The conclusion on the relationships between highly polyploid and diploid bluegrass species was tested using analysis of synapomorphic mutations in the 5.8S rRNA gene. It was demonstrated that genomes of Poa eminens (2n = 42) and P. schischkinii (2n = 70) (sect. Arctopoa both) were noticeably different in ITS regions from the genomes of the members of the type subgenus Poa. A comparison of the Arctopoa ITS regions showed that the differences between them constituted only 0.2%. At the same time, p distances between the Arctopoa ITS and those from the species belonging to other sections of the genus Poa varied from 5 to 14%. South American species P chonotica (sect. Andinae) (=Ncoraepoa chonotica) (2n = 42) was found to be related to Arctagrostis, Festucella, and Hookerochloa, being at the same time quite distant from the other species of the genus Poa. Polymorphic in chromosome number highly polyploid species of Northern Hemisphere, P. arctica (2n = 42 to 106), P. turneri (2n = 42, 63 to 64), and P. smirnovii (2n = 42, 70) (sect. Malacanthae) are relative to a large group of tetraploid (2n = 28) endemic bluegrass species from New Zealand and sub-Antarctic islands (P. novae-zelandiae and allied species).