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1.  Experimental evidence for the ancestry of allotetraploid Trifolium repens and creation of synthetic forms with value for plant breeding 
BMC Plant Biology  2012;12:55.
Background
White clover (Trifolium repens) is a ubiquitous weed of the temperate world that through use of improved cultivars has also become the most important legume of grazed pastures world-wide. It has long been suspected to be allotetraploid, but the diploid ancestral species have remained elusive. Putative diploid ancestors were indicated by DNA sequence phylogeny to be T. pallescens and T. occidentale. Here, we use further DNA evidence as well as a combination of molecular cytogenetics (FISH and GISH) and experimental hybridization to test the hypothesis that white clover originated as a hybrid between T. pallescens and T. occidentale.
Results
T. pallescens plants were identified with chloroplast trnL intron DNA sequences identical to those of white clover. Similarly, T. occidentale plants with nuclear ITS sequences identical to white clover were also identified. Reciprocal GISH experiments, alternately using labeled genomic DNA probes from each of the putative ancestral species on the same white clover cells, showed that half of the chromosomes hybridized with each probe. F1 hybrids were generated by embryo rescue and these showed strong interspecific chromosome pairing and produced a significant frequency of unreduced gametes, indicating the likely mode of polyploidization. The F1 hybrids are inter-fertile with white clover and function as synthetic white clovers, a valuable new resource for the re-incorporation of ancestral genomes into modern white clover for future plant breeding.
Conclusions
Evidence from DNA sequence analyses, molecular cytogenetics, interspecific hybridization and breeding experiments supports the hypothesis that a diploid alpine species (T. pallescens) hybridized with a diploid coastal species (T. occidentale) to generate tetraploid T. repens. The coming together of these two narrowly adapted species (one alpine and the other maritime), along with allotetraploidy, has led to a transgressive hybrid with a broad adaptive range.
doi:10.1186/1471-2229-12-55
PMCID: PMC3443075  PMID: 22530692
Trifolium repens; White clover; Allopolyploid; Interspecific hybridization
2.  Genetic transformation of western clover (Trifolium occidentale D. E. Coombe.) as a model for functional genomics and transgene introgression in clonal pasture legume species 
Plant Methods  2013;9:25.
Background
Western clover (Trifolium occidentale) is a perennial herb with characteristics compatible for its development as an attractive model species for genomics studies relating to the forage legume, white clover (Trifolium repens). Its characteristics such as a small diploid genome, self-fertility and ancestral contribution of one of the genomes of T. repens, facilitates its use as a model for genetic analysis of plants transformed with legume or novel genes.
Results
In this study, a reproducible transformation protocol was established following screening of T. occidentale accessions originating from England, Ireland, France, Spain and Portugal. The protocol is based upon infection of cotyledonary explants dissected from mature seed with the Agrobacterium tumefaciens strain GV3101 carrying vectors which contain the bar selection marker gene. Transformation frequencies of up to 7.5% were achieved in 9 of the 17 accessions tested. Transformed plants were verified by PCR and expression of the gusA reporter gene, while integration of the T-DNA was confirmed by Southern blot hybridisation and segregation of progeny in the T1 generation.
Conclusions
Development of this protocol provides a valuable contribution toward establishing T. occidentale as a model species for white clover. This presents opportunities for further improvement in white clover through the application of biotechnology.
doi:10.1186/1746-4811-9-25
PMCID: PMC3716983  PMID: 23841995
Agrobacterium-mediated transformation; Trifolium occidentale; Genetic transformation; Organogenic regeneration; Accession
3.  Evolutionary origin of highly repetitive plastid genomes within the clover genus (Trifolium) 
BMC Evolutionary Biology  2014;14(1):228.
Background
Some clover species, particularly Trifolium subterraneum, have previously been reported to have highly unusual plastomes, relative to closely related legumes, enlarged with many duplications, gene losses and the presence of DNA unique to Trifolium, which may represent horizontal transfer. In order to pinpoint the evolutionary origin of this phenomenon within the genus Trifolium, we sequenced and assembled the plastomes of eight additional Trifolium species widely sampled from across the genus.
Results
The Trifolium plastomes fell into two groups: those of Trifolium boissieri, T. strictum and T. glanduliferum (representing subgenus Chronosemium and subg. Trifolium section Paramesus) were tractable, assembled readily and were not unusual in the general context of Fabeae plastomes. The other Trifolium species (“core Trifolium”) proved refractory to assembly mainly because of numerous short duplications. These species form a single clade, which we call the “refractory clade” (comprising subg, Trifolium sections Lupinaster, Trifolium, Trichocephalum, Vesicastrum and Trifoliastrum). The characteristics of the refractory clade are the presence of numerous short duplications and 7-15% longer genomes than the tractable species. Molecular dating estimates that the origin of the most recent common ancestor (MRCA) of the refractory clade is approximately 13.1 million years ago (MYA). This is considerably younger than the estimated MRCA ages of Trifolium (c. 18.6 MYA) and Trifolium subg. Trifolium (16.1 MYA).
Conclusions
We conclude that the unusual repetitive plastome type previously characterized in Trifolium subterraneum had a single origin within Trifolium and is characteristic of most (but not all) species of subgenus Trifolium. It appears that an ancestral plastome within Trifolium underwent an evolutionary change resulting in plastomes that either actively promoted, were permissive to, or were unable to control, duplications within the genome. The precise mechanism of this important change in the mode and tempo of plastome evolution deserves further investigation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-014-0228-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12862-014-0228-6
PMCID: PMC4241210  PMID: 25403617
Trifolium; Clover; Plastome evolution; Plastid genome evolution; Repetitive DNA; Fabaceae
4.  Genome mapping of white clover (Trifolium repens L.) and comparative analysis within the Trifolieae using cross-species SSR markers 
Allotetraploid white clover (Trifolium repens L.), a cool-season perennial legume used extensively as forage for livestock, is an important target for marker-assisted breeding. A genetic linkage map of white clover was constructed using simple sequence repeat (SSR) markers based on sequences from several Trifolieae species, including white clover, red clover (T. pratense L.), Medicago truncatula (Gaertn.) and soybean (Glycine max L.). An F1 population consisting of 179 individuals, from a cross between two highly heterozygous genotypes, GA43 and Southern Regional Virus Resistant, was used for genetic mapping. A total of 1,571 SSR markers were screened for amplification and polymorphism using DNA from two parents and 14 F1s of the mapping population. The map consists of 415 loci amplified from 343 SSR primer pairs, including 83 from white clover, 181 from red clover, 77 from M. truncatula, and two from soybean. Linkage groups for all eight homoeologous chromosome pairs of allotetraploid white clover were detected. Map length was estimated at 1,877 cM with 87% genome coverage. Map density was approximately 5 cM per locus. Segregation distortion was detected in six segments of the genome (homoeologous groups A1, A2, B1, B2, C1, and D1). A comparison of map locations of markers originating from white clover, red clover, and alfalfa (M. sativa L.) revealed putative macro-colinearity between the three Trifolieae species. This map can be used to link quantitative trait loci with SSR markers, and accelerate the improvement of white clover by marker-assisted selection and breeding.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-007-0523-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-007-0523-3
PMCID: PMC1950584  PMID: 17356868
5.  Genome-wide identification of microsatellites in white clover (Trifolium repens L.) using FIASCO and phpSSRMiner 
Plant Methods  2008;4:19.
Background
Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover.
Results
Genomic libraries containing simple sequence repeat (SSR) sequences were constructed using a modified Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) procedure and phpSSRMiner was used to develop the microsatellite markers. SSR motifs were isolated using two biotin-labeled probes, (CA)17 and (ATG)12. The sequences of 6,816 clones were assembled into 1,698 contigs, 32% of which represented novel sequences based on BLASTN searches. Approximately 32%, 28%, and 16% of these SSRs contained hexa-, tri-, and di-nucleotide repeats, respectively. The most frequent motifs were the CA and ATG complementary repeats and the associated compound sequences. Primer pairs were designed for 859 SSR loci based on sequences from these genomic libraries and from GenBank white clover nucleotide sequences. A total of 191 SSR primers developed from the two libraries were tested for polymorphism in individual clones from the parental genotypes GA43 ('Durana'), 'SRVR' and six F1 progeny from a mapping population. Ninety two percent produced amplicons and 66% of these were polymorphic.
Conclusion
The combined approach of identifying SSR-enriched fragments by FIASCO coupled with the primer design and in silico amplification using phpSSRMiner represents an efficient and low cost pipeline for the large-scale development of microsatellite markers in plants.
The approach described here could be readily adapted and utilized in other non-related species with none or limited genomic resources.
doi:10.1186/1746-4811-4-19
PMCID: PMC2517061  PMID: 18631390
6.  Flooding and fragment size interact to determine survival and regrowth after fragmentation in two stoloniferous Trifolium species 
AoB Plants  2014;6:plu024.
Clonal plants are common in frequently flooded habitats because of their resilience to disturbance. We investigated if submergence prior to fragmentation of clones of two clover species reduced survival and regrowth of clonal fragments, and if these fitness parameters were different between genotypes from highly disturbed river forelands and less disturbed coastal dune slacks. We found that soil flooding severely decreased survival and regrowth, and that plants from the more disturbance-prone habitat were less negatively affected by fragmentation. However, internode size was, surprisingly, often negatively correlated with survival after fragmentation, but positively correlated with regrowth. Apparently, contrasting selection pressures exist on internode size in stoloniferous species growing in disturbed habitats.
Clonal plants, which reproduce by means of stolons and rhizomes, are common in frequently flooded habitats. Resilience to disturbance is an important trait enabling plants to survive in such highly disturbed habitats. Resource storage is thought to enable clonal plants to resume growth after clonal fragmentation caused by disturbance. Here we investigated if submergence prior to disturbance reduces survival and regrowth of clonal fragments and whether or not genotypes originating from highly disturbed riverine habitats are more resistant to mechanical disturbance than genotypes from less disturbed coastal dune slack habitats. We further tested if variation in survival and regrowth was affected by internode size. Clones from contrasting habitats of two closely related Trifolium species were first genotypically characterized by amplification fragment length polymorphism and then subjected to soil flooding and subsequent clonal fragmentation. These species differ with respect to their abundance in riverine and dune slack habitats, with Trifolium repens mainly occurring in riverine grasslands and Trifolium fragiferum in coastal dune slacks. Soil flooding decreased survival and regrowth by up to 80 %. Plants originating from riverine grasslands were less negatively affected by fragmentation than plants from dune slack habitats. Surprisingly, ramets did not always benefit from being attached to a larger internode, as internode size was often negatively correlated with survival after fragmentation. Regrowth, on the other hand, was generally positively correlated with internode size. These unexpected results indicate that there may be contrasting selection pressures on internode size in stoloniferous species growing in severely disturbed habitats.
doi:10.1093/aobpla/plu024
PMCID: PMC4062869  PMID: 24887003
Clonal growth; disturbance; fragmentation; soil flooding; Trifolium fragiferum; Trifolium repens.
7.  Genome sequence of Rhizobium leguminosarum bv trifolii strain WSM1689, the microsymbiont of the one flowered clover Trifolium uniflorum 
Standards in Genomic Sciences  2013;9(3):527-539.
Rhizobium leguminosarum bv. trifolii is a soil-inhabiting bacterium that has the capacity to be an effective N2-fixing microsymbiont of Trifolium (clover) species. R. leguminosarum bv. trifolii strain WSM1689 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Trifolium uniflorum collected on the edge of a valley 6 km from Eggares on the Greek Island of Naxos. Although WSM1689 is capable of highly effective N2-fixation with T. uniflorum, it is either unable to nodulate or unable to fix N2 with a wide range of both perennial and annual clovers originating from Europe, North America and Africa. WSM1689 therefore possesses a very narrow host range for effective N2 fixation and can thus play a valuable role in determining the geographic and phenological barriers to symbiotic performance in the genus Trifolium. Here we describe the features of R. leguminosarum bv. trifolii strain WSM1689, together with the complete genome sequence and its annotation. The 6,903,379 bp genome contains 6,709 protein-coding genes and 89 RNA-only encoding genes. This multipartite genome contains six distinct replicons; a chromosome of size 4,854,518 bp and five plasmids of size 667,306, 518,052, 341,391, 262,704 and 259,408 bp. This rhizobial genome is one of 20 sequenced as part of a DOE Joint Genome Institute 2010 Community Sequencing Program.
doi:10.4056/sigs.4988693
PMCID: PMC4149022  PMID: 25197438
root-nodule bacteria; nitrogen fixation; lupin-nodulating; rhizobia; Alphaproteobacteria
8.  The formation of diploid and triploid hybrids of female grass carp × male blunt snout bream and their 5S rDNA analysis 
BMC Genetics  2013;14:110.
Background
Hybridization is a useful strategy to alter the genotypes and phenotypes of the offspring. It could transfer the genome of one species to another through combing the different genome of parents in the hybrid offspring. And the offspring may exhibit advantages in growth rate, disease resistance, survival rate and appearance, which resulting from the combination of the beneficial traits from both parents.
Results
Diploid and triploid hybrids of female grass carp (Ctenopharyngodon idellus, GC, Cyprininae, 2n = 48) × male blunt snout bream (Megalobrama amblycephala, BSB, Cultrinae, 2n = 48) were successfully obtained by distant hybridization. Diploid hybrids had 48 chromosomes, with one set from GC and one set from BSB. Triploid hybrids possessed 72 chromosomes, with two sets from GC and one set from BSB.
The morphological traits, growth rates, and feeding ecology of the parents and hybrid offspring were compared and analyzed. The two kinds of hybrid offspring exhibited significantly phenotypic divergence from GC and BSB. 2nGB hybrids showed similar growth rate compared to that of GC, and 3nGB hybrids significantly higher results. Furthermore, the feeding ecology of hybrid progeny was omnivorous.
The 5S rDNA of GC, BSB and their hybrid offspring were also cloned and sequenced. There was only one type of 5S rDNA (designated type I: 180 bp) in GC and one type of 5S rDNA (designated type II: 188 bp) in BSB. However, in the hybrid progeny, diploid and triploid hybrids both inherited type I and type II from their parents, respectively. In addition, a chimera of type I and type II was observed in the genome of diploid and triploid hybrids, excepting a 10 bp of polyA insertion in type II sequence of the chimera of the diploid hybrids.
Conclusions
This is the first report of diploid and triploid hybrids being produced by crossing GC and BSB, which have the same chromosome number. The obtainment of two new hybrid offspring has significance in fish genetic breeding. The results illustrate the effect of hybridization and polyploidization on the organization and variation of 5S rDNA in hybrid offspring.
doi:10.1186/1471-2156-14-110
PMCID: PMC4222567  PMID: 24267392
Hybridization; Ployploidization; Fish genetic breeding; 5S rDNA
9.  Comparison of homoeolocus organisation in paired BAC clones from white clover (Trifolium repens L.) and microcolinearity with model legume species 
BMC Plant Biology  2010;10:94.
Background
White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised.
Results
A total of four paired homoeologous BACs were selected and sequenced to generate 173 kb of overlapping sequence between the O and P' sub-genomes. Equivalent gene content was generally observed, apart from small-scale deletions, in contrast to conservation of intergenic sequences, which varied between the four selected regions. Measurement of the number of synonymous substitutions between homoeologous genes led to estimation of a 4.2 million year divergence time between the two sub-genomes. Microsynteny was observed between the genomes of white clover and L. japonicus for all four targeted regions, but corresponding M. truncatula genomic regions were only identified for two BAC pairs.
Conclusions
This study describes the first analysis of sub-genome structural conservation across selected genomic regions in white clover. Although the high levels of sequence conservation between the O and P' sub-genomes would complicate efforts for whole genome sequence assembly, the conserved microsynteny with model legume genomes, especially that of L. japonicus, will be highly valuable for the future of white clover genomics and molecular breeding.
doi:10.1186/1471-2229-10-94
PMCID: PMC3095360  PMID: 20492736
10.  Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii 
Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.
Images
PMCID: PMC202882  PMID: 16347936
11.  Comparative Genetic Mapping and Discovery of Linkage Disequilibrium Across Linkage Groups in White Clover (Trifolium repens L.) 
G3: Genes|Genomes|Genetics  2012;2(5):607-617.
White clover (Trifolium repens L.) is an allotetraploid species (2n = 4X = 32) that is widely distributed in temperate regions and cultivated as a forage legume. In this study, we developed expressed sequence tag (EST)–derived simple sequence repeat (SSR) markers, constructed linkage maps, and performed comparative mapping with other legume species. A total of 7982 ESTs that could be assembled into 5400 contigs and 2582 singletons were generated. Using the EST sequences that were obtained, 1973 primer pairs to amplify EST-derived SSR markers were designed and used for linkage analysis of 188 F1 progenies, which were generated by a cross between two Japanese plants, ‘273-7’ and ‘T17-349,’ with previously published SSR markers. An integrated linkage map was constructed by combining parental-specific maps, which consisted of 1743 SSR loci on 16 homeologous linkage groups with a total length of 2511 cM. The primer sequences of the developed EST-SSR markers and their map positions are available on http://clovergarden.jp/. Linkage disequilibrium (LD) was observed on 9 of 16 linkage groups of a parental-specific map. The genome structures were compared among white clover, red clover (T. pratense L.), Medicago truncatula, and Lotus japonicus. Macrosynteny was observed across the four legume species. Surprisingly, the comparative genome structure between white clover and M. truncatula had a higher degree of conservation than that of the two clover species.
doi:10.1534/g3.112.002600
PMCID: PMC3362943  PMID: 22670230
comparative map; white clover; linkage disequilibrium; expressed sequence tag–simple sequence repeat
12.  Genome rearrangements and pervasive meiotic drive cause hybrid infertility in fission yeast 
eLife  2014;3:e02630.
Hybrid sterility is one of the earliest postzygotic isolating mechanisms to evolve between two recently diverged species. Here we identify causes underlying hybrid infertility of two recently diverged fission yeast species Schizosaccharomyces pombe and S. kambucha, which mate to form viable hybrid diploids that efficiently complete meiosis, but generate few viable gametes. We find that chromosomal rearrangements and related recombination defects are major but not sole causes of hybrid infertility. At least three distinct meiotic drive alleles, one on each S. kambucha chromosome, independently contribute to hybrid infertility by causing nonrandom spore death. Two of these driving loci are linked by a chromosomal translocation and thus constitute a novel type of paired meiotic drive complex. Our study reveals how quickly multiple barriers to fertility can arise. In addition, it provides further support for models in which genetic conflicts, such as those caused by meiotic drive alleles, can drive speciation.
DOI: http://dx.doi.org/10.7554/eLife.02630.001
eLife digest
It is widely thought that all of the billions of species on Earth are descended from a common ancestor. New species are created via a process called speciation, and nature employs various ‘barriers’ to keep closely related species distinct from one another. One of these barriers is called hybrid sterility. Horses and donkeys, for example, can mate to produce hybrids called mules, but mules cannot produce offspring of their own because they are infertile.
Hybrid sterility can occur for a number of reasons. Mules are infertile because they inherit 32 chromosomes from their horse parent, but only 31 chromosomes from their donkey parent—and so have an odd chromosome that they cannot pair-off when they make sperm or egg cells. However, even if a hybrid inherits the same number of chromosomes from each parent, if the chromosomes from the two parents have different structures, the hybrid may still be infertile.
Zanders et al. have now looked at two species of fission yeast—S. pombe and S. kambucha—that share 99.5% of their DNA sequence. Although hybrids of these two species inherit three chromosomes from each parent, the majority of spores (the yeast equivalent of sperm) that these hybrids produce fail to develop into new yeast cells. Zanders et al. identified two causes of this infertility: one of these was chromosomal rearrangement; the other was due to three different sites in the DNA of S. kambucha that interfere with the development of the spores that inherit S. pombe chromosomes.
Since these two yeast species are so closely related, the findings of Zanders et al. reveal how quickly multiple barriers to fertility can arise. In addition, these findings provide further support for models in which conflicts between different genes in genomes can drive the process of speciation.
DOI: http://dx.doi.org/10.7554/eLife.02630.002
doi:10.7554/eLife.02630
PMCID: PMC4066438  PMID: 24963140
speciation; meiotic drive; chromosomal rearrangements; recombination; S. pombe
13.  Worldwide Patterns of Ancestry, Divergence, and Admixture in Domesticated Cattle 
PLoS Genetics  2014;10(3):e1004254.
The domestication and development of cattle has considerably impacted human societies, but the histories of cattle breeds and populations have been poorly understood especially for African, Asian, and American breeds. Using genotypes from 43,043 autosomal single nucleotide polymorphism markers scored in 1,543 animals, we evaluate the population structure of 134 domesticated bovid breeds. Regardless of the analytical method or sample subset, the three major groups of Asian indicine, Eurasian taurine, and African taurine were consistently observed. Patterns of geographic dispersal resulting from co-migration with humans and exportation are recognizable in phylogenetic networks. All analytical methods reveal patterns of hybridization which occurred after divergence. Using 19 breeds, we map the cline of indicine introgression into Africa. We infer that African taurine possess a large portion of wild African auroch ancestry, causing their divergence from Eurasian taurine. We detect exportation patterns in Asia and identify a cline of Eurasian taurine/indicine hybridization in Asia. We also identify the influence of species other than Bos taurus taurus and B. t. indicus in the formation of Asian breeds. We detect the pronounced influence of Shorthorn cattle in the formation of European breeds. Iberian and Italian cattle possess introgression from African taurine. American Criollo cattle originate from Iberia, and not directly from Africa with African ancestry inherited via Iberian ancestors. Indicine introgression into American cattle occurred in the Americas, and not Europe. We argue that cattle migration, movement and trading followed by admixture have been important forces in shaping modern bovine genomic variation.
Author Summary
The DNA of domesticated plants and animals contains information about how species were domesticated, exported, and bred by early farmers. Modern breeds were developed by lengthy and complex processes; however, our use of 134 breeds and new analytical models enabled us to reveal some of the processes that created modern cattle diversity. In Asia, Africa, North and South America, humpless (Bos t. taurus or taurine) and humped (Bos t. indicus or indicine) cattle were crossbred to produce hybrids adapted to the environment and local production systems. The history of Asian cattle involves the domestication and admixture of several species whereas African taurines arose through the introduction of domesticated Fertile Crescent taurines and their hybridization with wild African aurochs. African taurine genetic background is commonly observed among European Mediterranean breeds. The absence of indicine introgression within most European taurine breeds, but presence within three Italian breeds is consistent with at least two separate migration waves of cattle to Europe, one from the Middle East which captured taurines in which indicine introgression had already occurred and the second from western Africa into Spain with no indicine introgression. This second group seems to have radiated from Spain into the Mediterranean resulting in a cline of African taurine introgression into European taurines.
doi:10.1371/journal.pgen.1004254
PMCID: PMC3967955  PMID: 24675901
14.  A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover 
BMC Genomics  2013;14:100.
Background
White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study.
Results
We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase.
Conclusions
In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for white clover genomics and genetics studies. We discuss the potential to extend the analysis to identify a “core set” of ancestrally derived homeolog specific variants in white clover.
doi:10.1186/1471-2164-14-100
PMCID: PMC3727989  PMID: 23402685
Allelic variants; Allotetraploid; Haplotype reconstruction; Next generation sequencing; Single nucleotide polymorphisms; Sub-genome; Transcript assembly; Trifolium; White clover
15.  Genetic Basis for Dosage Sensitivity in Arabidopsis thaliana 
PLoS Genetics  2007;3(4):e70.
Aneuploidy, the relative excess or deficiency of specific chromosome types, results in gene dosage imbalance. Plants can produce viable and fertile aneuploid individuals, while most animal aneuploids are inviable or developmentally abnormal. The swarms of aneuploid progeny produced by Arabidopsis triploids constitute an excellent model to investigate the mechanisms governing dosage sensitivity and aneuploid syndromes. Indeed, genotype alters the frequency of aneuploid types within these swarms. Recombinant inbred lines that were derived from a triploid hybrid segregated into diploid and tetraploid individuals. In these recombinant inbred lines, a single locus, which we call SENSITIVE TO DOSAGE IMBALANCE (SDI), exhibited segregation distortion in the tetraploid subpopulation only. Recent progress in quantitative genotyping now allows molecular karyotyping and genetic analysis of aneuploid populations. In this study, we investigated the causes of the ploidy-specific distortion at SDI. Allele frequency was distorted in the aneuploid swarms produced by the triploid hybrid. We developed a simple quantitative measure for aneuploidy lethality and using this measure demonstrated that distortion was greatest in the aneuploids facing the strongest viability selection. When triploids were crossed to euploids, the progeny, which lack severe aneuploids, exhibited no distortion at SDI. Genetic characterization of SDI in the aneuploid swarm identified a mechanism governing aneuploid survival, perhaps by buffering the effects of dosage imbalance. As such, SDI could increase the likelihood of retaining genomic rearrangements such as segmental duplications. Additionally, in species where triploids are fertile, aneuploid survival would facilitate gene flow between diploid and tetraploid populations via a triploid bridge and prevent polyploid speciation. Our results demonstrate that positional cloning of loci affecting traits in populations containing ploidy and chromosome number variants is now feasible using quantitative genotyping approaches.
Author Summary
Each eukaryotic genome is subdivided into a specific number of chromosome types, which in turn are present in a characteristic number of copies, usually the same for all chromosomes. In the condition called aneuploidy, copy number differs among chromosome types, disrupting their balance and that of their encoded factors. As a result, aneuploidy is associated with developmental defects and death. For example, most types of human aneuploids are unviable: the only autosomal aneuploidy compatible with protracted survival, Down syndrome, is caused by the presence of three copies, instead of two, of the very small Chromosome 21. In plants, aneuploidy is more common and less deleterious. This suggests that plants can more easily tolerate the effects of aneuploidy and can be used to investigate them. Here, we used the model plant Arabidopsis thaliana to produce and investigate populations of aneuploid individuals. By comparing genetically distinct aneuploid populations, we identified a chromosomal region that is associated with greater aneuploid survival. Characterizing the genetic mechanism modulating the response to changes in chromosomal dosage and aneuploid survival will help understand how genome organization affects biological processes and why aneuploidy results in such severe developmental defects.
doi:10.1371/journal.pgen.0030070
PMCID: PMC1857734  PMID: 17465685
16.  Expression and functional characterization of a white clover isoflavone synthase in tobacco 
Annals of Botany  2012;110(6):1291-1301.
Background and Aims
Trifolium repens (white clover) is a valuable component of pastures due to its ability to fix nitrogen. Productivity of T. repens is sometimes threatened by insect pests, and it has been suggested that phenylpropanoid-derived isoflavonoids such as formononetin can protect white clover from insect damage. The aim of this study was to isolate and functionally characterize an isoflavone synthase (IFS2_12) from T. repens by expressing it in Nicotiana tabacum (tobacco), a plant which does not naturally produce isoflavonoids.
Methods
To induce anthocyanin production and increase isoflavonoid precursors in tobacco, the tomato R2R3 MYB transcription factor ANT1 was expressed in tobacco (Nt-ANT1 plants). IFS2_12 was heterologously expressed in tobacco both transiently and stably, and isoflavonoids in leaf extracts were analysed by liquid chromatography (LC) coupled to mass spectrometry (MSn). As a positive control, a double construct of soybean IFS and alfalfa chalcone isomerase (IFS/CHI), which had been previously shown to induce isoflavonoid production in tobacco, was also expressed. Stable transformants expressing IFS2_12, soybean/alfalfa IFS/CHI and ANT1 were crossed and the resulting plants were analysed for isoflavonoid production.
Key results
Leaves of tobacco plants expressing ANT1 had a range of phenotypes from mainly green to uniformly bronze coloured. Both transient and stable expression of the IFS2_12 or IFS/CHI constructs resulted in the production of the isoflavonoid genistein and its conjugates. The highest levels (up to 19·2 mg g−1 d. wt) accumulated in a progeny of a cross between a purple ANT1 and a IFS/ CHI transformant, while the second highest concentration was found in a plant derived from a selfed IFS2-12 transformant.
Conclusions
It is concluded that the gene IFS2_12 isolated from T. repens encodes an isoflavone synthase. This study paves the way for engineering white clover plants with higher levels of isoflavonoids than naturally found in this species for sufficient insect protection.
doi:10.1093/aob/mcs168
PMCID: PMC3478045  PMID: 22915577
Trifolium repens; Nicotiana tabacum; isoflavone synthase; isoflavonoid biosynthesis; transient gene expression; liquid chromatography–mass spectrometry; genistein
17.  The Genomics of Speciation in Drosophila: Diversity, Divergence, and Introgression Estimated Using Low-Coverage Genome Sequencing 
PLoS Genetics  2009;5(7):e1000550.
In nature, closely related species may hybridize while still retaining their distinctive identities. Chromosomal regions that experience reduced recombination in hybrids, such as within inversions, have been hypothesized to contribute to the maintenance of species integrity. Here, we examine genomic sequences from closely related fruit fly taxa of the Drosophila pseudoobscura subgroup to reconstruct their evolutionary histories and past patterns of genic exchange. Partial genomic assemblies were generated from two subspecies of Drosophila pseudoobscura (D. ps.) and an outgroup species, D. miranda. These new assemblies were compared to available assemblies of D. ps. pseudoobscura and D. persimilis, two species with overlapping ranges in western North America. Within inverted regions, nucleotide divergence among each pair of the three species is comparable, whereas divergence between D. ps. pseudoobscura and D. persimilis in non-inverted regions is much lower and closer to levels of intraspecific variation. Using molecular markers flanking each of the major chromosomal inversions, we identify strong crossover suppression in F1 hybrids extending over 2 megabase pairs (Mbp) beyond the inversion breakpoints. These regions of crossover suppression also exhibit the high nucleotide divergence associated with inverted regions. Finally, by comparison to a geographically isolated subspecies, D. ps. bogotana, our results suggest that autosomal gene exchange between the North American species, D. ps. pseudoobscura and D. persimilis, occurred since the split of the subspecies, likely within the last 200,000 years. We conclude that chromosomal rearrangements have been vital to the ongoing persistence of these species despite recent hybridization. Our study serves as a proof-of-principle on how whole genome sequencing can be applied to formulate and test hypotheses about species formation in lesser-known non-model systems.
Author Summary
The transformation of populations into distinct species depends on whether hybridization, recombination, and subsequent gene introgression can be suppressed between diverging species. We use partial genome sequences to reconstruct this evolutionary process in the Drosophila pseudoobscura species subgroup, which includes the hybridizing species pair D. pseudoobscura pseudoobscura and D. persimilis. Recent models suggest that chromosomal inversions can facilitate the persistence of hybridizing species because of their effects on recombination, whereby inverted regions would exhibit higher nucleotide divergence than non-inverted regions. Indeed, D. pseudoobscura-D. persimilis nucleotide divergence outside these inverted regions is lower than within or near inversions, resembling D. ps. pseudoobscura levels of within-species nucleotide diversity. We also observe that recombination suppression in F1 hybrids extends greater than 2 Mbp outside the inversion breakpoints. Furthermore, when genomic sequence of D. persimilis is compared to two sister subspecies—the hybridizing subspecies, D. ps. pseudoobscura, and a non-hybridizing control subspecies, D. ps. bogotana—autosomal divergence is lower in the former, demonstrating recent gene exchange. These lines of evidence support a speciation model in which the two hybridizing species persist despite the presence of recent genic introgression in collinear regions of the genome because of the reduced recombinational effects of the inversions that distinguish them.
doi:10.1371/journal.pgen.1000550
PMCID: PMC2696600  PMID: 19578407
18.  Morphological Compatibility of White Clover and Perennial Ryegrass Cultivars Grown under Two Nitrate Levels in Flowing Solution Culture 
Annals of Botany  2003;92(2):247-258.
The effects of nitrate (NO3–) supply on shoot morphology, vertical distribution of shoot and root biomass and total nitrogen (N) acquisition by two perennial ryegrass (Lolium perenne L.) cultivars (AberElan and Preference) and two white clover (Trifolium repens L.) cultivars (Grasslands Huia and AberHerald) were studied in flowing nutrient culture. Cultivars were grown from seed as monocultures and the clovers inoculated with Rhizobium. The 6‐week measurement period began on day 34 (grasses) and day 56 (clovers) when the NO3– supply was adjusted to either 2 mmol m–3 (low nitrogen, LN) or 50 mmol m–3 (high nitrogen, HN). These treatments were subsequently maintained automatically. Plants were harvested at intervals to measure their morphology and N content. Cultivars of both species differed significantly in several aspects of their response to NO3– supply. In the grasses, the LN treatment increased the root : shoot ratio of AberElan but did not affect the distribution of root length in the root profile. In contrast, this treatment changed the root distribution of Preference compared with HN, resulting in a larger proportion of root length being distributed further down the root profile. The morphology of white clover Grasslands Huia was for the most part unaffected by the level of NO3– supply. In contrast, AberHerald exhibited different growth strategies, with LN plants increasing their stolon weight per unit length at the expense of leaf production, leaf area and stolon length, whereas HN plants showed reduced stolon thickness, greater leaf area production and stolon length per plant. Cultivars with different morphological/physiological strategies in response to NO3– supply may be of value in the construction of ‘compatible mixtures’ aimed at reducing oscillations in sward clover content by extending the range of conditions that allow balanced coexistence of species to occur.
doi:10.1093/aob/mcg128
PMCID: PMC4243651  PMID: 12876188
Lolium perenne L.; perennial ryegrass; Trifolium repens L.; white clover; nitrate supply; morphology; compatibility
19.  Recent Natural Hybridization between Two Allopolyploid Wheatgrasses (Elytrigia, Poaceae): Ecological and Evolutionary Implications 
Annals of Botany  2007;100(2):249-260.
Background and Aims
Natural hybridization was investigated between two predominantly allohexaploid wheatgrasses, weedy Elytrigia repens and steppic E. intermedia, with respect to habitats characterized by different degrees of anthropogenic disturbance.
Methods
Using flow cytometry (relative DNA content), 269 plants from three localities were analysed. Hybrids were further analysed using nuclear ribosomal (ITS1-5·8S-ITS2 region) and chloroplast (trnT-F region) DNA markers in addition to absolute DNA content and chromosome numbers.
Key Results
Weedy E. repens was rare in a steppic locality whereas E. intermedia was almost absent at two sites of agricultural land-use. Nevertheless, hybrids were common there whereas none were found at the steppic locality, underlining the importance of different ecological conditions for hybrid formation or establishment. At one highly disturbed site, > 16 % of randomly collected plants were hybrids. Hexaploid hybrids showed intermediate genome size compared with the parents and additive patterns of parental ITS copies. Some evidence of backcrosses was found. The direction of hybridization was highly asymmetric as cpDNA identified E. intermedia as the maternal parent in 61 out of 63 cases. Out of nine nonaploid cytotypes (2n = 9x = 63) which likely originated by fusion of unreduced and reduced gametes of hexaploids, eight were hybrids whereas one was a nonaploid cytotype of E. repens. The progeny of one nonaploid hybrid demonstrated gene flow between hexaploid and nonaploid cytotypes.
Conclusions
The results show that E. repens and E. intermedia frequently cross at places where they co-occur. Hybrid frequency is likely influenced by habitat type; sites disturbed by human influence sustain hybrid formation and/or establishment. Hexaploid and nonaploid hybrid fertility is not negligible, backcrossing is possible, and the progeny is variable. The frequent production of new at least partially fertile cyto- and genotypes provides ample raw material for evolution and adaptation.
doi:10.1093/aob/mcm093
PMCID: PMC2735307  PMID: 17562679
Triticeae; Poaceae; Elytrigia repens; Elytrigia intermedia; hybridization; polyploidy; chloroplast DNA; internal transcribed spacer; genome size; adaptation
20.  N2O induces mitotic polyploidization in anther somatic cells and restores fertility in sterile interspecific hybrid lilies 
Breeding Science  2011;61(4):327-337.
Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N2O) gas to obtain 2n male gametes. N2O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is un-known how N2O treatment restores fertility in sterile hybrids. To establish optimal N2O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N2O gas at different anther developmental stages from fertile and sterile hybrid lilies. N2O treatment using the optimal 1–4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N2O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC1 plants. Thus N2O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.
doi:10.1270/jsbbs.61.327
PMCID: PMC3406768  PMID: 23136469
chromosome doubling; diploid male gamete; hybrid sterility; interspecific hybridization; Lilium; nitrous oxide; polyploid
21.  Interallelic and Intergenic Incompatibilities of the Prdm9 (Hst1) Gene in Mouse Hybrid Sterility 
PLoS Genetics  2012;8(11):e1003044.
The Dobzhansky-Muller model of incompatibilities explains reproductive isolation between species by incorrect epistatic interactions. Although the mechanisms of speciation are of great interest, no incompatibility has been characterized at the gene level in mammals. The Hybrid sterility 1 gene (Hst1) participates in the arrest of meiosis in F1 males of certain strains from two Mus musculus subspecies, e.g., PWD from M. m. musculus and C57BL/6J (henceforth B6) from M. m. domesticus. Hst1 has been identified as a meiotic PR-domain gene (Prdm9) encoding histone 3 methyltransferase in the male offspring of PWD females and B6 males, (PWD×B6)F1. To characterize the incompatibilities underlying hybrid sterility, we phenotyped reproductive and meiotic markers in males with altered copy numbers of Prdm9. A partial rescue of fertility was observed upon removal of the B6 allele of Prdm9 from the azoospermic (PWD×B6)F1 hybrids, whereas removing one of the two Prdm9 copies in PWD or B6 background had no effect on male reproduction. Incompatibility(ies) not involving Prdm9B6 also acts in the (PWD×B6)F1 hybrids, since the correction of hybrid sterility by Prdm9B6 deletion was not complete. Additions and subtractions of Prdm9 copies, as well as allelic replacements, improved meiotic progression and fecundity also in the progeny-producing reciprocal (B6×PWD)F1 males. Moreover, an increased dosage of Prdm9 and reciprocal cross enhanced fertility of other sperm-carrying male hybrids, (PWD×B6-C3H.Prdm9)F1, harboring another Prdm9 allele of M. m. domesticus origin. The levels of Prdm9 mRNA isoforms were similar in the prepubertal testes of all types of F1 hybrids of PWD with B6 and B6-C3H.Prdm9 despite their different prospective fertility, but decreased to 53% after removal of Prdm9B6. Therefore, the Prdm9B6 allele probably takes part in posttranscriptional dominant-negative hybrid interaction(s) absent in the parental strains.
Author Summary
Disturbed gametogenesis in the progeny of two fertile parental forms is called hybrid sterility; it is an important part of reproductive barriers between species. The Dobzhansky-Muller model of incompatibilities explains reproductive isolation between species by incorrect interactions between genes. Hybrid sterility 1 (Hst1) is one of the genes causing meiotic arrest in F1 male hybrids between certain Mus musculus musculus (e.g., the PWD strain) and M. m. domesticus (C57BL/6J etc.) mice. Hst1, the first mammalian candidate for a speciation gene, was identified as a meiotic PR/SET-domain gene, Prdm9, but the mechanism causing sterility has remained unknown. While the F1 male offspring of C57BL/6J males and PWD females produce no sperm, the males from the reciprocal cross using PWD males and C57BL/6J females yield progeny. Here we show that the meiotic progress and fertility of hybrid males from both F1 crosses improved by removal as well as overexpression of the C57BL/6J allele of Prdm9, suggesting that Prdm9 interactions not present in the parental species (incompatibilities) play a role in hybrid sterility. Furthermore, the Prdm9 dosage also controlled fecundity in other F1 hybrids, indicating that this gene is an important regulator of mouse hybrid fertility.
doi:10.1371/journal.pgen.1003044
PMCID: PMC3486856  PMID: 23133405
22.  Longevity of White Clover (Trifolium repens) Leaves, Stolons and Roots, and Consequences for Nitrogen Dynamics under Northern Temperate Climatic Conditions 
Annals of Botany  2007;100(1):33-40.
Background and Aims
White clover (Trifolium repens) is, due to nitrogen (N) fixation, important to the N dynamics of several northern temperate agroecosystems. This study aimed at monitoring growth and death of major white clover plant organs to assess their potential contribution to within-season N input and risk of off-season N losses.
Methods
White clover (‘Snowy’) was studied in a plot and root window experiment in southeast Norway (60°42′N, 10°51′E). Leaves, stolons and roots were tagged for lifespan measurement in harvested and unharvested stands during two experimental years. The availability of soil inorganic N was measured by plant root simulator (PRS™) probes.
Key Results
The longevity of leaves and petioles ranged from 21 to 86 d (mean = 59 d), of main stolon sections from 111 to over 677 d (mean = 411 d) and of roots from 27 to 621 d (mean = 290 d). About 60 % of the leaves produced had turned over by the end of the growing season and another 30 % had died or disappeared by the subsequent spring. Harvesting reduced the longevity of stolons and increased plant fragmentation, but did not decrease leaf or root lifespan or increase soil N availability. From the plant organ turnover data, it was estimated that the gross N input to the soil–plant system from white clover in pure stand during two growing seasons corresponded to a 2·5-fold increase over the total N in harvestable shoots.
Conclusions
The short lifespan and poor over-wintering of leaves showed their potential importance as a nitrogen source in the soil–plant ecosystem but also their potential contribution to the risk of off-season N losses.
doi:10.1093/aob/mcm078
PMCID: PMC2735287  PMID: 17495980
Harvesting; inorganic nitrogen; lifespan; leaves; main stolons; roots; Trifolium repens; turnover; white clover
23.  Biodiversity effects on yield and unsown species invasion in a temperate forage ecosystem 
Annals of Botany  2009;103(6):913-921.
Background and Aims
Current agricultural practices are based on growing monocultures or binary mixtures over large areas, with a resultant impoverishing effect on biodiversity at several trophic levels. The effects of increasing the biodiversity of a sward mixture on dry matter yield and unsown species invasion were studied.
Methods
A field experiment involving four grassland species [two grasses – perennial ryegrass (Lolium perenne) and cocksfoot (Dactylis glomerata) – and two legumes – red clover (Trifolium pratense) and white clover (Trifolium repens)], grown in monocultures and mixtures in accordance with a simplex design, was carried out. The legumes were included either as single varieties or as one of two broad genetic-base composites. The experiment was harvested three times a year over three years; dry matter yield and yield of unsown species were determined at each harvest. Yields of individual species and interactions between all species present were estimated through a statistical modelling approach.
Key Results
Species diversity produced a strong positive yield effect that resulted in transgressive over-yielding in the second and third years. Using broad genetic-base composites of the legumes had a small impact on yield and species interactions. Invasion by unsown species was strongly reduced by species diversity, but species identity was also important. Cocksfoot and white clover (with the exception of one broad genetic-base composite) reduced invasion, while red clover was the most invaded species.
Conclusions
The results show that it is possible to increase, and stabilize, the yield of a grassland crop and reduce invasion by unsown species by increasing its species diversity.
doi:10.1093/aob/mcp008
PMCID: PMC2707887  PMID: 19168861
Cocksfoot; Dactylis glomerata; diversity effect; invasion; legumes; perennial ryegrass; Lolium perenne; red clover; Trifolium pratense; simplex design; statistical modelling; transgressive over-yielding; white clover; T. repens
24.  Evidence for Mito-Nuclear and Sex-Linked Reproductive Barriers between the Hybrid Italian Sparrow and Its Parent Species 
PLoS Genetics  2014;10(1):e1004075.
Studies of reproductive isolation between homoploid hybrid species and their parent species have rarely been carried out. Here we investigate reproductive barriers between a recently recognized hybrid bird species, the Italian sparrow Passer italiae and its parent species, the house sparrow P. domesticus and Spanish sparrow P. hispaniolensis. Reproductive barriers can be difficult to study in hybrid species due to lack of geographical contact between taxa. However, the Italian sparrow lives parapatrically with the house sparrow and both sympatrically and parapatrically with the Spanish sparrow. Through whole-transcriptome sequencing of six individuals of each of the two parent species we identified a set of putatively parent species-diagnostic single nucleotide polymorphism (SNP) markers. After filtering for coverage, genotyping success (>97%) and multiple SNPs per gene, we retained 86 species-informative, genic, nuclear and mitochondrial SNP markers from 84 genes for analysis of 612 male individuals. We show that a disproportionately large number of sex-linked genes, as well as the mitochondria and nuclear genes with mitochondrial function, exhibit sharp clines at the boundaries between the hybrid and the parent species, suggesting a role for mito-nuclear and sex-linked incompatibilities in forming reproductive barriers. We suggest that genomic conflict via interactions between mitochondria and sex-linked genes with mitochondrial function (“mother's curse”) at one boundary and centromeric drive at the other may best explain our findings. Hybrid speciation in the Italian sparrow may therefore be influenced by mechanisms similar to those involved in non-hybrid speciation, but with the formation of two geographically separated species boundaries instead of one. Spanish sparrow alleles at some loci have spread north to form reproductive barriers with house sparrows, while house sparrow alleles at different loci, including some on the same chromosome, have spread in the opposite direction to form barriers against Spanish sparrows.
Author Summary
Hybridization between two species has the potential to create a third, hybrid species. However this process, known as hybrid speciation, is thought to be unlikely because it requires reproductive barriers against both parent species to develop despite the barriers between parents being weak enough to allow for the formation of viable, fertile hybrids. The Italian sparrow, which occupies the entire Italian peninsula and some Mediterranean islands, is the product of past hybridization between house and Spanish sparrows and therefore represents one of the few documented cases of vertebrate hybrid speciation in nature. We show that reproductive barriers between Italian sparrows and their parent species exist and that genes on the sex (Z) chromosome and mitochondria are heavily involved. We suggest that speciation in this system may have been driven by dissociation of the sex (Z) chromosome into blocks of different parent species-specific genes, which have shifted alongside mitochondrial genes to form reproductive barriers where the hybrid now meets each of its parent species.
doi:10.1371/journal.pgen.1004075
PMCID: PMC3886922  PMID: 24415954
25.  Different genome-specific chromosome stabilities in synthetic Brassica allohexaploids revealed by wide crosses with Orychophragmus 
Annals of Botany  2009;104(1):19-31.
Background and Aims
In sexual hybrids between cultivated Brassica species and another crucifer, Orychophragmus violaceus (2n = 24), parental genome separation during mitosis and meiosis is under genetic control but this phenomenon varies depending upon the Brassica species. To further investigate the mechanisms involved in parental genome separation, complex hybrids between synthetic Brassica allohexaploids (2n = 54, AABBCC) from three sources and O. violaceus were obtained and characterized.
Methods
Genomic in situ hybridization, amplified fragment length polymorphism (AFLP) and single-strand conformation polymorphism (SSCP) were used to explore chromosomal/genomic components and rRNA gene expression of the complex hybrids and their progenies.
Key Results
Complex hybrids with variable fertility exhibited phenotypes that were different from the female allohexaploids and expressed some traits from O. violaceus. These hybrids were mixoploids (2n = 34–46) and retained partial complements of allohexaploids, including whole chromosomes of the A and B genomes and some of the C genome but no intact O. violaceus chromosomes; AFLP bands specific for O. violaceus, novel for two parents and absent in hexaploids were detected. The complex hybrids produced progenies with chromosomes/genomic complements biased to B. juncea (2n = 36, AABB) and novel B. juncea lines with two genomes of different origins. The expression of rRNA genes from B. nigra was revealed in all allohexaploids and complex hybrids, showing that the hierarchy of nucleolar dominance (B. nigra, BB > B. rapa, AA > B. oleracea, CC) in Brassica allotetraploids was still valid in these plants.
Conclusions
The chromosomes of three genomes in these synthetic Brassica allohexaploids showed different genome-specific stabilities (B > A > C) under induction of alien chromosome elimination in crosses with O. violaceus, which was possibly affected by nucleolar dominance.
doi:10.1093/aob/mcp099
PMCID: PMC2706731  PMID: 19403626
Synthetic Brassica allohexaploids; Orychophragmus violaceus; intergeneric hybrids; genomic in situ hybridization; amplified fragment length polymorphism; single-strand conformation polymorphism; chromosome elimination; chromosome stability; nucleolar dominance

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