Nuclear cyp73 intron fragment length polymorphism supports morphological analysis of Salix species and hybrids

Abstract

The polyploid Salix alba L.–Salix fragilis L. hybrid complex still presents major difficulties in morphological identification. Most of the measured characters show a low diagnostic value for unambiguously identifying the parental species and their hybrid Salix × rubens Schrank due to continuous variation creating a large overlap in leaf and catkin morphology. Fragment length polymorphism of nuclear cyp73 intron markers was used to identify species and hybrids. This multilocus genotyping could be applied in a morphological analysis of trees from hybrid zones and allowed to demonstrate that morphological features of leaves and catkins clearly separated S. alba from S. fragilis. The hybrid individuals largely overlapped with both parental species but appeared to be morphologically more similar to S. fragilis than to S. alba. Cyp73 analysis of 11 Salix taxa revealed intermediate positions of two hybrid taxa with S. alba, namely S. × rubens and S. × sepulcralis Simonkai with their respective parental species S. fragilis and S. babylonica L. Additionally, the cyp73 intron multilocus genotypes clustered tetraploid taxa separately from diploid willows. Cyp73 introns are valuable markers for fast, reliable and straightforward genotyping in willow species and hybrids.     

Linkage mapping in tetraploid willows: segregation of molecular markers and estimation of linkage phases support an allotetraploid structure for Salix alba x Salix fragilis interspecific hybrids

Salix alba-Salix fragilis complex includes closely related dioecious polyploid species, which are obligate outcrossers. Natural populations of these willows and their hybrids are represented by a mixture of highly heterozygous genotypes sharing a common gene pool. Since nothing is known about their genomic constitution, tetraploidy (2n=4x=76) in willow species makes basic and applied genetic studies difficult. We have used a two-way pseudotestcross strategy and single-dose markers (SDMs) to construct the first linkage maps for both pistillate and staminate willows. A total of 242 amplified fragment length polymorphisms (AFLPs) and 50 selective amplifications of microsatellite polymorphic loci (SAMPL) markers, which showed 1:1 segregation in the F(1) mapping populations, were used in linkage analysis. In S. alba, 73 maternal and 48 paternal SDMs were mapped to 19 and 16 linkage groups covering 708 and 339 cM, respectively. In S. fragilis, 13 maternal and 33 paternal SDMs were mapped in six and 14 linkage groups covering 98 and 321 cM, respectively. For most cosegregation groups, a comparable number of markers linked in coupling and repulsion was identified. This finding suggests that most of chromosomes pair preferentially as occurs in allotetraploid species exhibiting disomic inheritance. The detection of 10 pairs of marker alleles from single parents showing codominant inheritance strengthens this hypothesis. The fact that, of the 1122 marker loci identified in the two male and female parents, the vast majority (77.5%) were polymorphic and as few as 22.5% were shared between parental species highlight that S. alba and S. fragilis genotypes are differentiated. The highly difference between S. alba- and S. fragilis-specific markers found in both parental combinations (on average, 65.3 vs 34.7%, respectively) supports the (phylogenetic) hypothesis that S. fragilis is derived from S. alba-like progenitors.     

Isolation and Analysis of Cinnamic Acid 4-Hydroxylase Homologous Genes from a Hybrid Aspen,Populus kitakamiensis

Cinnamic acid 4-hydroxylase (CA4H) is the second enzyme involved in phenylpropanoid biosynthesis and is a member of the cytochrome P-450 superfamily. Three CA4H homologous genes, cyp73a, cyp73b, and cyp73c, and a cDNA clone of cyp73a were isolated from a genomic library and a cDNA library of a hybrid aspen; Populus kitakamiensis, and were characterized. They might be interrupted by two introns each. cyp73a and cyp73b were very similar to each other not only in coding regions but also in non-coding regions. Southern blot analysis showed that four homologous genes for CA4H constructed a small gene family in the diploid genome of P. kitakamiensis. In the promoter regions, there were many common m-element-like sequences in phenylpropanoid biosynthesis genes.     

Patterns of hybridization and introgression between invasive Ulmus pumila (Ulmaceae) and native U. rubra

Natural hybridization between introduced species and their native congeners occurs frequently and can create serious conservation concerns. Ulmus pumila (Siberian elm) is an introduced Asian elm species that has naturalized in the United States and is now considered invasive in 41 states. Red elm (U. rubra), a native to the eastern United States, often occurs in sympatry with Siberian elm, and the two species are thought to hybridize. Here, we genetically characterized reference populations of the two elm species to identify species-specific microsatellite alleles. These markers were used to classify individuals in putative hybrid zones as parental species or hybrids, assess the extent of hybridization, and track patterns of introgression. We identified nine U. rubra, 32 U. pumila, and 51 hybrid individuals in our hybrid zones. Of the 51 hybrids, 35 were classified as first-generation hybrids and 16 as backcrosses. The majority of the backcrosses (88%) were introgressed toward U. pumila. Our classification of genotypes was consistent whether we used manual classification, principal coordinate analyses or Bayesian clustering. We observed greater genetic diversity and new combination of alleles in the hybrids. Our study indicates widespread hybridization between U. pumila and U. rubra and an asymmetric pattern of introgression toward U. pumila.     

The genetic architecture of reproductive isolation in Louisiana irises: flowering phenology

Despite the potential importance of divergent reproductive phenologies as a barrier to gene flow, we know less about the genetics of this factor than we do about any other isolating barrier. Here, we report on the genetic architecture of divergent flowering phenologies that result in substantial reproductive isolation between the naturally hybridizing plant species Iris fulva and I. brevicaulis. I. fulva initiates and terminates flowering significantly earlier than I. brevicaulis. We examined line crosses of reciprocal F1 and backcross (BC1) hybrids and determined that flowering time was polygenic in nature. We further defined quantitative trait loci (QTL) that affect the initiation of flowering in each of these species. QTL analyses were performed separately for two different growing seasons in the greenhouse, as well as in two field plots where experimental plants were placed into nature. For BCIF hybrids (BC1 toward I. fulva), 14 of 17 detected QTL caused flowering to occur later in the season when I. brevicaulis alleles were present, while the remaining 3 caused flowering to occur earlier. In BCIB hybrids (BC1 toward I. brevicaulis), 11 of 15 detected QTL caused flowering to occur earlier in the season when introgressed I. fulva alleles were present, while the remaining 4 caused flowering to occur later. These ratios are consistent with expectations of selection (as opposed to drift) promoting flowering divergence in the evolutionary history of these species. Furthermore, epistatic interactions among the QTL also reflected the same trends, with the majority of epistatic effects causing later flowering than expected in BCIF hybrids and earlier flowering in BCIB hybrids. Overlapping QTL that influenced flowering time across all four habitat/treatment types were not detected, indicating that increasing the sample size of genotyped plants would likely increase the number of significant QTL found in this study.     

A suite of molecular markers for identifying species, detecting introgression and describing population structure in spadefoot toads (Spea spp.)

Two congeneric species of spadefoot toad, Spea multiplicata and Spea bombifrons, have been the focus of hybridization studies since the 1970s. Because complex hybrids are not readily distinguished phenotypically, genetic markers are needed to identify introgressed individuals. We therefore developed a set of molecular markers (amplified fragment length polymorphism, polymerase chain reaction-restriction fragment length polymorphism and single nucleotide polymorphism) for identifying pure-species, F1 hybrids and more complex introgressed types. To do so, we tested a series of markers across both species and known hybrids using populations in both allopatry and sympatry. We retained those markers that differentiated the two pure-species and also consistently identified known species hybrids. These markers are well suited for identifying hybrids between these species. Moreover, those markers that show variation within each species can be used in conjunction with existing molecular markers in studies of population structure and gene flow.     

Willow hybrids and herbivory: a test of hypotheses of phytophage response to hybrid plants using the generalist leaf-feeder Lochmaea caprea (Chrysomelidae)

To test hypotheses of herbivore response to hybrid plants, I determined the preference of the generalist beetle Lochmeae capreae for individuals of Salix caprea, Salix phylicifolia and F1 hybrids between these species. F1 hybrids and pure individuals were created by hand-pollination of willow individuals in the field. The preference tests were performed in 1994 and 1995 and in both years there was a tendency for higher leaf area consumption from hybrids than from the parental species. However, statistically significant differences in consumption were only found in 1995 between hybrids and S. phylicifolia. The result from this study therefore lends most support to the dominance hypothesis: hybrid susceptibility was similar to that of one of the parental species. This is one of the first strict preference tests showing that a generalist herbivore can detect differences in palatability between hybrids and parental species. Furthermore, the experimental design used in the study minimized environmental variation. The results should therefore primarily reflect genetically based quality differences between hybrids and parental species. Received: 4 March 1996 / Accepted: 12 August 1996     

Detecting adaptive trait introgression between Iris fulva and I. brevicaulis in highly selective field conditions

The idea that natural hybridization has served as an important force in evolutionary and adaptive diversification has gained considerable momentum in recent years. By combining genome analyses with a highly selective field experiment, we provide evidence for adaptive trait introgression between two naturally hybridizing Louisiana Iris species, flood-tolerant Iris fulva and dry-adapted I. brevicaulis. We planted reciprocal backcross (BC1) hybrids along with pure-species plants into natural settings that, due to a flooding event, favored I. fulva. As expected, I. fulva plants survived at much higher rates than I. brevicaulis plants. Backcross hybrids toward I. fulva (BCIF) also survived at significantly higher rates than the reciprocal backcross toward I. brevicaulis (BCIB). Survivorship of BCIB hybrids was strongly influenced by the presence of a number of introgressed I. fulva alleles located throughout the genome, while survivorship in the reciprocal BCIF hybrids was heavily influenced by two epistatically acting QTL of opposite effects. These results demonstrate the potential for adaptive trait introgression between these two species and may help to explain patterns of genetic variation observed in naturally occurring hybrid zones.     

A genetic test of bioactive gibberellins as regulators of heterosis in maize

This study tested the hypothesis that gibberellin levels were responsible for the superior growth habit of hybrids (i.e., heterosis). If this were true, plants reduced in their capacity to produce gibberellin, such as maize plants homozygous for dwarf1 (d1), should display a lesser heterotic response. The d1 mutation was introgressed into two inbred lines of maize, B73 and Mo17, for seven generations. Plants segregating for the dwarf phenotype were produced both by self-fertilizing the introgressed inbred lines and by making reciprocal crosses between them to produce hybrids. Measurements were made of several physical traits. The results indicated that the hybrid dwarf plants experienced no loss of heterosis relative to their normal siblings. These results exclude the possibility that modulation of bioactive gibberellins is a major underlying basis of the heterotic response.     

Microsatellite markers for diverse Salix species

Forty‐six microsatellites were isolated from an enriched library of Salix burjatica and tested on 20 individuals (of nine species/hybrids) from the National Willows Collection (IACR‐Long Ashton Research Station, UK). Twenty‐nine were monomorphic, gave multilocus or unscorable patterns, or were duplicates. The remaining 17 microsatellites gave 2–22 alleles/locus. Three microsatellites successfully cross‐amplified in 31 additional Salix species. A further six were tested on panels comprising 6–25 individuals from the 31 species. Cross‐amplification was successful in all cases. These results suggest that the microsatellites isolated here should prove useful for population studies in a wide range of Salix species.     

Alien introgression in rice

Rice (Oryza sativa L.) productivity is affected by several biotic and abiotic stresses. The genetic variability for some of these stresses is limited in the cultivated rice germplasm. Moreover, changes in insect biotypes and disease races are a continuing threat to increased rice production. There is thus an urgent need to broaden the rice gene pool by introgressing genes for such traits from diverse sources. The wild species of Oryza representing AA, BB, CC, BBCC, CCDD, EE, FF, GG and HHJJ genomes are an important reservoir of useful genes. However, low crossability and limited recombination between chromosomes of cultivated and wild species limit the transfer of such genes. At IRRI, a series of hybrids and monosomic alien addition lines have been produced through embryo rescue following hybridization between rice and several distantly related species. Cytoplasmic male sterility and genes for resistance to grassy stunt virus and bacterial blight have been transferred from A genome wild species into rice. Similarly, genes for resistance to brown planthopper, bacterial blight and blast have also been introgressed across crossability barriers from distanly related species into rice. Some of the introgressed genes have been mapped via linkage to molecular markers. One of the genes Xa-21 introgressed from O. longistaminata has been cloned and physically mapped on chromosome 11 of rice using BAC library and flourescence in-situ hybridization. RFLP analysis revealed introgression from 11 of the 12 chromosomes of C genome species into rice. Introgression has also been obtained from other distant genomes (EE, FF, GG) into rice and in majority of the cases one or two RFLP markers were introgressed. Reciprocal replacement of RFLP alleles of wild species with the alleles of O. sativa indicates alien gene transfer through crossing over. The rapid recovery of recurrent phenotypes in BC₂ and BC₃ generations from wide crosses is an indication of limited recombination. Further cytogenetic and molecular investigations are required to determine precisely the mechanism of introgression of small chromosome segments from distant genomes in the face of limited homoeologous chromosome pairing. Future research should focus on enhancing recombination between homoeologous chromosomes. Introgression of QTL from wild species should be attempted to increase the yield potential of rice.     

Investigating temporal changes in hybridization and introgression in a predominantly bimodal hybridizing population of invasive sika (Cervus nippon) and native red deer (C. elaphus) on the Kintyre Peninsula,Scotland

We investigated temporal changes in hybridization and introgression between native red deer (Cervus elaphus) and invasive Japanese sika (Cervus nippon) on the Kintyre Peninsula, Scotland, over 15 years, through analysis of 1513 samples of deer at 20 microsatellite loci and a mtDNA marker. We found no evidence that either the proportion of recent hybrids, or the levels of introgression had changed over the study period. Nevertheless, in one population where the two species have been in contact since ～1970, 44% of individuals sampled during the study were hybrids. This suggests that hybridization between these species can proceed fairly rapidly. By analysing the number of alleles that have introgressed from polymorphic red deer into the genetically homogenous sika population, we reconstructed the haplotypes of red deer alleles introduced by backcrossing. Five separate hybridization events could account for all the recently hybridized sika‐like individuals found across a large section of the Peninsula. Although we demonstrate that low rates of F1 hybridization can lead to substantial introgression, the progress of hybridization and introgression appears to be unpredictable over the short timescales.     

Diversity of cuticular wax among Salix species and Populus species hybrids

The leaf cuticular waxes of three Salix species and two Populus species hybrids, selected for their ability to produce high amounts of biomass, were characterized. Samples were extracted in CH(2)Cl(2) three times over the growing season. Low kV SEM was utilized to observe differences in the ultrastructure of leaf surfaces from each clone. Homologous series of wax components were classified into organic groups, and the variation in wax components due to clone, sample time, and their interaction was identified. All Salix species and Populus species hybrids showed differences in total wax load at each sampling period, whereas the pattern of wax deposition over time differed only between the Salix species. A strong positive relationship was identified between the entire homologous series of alcohols and total wax load in all clones. Similarly strong relationships were observed between fatty acids and total wax load as well as fatty acids and alcohols in two Salix species and one Populus species hybrid. One Salix species, S. dasyclados, also displayed a strong positive relationship between alcohols and alkanes. These data indicate that species grown under the same environmental conditions produce measurably different cuticular waxes and that regulation of wax production appears to be different in each species. The important roles cuticular waxes play in drought tolerance, pest, and pathogen resistance, as well as the ease of wax extraction and analysis, strongly suggest that the characteristics of the cuticular wax may prove to be useful selectable traits in a breeding program.     

新疆主要造林树种对光肩星天牛的抗性

利用网室接种和树干套笼接种光肩星天牛Anoplophora glabripennis(Motschulsky)成虫的方法,于2005年～2006年在新疆光肩星天牛的发生疫区,进行10种主要造林树种对其抗性的测定试验。结果显示:2种接种试验结果基本一致,各试验树种都不同程度地受到光肩星天牛的危害,表现出不同的抗虫性。根据孵化幼虫数综合分析,判断出试验树种的抗性高低的顺序为:桑树、沙枣>胡杨、圆冠榆、新疆杨、白榆、箭杆杨>复叶槭、旱柳、馒头柳。另外根据网室内试验树种上出现的羽化孔判断,光肩星天牛在新疆巴音郭楞蒙古自治州1年发生1代。     

Evaluation of somatic hybrids of potato with Solanum stenotomum after a long-term in vitro conservation.

Somatic hybrids of potato with a cultivated relative, Solanum stenotomum also called Solanum tuberosum Stenotomum group, were evaluated for their physiological and agronomical characteristics as well as the stability of the introgressed resistance to bacterial wilt, caused by Ralstonia solanacearum, after a long-term in vitro conservation for more than 5 years. Analysis of photosynthesis showed that the PEPC/Rubisco ratio remained lower than 0.5 for all vitroplants of potato and the somatic hybrids, except for the relative species. This indicates that the carbon metabolism is heterotrophic (ratio>1) for S. stenotomum, and autotrophic for potato and the somatic hybrids (ratio<1). In both in vitro and greenhouse conditions, potato and the somatic hybrids produced few bigger tubers, while many small tubers were obtained from the relative. The hybrid tubers were morphologically intermediate. The starch content of hybrid tubers was much lower than that of potato, but similar to that of the relative species. Interestingly, the level of bacterial resistance, introgressed from S. stenotomum into potato, was shown to be very stable and remained as high as that of the relative after a long-term period of in vitro conservation.     

Widespread hybridization within mound‐building wood ants in Southern Finland results in cytonuclear mismatches and potential for sex‐specific hybrid breakdown

Hybridization and gene flow between diverging lineages are increasingly recognized as common evolutionary processes, and their consequences can vary from hybrid breakdown to adaptive introgression. We have previously found a population of wood ant hybrids between Formica aquilonia and F. polyctena that shows antagonistic effects of hybridization: females with introgressed alleles show hybrid vigour, whereas males with the same alleles show hybrid breakdown. Here, we investigate whether hybridization is a general phenomenon in this species pair and analyse 647 worker samples from 16 localities in Finland using microsatellite markers and a 1200‐bp mitochondrial sequence. Our results show that 27 sampled nests contained parental‐like gene pools (six putative F. polyctena and 21 putative F. aquilonia) and all remaining nests (69), from nine localities, contained hybrids of varying degrees. Patterns of genetic variation suggest these hybrids arise from several hybridization events or, instead, have backcrossed to the parental gene pools to varying extents. In contrast to expectations, the mitochondrial haplotypes of the parental species were not randomly distributed among the hybrids. Instead, nests that were closer to parental‐like F. aquilonia for nuclear markers preferentially had F. polyctena's mitochondria and vice versa. This systematic pattern suggests there may be underlying selection favouring cytonuclear mismatch and hybridization. We also found a new hybrid locality with strong genetic differences between the sexes similar to those predicted under antagonistic selection on male and female hybrids. Further studies are needed to determine the selective forces that act on male and female genomes in these newly discovered hybrids.     

Admixture in European Populus hybrid zones makes feasible the mapping of loci that contribute to reproductive isolation and trait differences

The use of admixed human populations to scan the genome for chromosomal segments affecting complex phenotypic traits has proved a powerful analytical tool. However, its potential in other organisms has not yet been evaluated. Here, we use DNA microsatellites to assess the feasibility of this approach in hybrid zones between two members of the 'model tree' genus Populus: Populus alba (white poplar) and Populus tremula (European aspen). We analyzed samples of both species and a Central European hybrid zone (N=544 chromosomes) for a genome-wide set of 19 polymorphic DNA microsatellites. Our results indicate that allele frequency differentials between the two species are substantial (mean delta=0.619+/-0.067). Background linkage disequilibrium (LD) in samples of the parental gene pools is moderate and should respond to sampling schemes that minimize drift and account for rare alleles. LD in hybrids decays with increasing number of backcross generations as expected from theory and approaches background levels of the parental gene pools in advanced generation backcrosses. Introgression from P. tremula into P. alba varies strongly across marker loci. For several markers, alleles from P. tremula are slightly over-represented relative to neutral expectations, whereas a single locus exhibits evidence of selection against P. tremula genotypes. We interpret our results in terms of the potential for admixture mapping in these two ecologically divergent Populus species, and we validate a modified approach of studying genotypic clines in 'mosaic' hybrid zones.     

Comparative leaf anatomy of Salix species and hybrids

Epidermal features, mesophyll differentiation and calcium oxalate characteristics of 19 species and 12 hybrids of Salix are described. The species and hybrids can be distinguished by the presence or absence of the following epidermal features: striated cuticle; stomata; covering trichomes; beaded anticlinal walls, and diosmin-like njstals. In or near marginal teeth, glandular trichomes are present in all cases. The leaf veins of all specimens examined have calcium oxalate prism sheaths and, with the exception of S. herbacea , cluster crystals in some cells of the mesophyll. Most sprcies studied in the subgenus Salix show: both adaxial and abaxial stomata; striated cuticle metopllyll of palisade cells, with little or no spongy mesophyll, but with a well-defined hypodermis, and absence of thick-walled, sinuous trichomes. Characteristic features of the subgenus Caprisalix are: abaxial stomata only; epidermal crystals; smooth cuticle; mesophyll diflerentiated into palisade cells and spongy mesophyll and without a hypodermis, and trichomes more numerous and varied than those of the subgenus Salix . Leaves of the two species of the subgenus Chaemelia examined and those of S. lapponum , have predominantly anomocytic stomata, whereas all the other leaves studied have predominantly paracytic stomata. The anatomical features described, in conjunction with the morphologiral characters, enable the species and hybrids of Salix studied to be autheenticated.     

Characterisation and inheritance of nuclear microsatellite loci for use in population studies of the allotetraploid <Emphasis Type="Italic">Salix alba</Emphasis>–<Emphasis Type="Italic">Salix fragilis</Emphasis> complex

We present nine polymorphic di- and tri-nucleotide repeat nuclear microsatellite markers selected specifically for their use in high throughput studies concerning the dioecious allotetraploid Salix alba–Salix fragilis willow complex. These taxa and their hybrids are difficult to discriminate using morphological characters. Thus, multiplex reactions were developed for these microsatellite loci and their effectiveness to distinguish individuals, especially hybrids, and their inheritance patterns in controlled crosses were determined. All loci displayed disomic–monogenic inheritance which allowed for the genotype data to be analysed as for a diploid organism. The nine loci produced a total of 67 alleles (mean, 7.4 alleles per locus; range, 3–11 alleles) in a reference panel of 57 individuals from two germplasm collections and natural populations. Gene diversity values (as measured by the expected heterozygosity) ranged from 0.000–0.820. A total of 53 distinct multilocus genotypes were observed, and ordination analysis revealed three separate clusters corresponding to S. alba, S. fragilis and hybrids. The microsatellite loci described here will be used in population genetic studies to investigate genetic variation, gene flow, levels of hybridisation and the extent of introgression in natural populations of the S. alba–S. fragilis complex. They are also useful for clonal identification, conservation and sustainable management of germplasm collections, genetic mapping and the selection of individuals and/or certification of controlled crosses for breeding programmes.