Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information

DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum × F. pratensis (2n=4x=28) with L. multiflorum (2n=2x=14), the technique identified chromosomes with interspecific recombination. Also, in an introgressed line of L. multiflorum which was homozygous for the recessive sid (senescence induced degradation) allele from F. pratensis, a pair of chromosome segments carrying the sid gene could be identified, indicating the suitability of GISH in showing the presence and location of introgressed genes. By screening backcross progeny for the presence of critical alien segments and the absence of other segments the reconstitution of the genome of the recipient species can be accelerated.     

Genomic constitution of <Emphasis Type="Italic">Festuca</Emphasis> × <Emphasis Type="Italic">Lolium</Emphasis> hybrids revealed by the DArTFest array

Complementary attributes of Festuca and Lolium grasses can be combined in hybrid cultivars called Festuloliums, which are becoming increasingly popular fodder crops and amenity plants. Genomic constitution of commercially available Festuloliums was reported to vary from almost equal representation of parental genomes to apparent lack of one of them based on molecular cytogenetic analyses and screening with a small set of DNA markers, both approaches with limited resolution. Here, we describe the use of the DArTFest array comprising 3,884 polymorphic DArT markers for characterization of genomes in five Festulolium cultivars. In any of the cultivars, the minimum number of informative markers, which discriminated the parental Lolium and Festuca genomes was 361 and 171, respectively. Using the DArTFest array, it was possible to determine hybrid genome constitution at resolution which has never been achieved before and the analysis of a set of randomly selected plants from each cultivar provided information on genetic structure of outcrossing Festulolium cultivars. In addition to a core set of markers typical for each hybrid cultivar, markers occurring at low frequency among the plants within each cultivar were identified. Biological significance of genomic loci associated with the rare markers is yet to be determined. Finally, with the aim to simplify the use of DArTFest arrays to characterize Festuca × Lolium hybrids, various bulking strategies were compared. While all bulks were suitable for identification of hybrids, only bulks of few plants have been found to reveal the rare markers.     

Introgression of chromosomes of Festuca arundinacea var. glaucescens into Lolium multiflorum revealed by genomic in situ hybridisation (GISH)

An F₁ hybrid (n=4x=28) between the tetraploid species Festuca arundinacea var. glaucescens (GGG′G′) and a synthetic tetraploid Lolium multiflorum (LmLmLmLm) was backcrossed to diploid L. multiflorum to produce triploid (2n=3x=21) BC₁ hybrids (LmLmG). At metaphase I of meiosis the triploids had a preponderance of ring bivalents and univalents with some linear and frying-pan trivalents. Genomic in situ hybridisation (GISH) differentiated the Festuca chromosomes from Lolium and revealed that the bivalents were exclusively between Lolium homologues, while the univalents were Festuca. Despite the limited amount of homoeologous chiasmata pairing in the triploids, some recombinant chromosomes were recovered in the second backcross when the hybrids were further crossed to diploid L. multiflorum. The progeny from the second backcross was predominantly diploid. Genotypes with recombinant chromosomes and chromosome additions involving an extra Festuca chromosome were identified using GISH. Changes in plant phenotype were related to the presence of Festuca chromatin. Received: 20 September 2000 / Accepted: 05 January 2001     

Intergeneric relationships ofLolium,Festuca, andVulpia (Poaceae) and their phylogeny

Morphological and seed protein analyses of 26 species of the generaLolium, Festuca andVulpia confirmed their close systematic affinities. Six inflorescence characters readily differentiatedFestuca fromLolium. Protein similarities betweenFestuca of sect.Bovinae and cross-pollinated species ofLolium, coupled with cytogenetic and crossability data, substantiate that they should be united into one genus.Vulpia had phenetic similarities with sect.Scariosae, Montanae andOvinae ofFestuca. Lolium, Festuca, andVulpia are most likely derived from a common ancestral form which was close toFestuca pratensis andLolium perenne.     

Phylogenetic relationships and genetic diversity among members of theFestuca-Lolium complex (Poaceae) based on ITS sequence data

There have been few DNA sequencebased studies of phylogenetic relationships within theFestuca-Lolium complex. Here we infer the phylogeny of 31Festuca-Lolium taxa with a dataset of 116 ITS sequences. The results are consistent with previous studies that resolved two majorFestuca clades: one clade of fine fescues and another clade that containsLolium and associatedFestuca species. This study is unique in suggesting a third, basalFestuca clade, but support for the basal position of this group is low. Extensive sampling permitted investigation of the effects of lineage sorting and reticulate events on the evolution of the complex. Roughly half of the taxa show evidence of lineage sorting or reticulation, and the monophyly ofLolium has likely been obscured by reticulate events. Overall, polyploid species harbor higher levels of ITS sequence diversity than diploids; ITS sequence variants may provide clues to the identity of allopolyploid parents.     

Reticulate hybridization of <Emphasis Type="Italic">Alpinia</Emphasis> (Zingiberaceae) in Taiwan

Reticulate hybridization is a complicated and creative mechanism in plant evolution that can cause interference in phylogenetic studies. Based on observations of intermediate morphology, low pollen fertility, and overlapping distributions of putative parent species, Yang and Wang (Proceedings of the cross-strait symposium on floristic diversity and conservation. National Museum of Natural Science, Taichung, Taiwan, pp 183–197, 1998) first proposed reticulate hybridization of Alpinia in Taiwan. In the present study, molecular tools were used to explore relationships between four parental species and their homoploidy hybrids, and the impact of hybridization on phylogeny reconstruction. Based on DNA markers, maternal heritance of the chloroplast genome, and additivity of nuclear ribosomal internal transcribed spacer, the present results provide strong support for the hybridization hypothesis. Co-existence of parental ribotypes within hybrids revealed that these hybridization events were current, while reciprocal and introgressive hybridization were inferred from chloroplast DNA data. Furthermore, iterative hybridizations involving more than two parental species may occur in notorious hybrid zones. Ecological, phenological, and physiological evidence provides insight into why such frequent hybridization occurs in Taiwanese Alpinia. In the phylogenetic tree of the Zerumbet clade reconstructed in this study, the chloroplast sequences from one hybrid species were not grouped into a subclade, implying instability caused by hybridization. Failure to find morphological apomorphies and biogeographical patterns in this clade was likely partially due to reticulate hybridization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Somatic hybrids between Solanum etuberosum and diploid,tuber bearing Solanum clones

Electrofusion was used to obtain somatic hybrids between Solanum etuberosum (2n=2x=24) and two diploid potato lines. These hybridizations were conducted to determine if haploidxwild species hybrids are better fusion partners than conventional S. tuberosumGp. Tuberosum haploids. Restriction fragment length polymerase (RFLP) analyses of the putative somatic hybrids confirmed that each parental genome was present. The somatic hybrids between S. etuberosum and a haploid S. tuberosum clone, US-W730, were stunted and had curled, purple leaves. In contrast, somatic hybrids between S. etuberosum and a haploidxwild species hybrid (US-W 730 haploidx S. berthaultii), were vigorous and generally tuberized under field conditions. These hybrids were designated as E+BT somatic hybrids. Analyses of 23 E+BT somatic hybrids revealed a statistically significant bias towards the retention of S. etuberosum chloroplasts. Stylar incompatibilities were observed when the E+BT somatic hybrids were used as pollen donors in crosses with S. tuberosum cultivars. Reciprocal crosses did not show this incompatibility. The progeny were vigorous and had improved tuber traits when compared to the maternal E+BT parent. RFLP analyses of three sexual progeny lines confirmed the presence of all 12 S. etuberosum chromosomes. In two of these lines, RFLPs that marked each of the 24 chromosome arms of S. etuberosum were present. However, RFLP markers specific for regions on chromosomes 2, 7, and 11 were missing from the third clone. Because other markers for these chromosomes were present in the progeny line, these results indicated the likelihood of pairing and recombination between S. etuberosum and S. tuberosum chromosomes.     

Genome balance in six successive generations of the allotetraploid Festuca pratensis × Lolium perenne

In the allotetraploid, Festuca pratensis Huds. (2n = 4x = 28) × Lolium perenne L. (2n = 4x = 28) the balance of chromatin, as determined by GISH, changes over successive generations of open pollination in favour of L. perenne. There is extensive recombination between chromosomes of the two parental genomes, as well as substitution of whole Festuca chromosomes by whole Lolium chromosomes. The total number of Lolium chromosomes increased from a mean 14.36 in the F₂ to 16.26 in the F₆, and the total number of Festuca chromosomes decreased correspondingly from a mean of 13.57 to a value of 11.56. The number of recombinant chromosomes and recombination breakpoints per genotype also increased from generation to generation, although the respective values of both characters were higher for Festuca (0.86–8.41 and 1.14–15.22) than for Lolium (0.68–4.59 and 0.68–6.0). The proportion of total genome length contributed by the L. perenne chromatin increased from about 50% in F₂ to 59.5% in F₆. The results are based on the sample of 134 plants studied (26–28 plants per generation), and are discussed in terms of the dominance of Lolium chromosomes over those of Festuca, and possible mechanisms underlying this phenomenon of chromatin substitution.     

Reciprocal allopolyploid grasses (Festuca × Lolium) display stable patterns of genome dominance

Allopolyploidization entailing the merger of two distinct genomes in a single hybrid organism, is an important process in plant evolution and a valuable tool in breeding programs. Newly established hybrids often experience massive genomic perturbations, including karyotype reshuffling and gene expression modifications. These phenomena may be asymmetric with respect to the two progenitors, with one of the parental genomes being “dominant.” Such “genome dominance” can manifest in several ways, including biased homoeolog gene expression and expression level dominance. Here we employed a k-mer–based approach to study gene expression in reciprocal Festuca pratensis Huds. × Lolium multiflorum Lam. allopolyploid grasses. Our study revealed significantly more genes where expression mimicked that of the Lolium parent compared with the Festuca parent. This genome dominance was heritable to successive generation and its direction was only slightly modified by environmental conditions and plant age. Our results suggest that Lolium genome dominance was at least partially caused by its more efficient trans-acting gene expression regulatory factors. Unraveling the mechanisms responsible for propagation of parent-specific traits in hybrid crops contributes to our understanding of allopolyploid genome evolution and opens a way to targeted breeding strategies.     

Neotyphodium Endophyte Changes Phytoextraction of Zinc in Festuca arundinacea and Lolium perenne

The effect of Neotyphodium endophytes on growth parameters and zinc (Zn) tolerance and uptake was studied in two grass species of Festuca arundinacea and Lolium perenne. Plants were grown under different Zn concentrations (control, 200, 400, 800, and 1800 mg kg^?1) in potted soil for 5 months. The results showed that the number of plant tillers was 85 and 51% greater in endophyte infected Festuca (FaEI) and Lolium (LpEI), respectively, compared to their endophyte free (EF) plants. Roots and shoots dry weights in infected Festuca were 87 and 9% greater than non-infected counterparts but in opposite, EF Lolium had 47 and 8% greater root and shoot dry weights than LpEI. Endophyte infected Festuca and Lolium improved chlorophyll fluorescence as Fv/Fm at high concentrations of Zn, showing their better chlorophyll functions and significant reduction of Zn stress in endophyte infected plants. Shoots of endophyte infectedFestuca had 82% greater concentration of Zn than EF Festuca when grown in soil containing 1800 mg kg^?1 Zn. Festuca and Lolium may tolerate high Zn concentration in soil without reduction in shoot and root growth. Endophyte infection in Festuca may help the grass accumulate and transport more Zn in aboveground parts under Zn-stress, thereby aiding phytoremediation of contaminated soils.     

Advances in introgression technologies for precision breeding within the Lolium - Festuca complex

The anticipated complexity of multifunctional grasslands with environment‐friendly and sustainable management practices demands better understanding of traits, their interactions, and their genetic control. Intergeneric hybrids between closely related Lolium and Festuca species are being used to broaden the gene pool and provide the plant breeder with options to combine complementary traits aimed at high quality but more robust grass varieties for the future. New techniques in introgression mapping provide opportunities for precision breeding whereby desirable gene combinations transferred from one species into another are selected preferentially, with the exclusion of deleterious alien genes. The close homology between genomes of Lolium and Festuca species allows high levels of chromosome pairing and recombination. Using genomic in situ hybridisation (GISH) on Lolium/Festuca hybrids and their derivatives, recombination between Lolium and Festuca chromosomes is observed at any point along the chromosome. The system provides unlimited access to any combination of Lolium and Festuca DNA sequence. Moreover, genes transferred between homoeologous chromosome sites are expected to function normally at their new locations. Alien chromosome segments may be reduced in size by further recombination events thereby reducing linkage drag. Molecular markers such as AFLPs, SSRs, SNPs, or RFLPs are being targeted to genes of interest to allow their selection through different generations in plant breeding programmes. Relatively simple PCR‐based marker systems are used for specific traits as breeders' toolkits in plant breeding programmes.     

Expression of nuclear and chloroplastic genes coding for fraction-1 protein in somatic hybrids of Nicotiana tabacum + rustica

In the sexual interspecific cross, Nicotiana rustica L.xN. tabacum L., N. rustica can serve as the female but not as the male parent. By fusion of protoplasts, the barrier to fertilization was overcome and somatic hybrids containing N. tabacum cytoplasm were produced as shown by isoelectric focusing of the Fraction-1 protein (F-1-protein). All somatic hybrids displayed polypeptides of the large subunit of F-1 protein (which is coded by the chloroplast genome) characteristic of only one or the other parental species. Two hybrids had large subunits of the N. tabacum type and two hybrids had those of the N. rustica type. Three hybrids contained three smallsubunit polypeptides (coded by the nuclear genome), one being characteristic of N. rustica, one characteristic of N. tabacum, and one with an isoelectric point common to both species. A fourth hybrid contained only two small-subunit polypeptides of the N. tabacum type but in a F-1 protein macromolecule whose large subunits were of the N. rustica type. One somatic hybrid was self-fertile and its F₂ progeny contained large- and small-subunit polypeptides indistinguishable in their isoelectric points from those in the parent F₁ hybrid. All somatic hybrids showed an aneuploid chromosome number and morphological characteristics intermediate between those of N. rustica and N. tabacum.     

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune by PEG-induced protoplast fusion

Intergeneric hybridization between Pleurotus ostreatus and Schizophyllum commune was studied using PEG-induced fusion. The fusion of protoplasts from auxotrophic mutant strains resulted in the formation of fusion hybrids in the frequencies of 3.6 to 7.3×10^–5. Most of these fusion hybrids were monokaryotic and sterile and no heterokaryosis occurred. Most fusants showed a significantly higher nuclear DNA content when compared to parental strains and no diploids (parent 1 genome plus parent 2 genome) were found. Some fusion hybrids revealed both parental fragments in nuclear and mitochondrial rDNA PCR profiles. AP-PCR (Arbitrarily-primed Polymerase Chain Reaction) fingerprints also indicated that most of the fusion products were recombinant hybrids.     

Effects of genotype and plant growth regulator on callus induction and plant regeneration in four important turegrass genera: A comparative study

Summary Turfgrass, like other major crop species, is recalcitrant to manipulation in vitro. To perform efficient genetic transformation of turfgrass, it is necessary to optimize tissue culture conditions. In most reports, plant tissue culture techniques have been applied to propagate a single cultivar or several cultivars in one species of turfgrass. In this experiment, four turfgrass genera were used, namely common bermudagrass, Cynodon dactylon [L.] Pers. (California origin); red fescue, Festuca rubra L. var. rubra ‘Shadow’; perennial ryegrass, Lolium perenne L. ‘Barbal’; and Kentucky bluegrass, Poa pratensis L. ‘Merion.’ Mature seeds were surface-sterilized and cultured on basal Murashige and Skoog (MS) media supplemented with 30–250 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction. Regeneration media consisted of MS supplemented with 5–10 μM 6-benzyladenine (BA). Among the genera, Poa had the higest callus induction percentage (CIP) regardless of 2,4-D concentration, followed by Cynodon, Lolium, and Festuca, respectively. Cynodon and Lolium had the highest callus regeneration percentage (CRP) and overall regeneration rate (ORR). Festuca had a poor CIP, CRP, and ORR compared to other studied genera. Cynodon produced the highest shoot number per explant. Based on the results of the present study, MS medium supplemented with 60 μM 2,4-D (for callus induction) and 7.5 μM BA (for regeneration) can be used in multi-generic transformation studies with the genera used.     

The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny

Genomic in-situ hybridization (GISH) was used to monitor the behaviour of parental genomes, and the fate of intergenomic chromosome translocations, through meiosis of plants regenerated from asymmetric somatic hybrids between Nicotiana sylvestris and N. plumbaginifolia. Meiotic pairing in the regenerants was exclusively between chromosomes or chromosome segments derived from the same species. Translocation (recombinant) chromosomes contained chromosome segments from both parental species, and were detected at all stages of meiosis. They occasionally paired with respectively homologous segments of N. sylvestris or N. plumbaginifolia chromosomes. Within hybrid nuclei, the meiotic division of N. plumbaginifolia lagged behind that of N. sylvestris. However, normal and recombinant chromosomes were eventually incorporated into dyads and tetrads, and the regenerants were partially pollen fertile. Recombinant chromosomes were transmitted through either male or female gametes, and were detected by GISH in sexual progeny obtained on selfing or backcrossing the regenerants to N. sylvestris. A new recombinant chromosome in one plant of the first backcross generation provided evidence of further chromosome rearrangements occurring at, or following, meiosis in the original regenerants. This study demonstrates the stable incorporation of chromosome segments from one parental genome of an asymmetric somatic hybrid into another, via intergenomic translocation, and reveals their transmission to subsequent sexual progeny.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.     

送春与多花兰种间杂交后代的ISSR分析

该文使用简单重复序列间( ISSR)分子标记,对送春与多花兰种间杂交后代进行了研究。结果表明：从80个ISSR引物中筛选出14个扩增效果稳定的ISSR引物,对两亲本和59个F1代个体进行了ISSR扩增,得到107个扩增位点,扩增的片段大小位于90~2100 bp之间,平均每个引物扩增7.64条条带,得到11种类型的带。 ISSR标记在送春×多花兰的F1代中表现出一定的多态性,分离频率为44.86％,分离位点有83.33％符合孟德尔1︰1或3︰1的分离规律,产生偏孟德尔分离的位点占12.50％,余下的4.17％属于特殊分离带型。可能导致后代变异的位点为偏孟德尔分离的6条带、缺失的8条带或新生成的2条带。聚类图中父本和母本与F1代个体间的遗传距离较远,59个杂交后代先聚集成一组,再同母本相聚为一组,最后才同父本聚在一起,59个杂种均偏母本型。送春与多花兰的杂交后代在植株形态、染色体、遗传物质方面都具备双亲特点,61个个体间的ISSR分子量标记结果和植株形态学特征都说明,59个F1代杂种包含送春和多花兰的遗传特性是真杂种;F1代杂种既有双亲的互补特征带,又有双亲的重组片断即产生新的特异带,这说明送春与多花兰的杂交后代具有遗传变异的特点。该研究结果可以有效地对杂交后代进行定向选择,为兰花的杂交育种提供了分子依据。     

Consistent detection of QTLs for crown rust resistance in Italian ryegrass (<Emphasis Type="Italic">Lolium multiflorum</Emphasis> Lam.) across environments and phenotyping methods

Crown rust, caused by Puccinia coronata f. sp. lolii, is one of the most important diseases of temperate forage grasses, such as ryegrasses (Lolium spp.), affecting yield and nutritional quality. Therefore, resistance to crown rust is a major goal in ryegrass breeding programmes. In a two-way pseudo-testcross population consisting of 306 Lolium multiflorum individuals, multisite field evaluations as well as alternative methods based on artificial inoculation with natural inoculate in controlled environments were used to identify QTLs controlling resistance to crown rust. Disease scores obtained from glasshouse and leaf segment test (LST) evaluations were highly correlated with scores from a multisite field assessment (r = 0.66 and 0.79, P < 0.01, respectively) and thus confirmed suitability of these methods for crown rust investigations. Moreover, QTL mapping based on a linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed similar results across different phenotyping methods. Two major QTLs were consistently detected on linkage group (LG) 1 and LG 2, explaining up to 56% of total phenotypic variance (V _p). Nevertheless, differences between position and magnitude of QTLs were observed among individual field locations and suggested the existence of specific local pathogen populations. The present study not only compared QTL results among crown rust evaluation methods and environments, but also identified molecular markers closely linked to previously undescribed QTLs for crown rust resistance in Italian ryegrass with the potential to be applied in marker-assisted forage crop breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production and cytogenetic characterization of intertribal somatic hybrids between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Isatis indigotica</Emphasis> and backcross progenies

Intertribal somatic hybrids between Brassica napus (2n = 38, AACC) and a dye and medicinal plant Isatis indigotica (2n = 14, II) were obtained by fusions of mesophyll protoplasts. From a total of 237 calli, only one symmetric hybrid (S2) and five asymmetric hybrids (As1, As4, As6, As7 and As12) were established in the field. These hybrids showed some morphological variations and had very low pollen fertility. Hybrids S2 and As1 possessed 2n = 52 (AACCII), the sum of the parental chromosomes, and As12 had 2n = 66 (possibly AACCIIII). Hybrids As4, As6 and As7 were mixoploids (2n = 48–62). Genomic in situ hybridization analysis revealed that pollen mother cells at diakinesis of As1 contained 26 bivalents comprising 19 from B. napus and 7 from I. indigotica and mainly showed the segregation 26:26 at anaphase I (AI) with 7 I. indigotica chromosomes in each polar group. Four BC₁ plants from As1 after pollinated by B. napus resembled mainly B. napus in morphology but also exhibited some characteristics from I. indigotica. These plants produced some seeds on selfing or pollination by B. napus. They had 2n = 45 (AACCI) and underwent pairing among the I. indigotica chromosomes and/or between the chromosomes of two parents at diakinesis. All hybrids mainly had the AFLP banding patterns from the addition of two parents plus some alterations. B. napus contributed chloroplast genomes in majority of the hybrids but some also had from I. indigotica. Production of B. napus–I. indigotica additions would be of considerable importance for genome analysis and breeding.