Tetraploid Varieties Research Articles

ОСНОВНЫЕ ИТОГИ И ПЕРСПЕКТИВЫ СЕМЕНОВОДСТВА КОРМОВЫХ ТРАВ В ФГУП «ПОЙМА»

Presents the historical aspects and the current state of breeding and seed production in the Federal State Unitary Enterprise "Poima", as well as the prospects for the production of seeds of forage herbs, including their higher reproduction in order to provide farmers and other consumers with high-quality seed. The stages of seed production of perennial grasses in the economy are reflected, the analysis and the importance of seed production for agricultural production, as well as prospects. Special attention is paid to the creation and reproduction of high-yielding varieties of perennial grasses for floodplain conditions, such as awnless brome, fescue, timothy-grass, cocksfoot and other species. It is noted that the special variety of fescue meadow hayfields and pasture ecotype Krasnopoimskaya 92, well adapted to the conditions of flooding in the underground conditions, as well as when growing it on watershed areas, is of great importance in modern conditions of forage production. The article also notes a great prospect of developing tetraploid variety of ryegrass annual Rapid in the system of feed production in floodplain conditions, which can replace to a large extent in the Central, Northern and North-Western regions of unstable productivity of corn, even its early-maturing hybrids. In addition, the publication presents the basic agrotechnical methods of cultivation of seeds of such most sought-after perennial grasses as perennial ryegrass, festulolium, fescue reed, allowing them to obtain high and stable yields.

Resistance of Multiple Diploid and Tetraploid Perennial Ryegrass (Lolium perenne L.) Varieties to Three Projected Drought Scenarios for the UK in 2080

Forage plants underpin the livestock industry. Selective breeding, including polyploidization, where genome size is increased by whole genome duplication, changes the productivity and stress tolerance of new varieties. We conducted a growth chamber experiment to investigate the likely responses of Lolium perenne L. to drought, testing four diploid and four tetraploid varieties. We simulated projected spring and summer temperatures for the South-West of England in 2080, applying three projected rainfall scenarios, which varied in drought severity. Drought caused a reduction in productivity, but there was substantial variation between varieties (up to 82%), with the optimal variety changing depending on drought severity. Across three harvests, productivity declined by 43% and 27% (dry biomass) for the severe and likely drought scenarios, respectively. In the final harvest, tetraploids exhibited a greater biomass under severe drought, whereas diploids had a greater biomass under the current rainfall and likely drought scenarios. Longer stomata were observed in tetraploids; however, stomatal conductance was not significantly different between ploidy levels. Trait selection will be important for future drought adaptation. Local climate projections will need to be consulted when selecting L. perenne varieties to tolerate the spatially variable reductions in future rainfall.

Comparison of Methods to Distinguish Diploid and Tetraploid Potato in Applied Diploid Breeding

Diploid (2n = 2x = 24) and tetraploid (2n = 4x = 48) germplasm are commonly used in potato breeding programs. Potato breeders need to efficiently and inexpensively differentiate between diploid and tetraploid progeny in dihaploid induction crosses as well as 2x-2x crosses where 2n gametes may occur in the parents. In this study, we compared the chloroplast count, genome-wide SNP genotyping and flow cytometry methods to determine ploidy. Twenty-eight clones were used as reference samples (14 diploid lines (2n = 2x = 24), 14 tetraploid (2n = 4x = 48) varieties and advanced breeding lines) to compare the three ploidy determination methods. The chloroplast count method was used to determine the ploidy level in the 28 reference samples, and 102 potato breeding lines derived from diploid (2x-2x) crosses. The Infinium 12 K V2 Potato Single Nucleotide Polymorphism (SNP) Array was used to examine the 28 reference samples and the 102 breeding lines. The results obtained from both chloroplasts counts and SNP genotyping techniques determined that there was a total of 84 diploid lines and 18 tetraploid lines. Flow cytometry was also used to determine ploidy level in a subset of 42 lines (28 reference lines set and 14 breeding lines). All three methods of ploidy determination (chloroplast counting, SNP genotyping and flow cytometry) agreed for all samples evaluated. These results demonstrate the usefulness of chloroplast counting as an efficient and inexpensive method for breeders to differentiate ploidy between diploid and tetraploid potato in applied breeding programs.

Сравнителна оценка на ди- и тетраплоидна генплазма от червена детелина при равнинните условия на Северна България

Abstract From the first to third growing season in the field test carried out at the Experimental Station of Soybean (Pavlikeni, Bulgaria) was observed 12 accessions of red clover, including two Bulgarian diploid varieties Nika 11 (standard) and Sofia 52, four local populations and next six foreign tetraploid varieties, i.e. Kvarta, Astur, Carbo, Elanus, Fregata and Larus. The goal of study was to determine the effect of ploidity level of the varieties as well as the effect of adaptive variability of local populations on fresh mass yield, dry mass yields and their persistence in an agro-ecological area with lower summer moisture assurance for red clover. The highest total dry mass yield was observed for Sofia 52 variety (2n), with insignificant differences being inferior to the standard variety Nika 11 (2n) and tetraploid varieties Kvarta and Astur. The productivity of these tetraploids strongly varies depending on the season and the year, as well as the age of sward. According to the obtained comparative production data for cuts and years, the tetraploid germplasm has a stronger adaptation to the limiting moisture. In summer drought, typical for the plains of Northern Bulgaria, tetraploids stops growing and does not compensate for its limited vegetative growth, despite the excellent moisture guarantee in the late summer, as well as in the spring the next growing season. Local wild-type germplasm of the species is characterized by a rapid development and lower yield of fresh and dry forage mass compared to the diploid standard when growing in conditions of optimum moisture but relatively high productivity in the dry conditions of the summer months. The differences in the genotypic productivity response under dry conditions reported in this experiment also suggest that the abiotic limit studied can be successfully responded by the genotype factor.

Endophytic fungal communities of ancient wheat varieties

The fungal community composition and structure of two ancient tetraploid wheat varieties, native to the Sicilian territory of Italy, Perciasacchi (winter wheat) and Tumminia (spring wheat) were investigated using High Throughput Sequencing (HTS). This showed a predominance of Ascomycetes and Basidiomycetes including Alternaria , Fusarium , Mycosphaerella, Filobasidium, Cystofilobasidium, Cryptococcus , Leucosporidium , Dioszegia, Puccinia , Sporobolomyces, Aureobasidium, Cladosporium, Holtermanniella and Gibberella . Principal Coordinates Analysis (PCoA) and Linear discriminant analysis Effect Size (LEfSe) showed that Aureobasidium, Leucosporidium and Puccinia differentiated between the two wheat varieties. In addition, the microbial association analysis suggested that some endophytic taxa play important roles within the wheat fungal community. Genera such as Cryptococcus and Cystofilobasidium were shown to have consistent antagonistic activity against Gibberella spp., while, Acremonium and a group of unidentified ascomycetes had mutual exclusion relationships with Puccinia. Since both Gibberella and Puccinia contain several economically important pathogens of wheat, the detected fungal interactions may indicate microbial-mediated resistance in these wheat varieties.

A new tetraploid variety of Cryptocoryne ciliata (Araceae) from Sarawak

A new, tetraploid variety of Cryptocoryne ciliata (Roxb.) Fisch. ex Schott from Sarawak, Malaysia, is described and illustrated: C. ciliata var. bogneri N. Jabobsen. The ecology, morphology and chromosome numbers of C. ciliata are discussed. The new variety is compared with the other two varieties of the species, C. ciliata var. ciliata and C. ciliata var. latifolia Rataj, and an identification key is provided. Citation: Jacobsen N., Bastmeijer J. D., Jensen K. R. & Ørgaard M. 2018: A new tetraploid variety of Cryptocoryne ciliata (Araceae) from Sarawak. – Willdenowia 48: 425–431. doi: https://doi.org/10.3372/wi.48.48312 Version of record first published online on 1 December 2018 ahead of inclusion in December 2018 issue.

Electromagnetic radiation of low intensity as a factor of change of phenolic compounds content

The influence of low-intensity electromagnetic radiation (EMR) on the content of phenolic compounds (PC) in plants on different stages of ontogeny is discussed. A medicinal plant (Calendula officinalis L.) and agricultural crop Fagopyrum sagittatum G., diploid varieties and tetraploid varieties were chosen as objects of study. Microwave EMR in two frequency bands, 53.57-78.33 GHz (wide range mode) and 64.00-66.00 GHz (narrow range mode), has been selected to learn the physical effects on seeds, with a treatment exposure time of 20, 12, or 8 min. The experiments were conducted under laboratory and field conditions. Treated and control seeds were germinated by 100 pcs in a growth chamber on moistened filter paper at 22–24˚C in the dark and placed on the daylight from the third day. The level of PC in the seedlings was measured with the Folin-Ciocalteu reagent. It was found that in the EMR pretreated C. officinalis seedlings the level of PC is maximal on 14th day of germination. The wide mode treatment for 12 min results in a two-fold increase in the level of PC in juvenile shoots with respect to control. Different constitutional levels of phenolic compounds in the leaves of diploid and tetraploid varieties of F. sagittatum in the most critical phases of ontogeny were found. It is recommended to use EMR with a 12 min exposure time for C. officinalis. The narrow frequency mode with an exposure time of 20 min is preferable to use for most varieties of F. sagittatum.

Improved genetic resolution for linkage mapping of resistance to potato wart in monoparental dihaploids with potential diagnostic value in tetraploid potato varieties

Key messageWe achieved improved mapping resolution of the major wart resistance locus Xla-TNL containing also Sen1 in a dihaploid population using SNP data and developed additional markers with diagnostic value in tetraploid varieties.We analyzed a segregating monoparental dihaploid potato population comprising 215 genotypes derived from a tetraploid variety that is highly resistant to Synchytrium endobioticum pathotypes 18 and 6. The clear bimodal segregation for both pathotypes indicated that a major dominant resistance factor in a simplex allele configuration was present in the tetraploid donor genotype. Compared to that in previous analyses of the same tetraploid donor in conventional crosses with susceptible tetraploid genotypes, a segregation pattern with a reduced genetic complexity of resistance in dihaploids was observed here. Using the 12.8 k SolCAP SNP array, we mapped a resistance locus to the Xla-TNL region containing also Sen1 on potato chromosome 11. The improved mapping resolution provided by the monoparental dihaploids allowed for the localization of the genes responsible for the resistance to both pathotypes in an interval spanning less than 800 kbp on the reference genome. Furthermore, we identified eight molecular markers segregating without recombination to pathotype 18 and pathotype 6 resistance. Also, two developed markers display improved diagnostic properties in an independent panel of tetraploid varieties. Overall, our data provide the highest resolution mapping of wart resistance genes at the Xla-TNL locus thus far.

The early flowering trait of an emmer wheat accession (Triticum turgidum L. ssp. dicoccum) is associated with the cis-element of the Vrn-A3 locus.

We identified a novel allele of the Vrn-A3 gene that is associated with an early flowering trait in wheat. This trait is caused by a cis-element GATA box in Vrn-A3. To identify novel flowering genes in wheat, we investigated days from germination to heading (DGH) in tetraploid wheat accessions. We found that the tetraploid variety Triticum turgidum L. ssp. dicoccum (TN26) harbors unknown genes that surpass the earliness effect of the early flowering allele Ppd-A1a harbored by TN28 (T. turgidum L. ssp. turgidum conv. pyramidale). Using recombinant inbred lines resulting from a cross between TN26 and TN28, we performed a quantitative trait locus (QTL) analysis for DGH. We identified a QTL for earliness in TN26 on chromosome 7AS, the chromosome on which Vrn-A3 is located. By sequence analysis for the Vrn-A3 locus in both TN26 and TN28, we identified a 7-bp insertion that included a cis-element GATA box sequence at the promoter region of the Vrn-A3 locus of TN26. Based on an expression analysis using sister lines for Vrn-A3, we suggest that the early flowering trait of TN26 was caused by the GATA box in Vrn-A3. In addition, we identified tetraploid wheat as a useful genetic resource for wheat breeding.

Autopolyploids in fodder grass breeding: induction and field performance

Doubling of chromosome set directly affects plant performance through increase of organ size, higher feeding value and increased resistance to adverse environmental factors. Therefore efficient methods of polyploid induction are needed in order to develop new varieties of naturally diploid fodder grass species. The efficiency of antimitotic agents as colchicine, amiprophos-methyl, trifluralin and oryzalin was compared in a series of tetraploid induction experiments in Lolium multiflorum, L. perenne and Festuca pratensis, while newly developed tetraploid plants were compared to standard tetraploid varieties in the field trials. Colchicine treatment proved to be the most efficient method for in vitro cultured embryos in comparison with the other agents. Induced tetraploids of F. pratensis produced higher dry matter and seed yield and could be used for the development of new varieties. Induced tetraploid plants of Lolium spp. were equal to the standard varieties in field trials, therefore they could be used as parental genotypes in crosses. Induced tetraploids of F. pratensis produced higher dry matter and seed yield and could be used for development of new variety.

Red clover varieties show nitrogen fixing advantage during the early stages of seedling development

Plant and environmental factors affect root nitrogen (N) exudation dynamics in legumes. To better understand the genotypic variability and plant factors affecting root N release nodulation, plant growth, tissue N content, and root N exudation, six (three diploid and three tetraploid) red clover (Trifolium pratense L.) varieties were evaluated under controlled environmental conditions during the first 8 wk of plant growth after rhizobia inoculation. Genotypic differences were found for nodulation, plant dry weight (DW), leaf area, root attributes (root length, surface area, volume, and diameter), shoot and root N concentration, and N content. Genotypic differences were also found for root exudate N content in terms of NO3−-N, NH4+-N, and dissolved organic N (DON). In general, root exudate inorganic N content was greater in tetraploid varieties than in the diploids throughout the growth period. Root exudate DON content was greater than the inorganic N content. The NO3−-N content in root exudate was positively correlated with root growth attributes and root N concentration, whereas NH4+-N content was positively correlated with nodule number. Root exudate DON was positively correlated with shoot N concentration and average nodule DW. These results highlight the existence of genotypic differences among red clover varieties for plant morphological factors affecting root N release during the early stages of plant development.

A System for Identification of Potato Varieties Using SNP Dosage

As in autoteraploids, five genotypes are possible in a biallelic locus, allele dosage could be useful for variety identification in potato. DNA sequences of 13 genes associated with tuber yield, tuber starch content or late blight resistance were surveyed for single nucleotide polymorphism (SNP) in eight tetraploid varieties. A total of 193 potential SNPs was found by Sanger-sequencing, of which 58 were measured for nucleotide frequencies using a pyrosequencer. Of the 58 SNPs, 35 yielded distinct clusters, corresponding to allele dosages. Among these, six were highly correlated with another, leaving 29 independent SNP loci for use in variety discrimination. By using dosage scores at the 29 SNP loci, it was possible to differentiate 115 varieties, except for a sport with red tuber skin color, with at least three different SNP dosages. Therefore, using SNP dosages is a simple, fast and reliable tool for variety identification.

Isolation and Culture of Pollen Tetrad Protoplasts from Solanum tuberosum

Pollen protoplasts provide a sexual and haploid system for haploid production, cell fusion and mutation studies used in plant improvement. Due to the multiploidy, heterozygosity, and often self-incompatibility in tetraploid genotypes, haploid potatoes are desirable for breeding schemes via ploidy manipulations. In this study, two tetraploid varieties and two dihaploid lines of potato were used for pollen tetrad protoplast isolation and culture. The meiotic tetrad buds were first pre-treated at 5 °C for 0–12 days, then the tetrads were transferred into enzyme solutions containing different concentrations of snailase (0.5–1.5%), 0.3 M osmolites (sucrose, mannitol, glucose or sorbitol), 1.0% Cellulose, 0.5% Hemicellulase, 0.5% Pectolyase, 0.3% Sucrose, 3 mM 2-(N-Morpholino) ethane sulfonic acid, 1% polyvinyl pyrrolidone, 0.01% casein hydrolysate and K3 medium compositions. Among the four donor materials, tetraploid cv. Gannongshu No. 3 (‘GNS No.3’) showed the greatest protoplast yield (74.6 ± 2.4%). In this variety, most of the tetrad protoplasts regenerated a cell wall and continued cell divisions were observed when they were inoculated in K3 basic medium supplemented with (0.5–1.0) mg/L 2,4-D + (0.1–0.5) mg/L KT + 0.4 mg/L 6-BA +800 mg/L glutamine +100 mg/L serine. ‘GNS No.3’ also showed the greatest first division frequency (21.6 ± 1.5%) and sustained division to form multicellular structures. The study findings suggested that cultured tetrad pollen protoplasts could reverse the gametophytic developmental pattern programmed in vivo to a sporophytic pathway leading to multicellular microspore-derived colonies.

Proteome Profiling of Wheat Shoots from Different Cultivars.

Wheat is a cereal grain and one of the world’s major food crops. Recent advances in wheat genome sequencing are by now facilitating its genomic and proteomic analyses. However, little is known about possible differences in total protein levels of hexaploid versus tetraploid wheat cultivars, and also knowledge of phosphorylated wheat proteins is still limited. Here, we performed a detailed analysis of the proteome of seedling leaves from two hexaploid wheat cultivars (Triticum aestivum L. Pavon 76 and USU-Apogee) and one tetraploid wheat (T. turgidum ssp. durum cv. Senatore Cappelli). Our shotgun proteomics data revealed that, whereas we observed some significant differences, overall a high similarity between hexaploid and tetraploid varieties with respect to protein abundance was observed. In addition, already at the seedling stage, a small set of proteins was differential between the small (USU-Apogee) and larger hexaploid wheat cultivars (Pavon 76), which could potentially act as growth predictors. Finally, the phosphosites identified in this study can be retrieved from the in-house developed plant PTM-Viewer (bioinformatics.psb.ugent.be/webtools/ptm_viewer/), making this the first searchable repository for phosphorylated wheat proteins. This paves the way for further in depth, quantitative (phospho)proteome-wide differential analyses upon a specific trigger or environmental change.

Karyotype and Stomatal Studies on Three Genotypes of <i>Morus</i> spp.

Three mulberry genotypes viz., MR2, C763 and Morus macroura were analyzed for the stomatal frequency and karyotypic attributes. MR2 and C763 were found to be diploids with somatic chromosome number of 2n=28, while variety Morus cathyana revealed tetraploid chromosome number of 2n=56. Somatic chromosomes measured 1.26 to 2.86µm in length. Karyotypes of these taxa are symmetric. Only metacentric and sub-metacentric chromosomes are found in the complement. Stomatal frequency was found to be high in diploid mulberry varieties compared to tetraploid variety. © 2017 The Japan Mendel Society.

Evaluation of LD decay and various LD-decay estimators in simulated and SNP-array data of tetraploid potato

Key messageThe number of SNPs required for QTL discovery is justified by the distance at which linkage disequilibrium has decayed. Simulations and real potato SNP data showed how to estimate and interpret LD decay.The magnitude of linkage disequilibrium (LD) and its decay with genetic distance determine the resolution of association mapping, and are useful for assessing the desired numbers of SNPs on arrays. To study LD and LD decay in tetraploid potato, we simulated autotetraploid genotypes and used it to explore the dependence on: (1) the number of haplotypes in the population (the amount of genetic variation) and (2) the percentage of haplotype specific SNPs (hs-SNPs). Several estimators for short-range LD were explored, such as the average r2, median r2, and other percentiles of r2 (80, 90, and 95 %). For LD decay, we looked at LD½,90, the distance at which the short-range LD is halved when using the 90 % percentile of r2 at short range, as estimator for LD. Simulations showed that the performance of various estimators for LD decay strongly depended on the number of haplotypes, although the real value of LD decay was not influenced very much by this number. The estimator LD½,90 was chosen to evaluate LD decay in 537 tetraploid varieties. LD½,90 values were 1.5 Mb for varieties released before 1945 and 0.6 Mb in varieties released after 2005. LD½,90 values within three different subpopulations ranged from 0.7 to 0.9 Mb. LD½,90 was 2.5 Mb for introgressed regions, indicating large haplotype blocks. In pericentromeric heterochromatin, LD decay was negligible. This study demonstrates that several related factors influencing LD decay could be disentangled, that no universal approach can be suggested, and that the estimation of LD decay has to be performed with great care and knowledge of the sampled material.

Acorus calamus Linn.: phytoconstituents and bactericidal property

Acorus calamus Linn. of the family Araceae (Acoraceae), commonly known as Sweet Flag and Vacha. The rhizome of this plant has medicinal properties against bugs, moths, lice and emetic stomach in dyspepsia. Chemical composition of the hydro-distilled essential oil obtained from the rhizomes of A. calamus was analyzed by gas chromatography equipped with flame ionization detector and gas chromatography coupled with mass spectrometry. The essential oil of A. calamus and its major compound β-asarone were tested against five Gram-positive, eight Gram-negative bacteria, and three fungi by the tube-dilution method at a concentration rang of 5.0-0.009mg/mL. Forty constituents were identified which comprised 98.3% of the total oil. The major compound β-asarone (80.6%) was identified and confirm by NMR ((1)H- & (13)C-) in rhizome oil of A. calamus. The organism Micrococcus luteus was found to be more susceptible to the oil with minimum bactericidal concentration (MBC) value of 0.032±0.004mg/mL, followed by Aspergillus fumigatus, Aspergillus niger and Micrococcus flavus with MBC values of 0.104±0.016, 0.117±0.017 and 0.143±0.013mg/mL, respectively. The compound β-asarone was susceptible to the microorganism A. niger with MBC value 0.416±0.065mg/mL. The present study revealed that tetraploid variety of A. calamus is growing in this region with substantial amount of β-asarone. The oil showed bactericidal property against tested bacteria and fungi. The β-asarone exhibited poorer bactericidal activity against test microorganisms.

GENETIC RELATIONSHIPS AMONG WHEAT VARIETIES BASED ON ISSR MARKERS

DNA based molecular markers allow precise, objective and rapid cultivar identification and discrimination. In the present study, the genetic variability and relationships among 65 hexaploid (Triticum aestivum L.) and 8 tetraploid (Triticum durum L.) wheat varieties were evaluated using five inter simple sequence repeats (ISSR) primers. The used primers generated 63 DNA fragments with an average of 12.6 bands per primer. Polymorphism percentage ranged from 53.33 % (ISSR-844) to 78.57 % (HB12) with an average of 64.95 %. The 308bp (HB12) DNA fragment was unique marker for durum wheat varieties while the 855bp (ISSR-814) fragment was unique maker for bread wheat genotypes. Low PIC values were found indicating the wide differences between the p and q alleles tested by the ISSR markers. The ISSR primers (HB12 and ISSR-844) possessed high RP values and therefore seem to be the most informative primers for distinguishing wheat varieties. The dendrogram indicated that the ISSR markers succeeded in distinguishing the tested varieties in relation to their ploidy level and location, which the tetraploid varieties were put together in one group as well as the hexaploid varieties.

Models with only two predictor variables can accurately predict seed yield in diploid and tetraploid red clover

Red clover (Trifolium pratense L.) is a valuable protein-rich forage crop, but poor seed yields are often a setback, especially in tetraploid varieties. Published work on factors underlying seed yield report contradictory or (sometimes) inconclusive results. Here we report on the effect of 10 traits related to seed yield and flowering on seed yield in 600 genotypes from 15 diploid and 15 tetraploid cultivars from diverse origins. Multiple linear regression (MLR) models indicated that the variation in seed number per plant was well explained by (1) the number of ripe flower heads per plant and (2) the seed number per ripe flower head in diploids (R2 = 0.953) and tetraploids (R2 = 0.919). Flower color was not significantly related to seed yield, and corolla tube dimensions were not (or only borderline) significantly related to seed yield. The applicability of our models to predict seed yield was validated on the same set of plants in the following harvest year, and on a totally different dataset available from Vleugels et al. (Plant Breed 134:56–61, 2015). In the 600-plant dataset and the separate Vleugels et al. dataset, the diploid model explained 94.8 and 53.2 %, respectively, and the tetraploid model explained 88.1 and 64.9 %, respectively, of the variation in seed yield. Our models can therefore predict seed yield with good to fair accuracy in red clover plants grown under various conditions. Breeders can increase seed yield by selecting plants that have numerous ripe flower heads and a high seed number per head. Flower color and corolla tube dimensions can be excluded as breeding targets.

Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers.

The origin of limes and lemons has been a source of conflicting taxonomic opinions. Biochemical studies, numerical taxonomy and recent molecular studies suggested that cultivated Citrus species result from interspecific hybridization between four basic taxa (C. reticulata,C. maxima,C. medica and C. micrantha). However, the origin of most lemons and limes remains controversial or unknown. The aim of this study was to perform extended analyses of the diversity, genetic structure and origin of limes and lemons. The study was based on 133 Citrus accessions. It combined maternal phylogeny studies based on mitochondrial and chloroplastic markers, and nuclear structure analysis based on the evaluation of ploidy level and the use of 123 markers, including 73 basic taxa diagnostic single nucleotide polymorphism (SNP) and indel markers. The lime and lemon horticultural group appears to be highly polymorphic, with diploid, triploid and tetraploid varieties, and to result from many independent reticulation events which defined the sub-groups. Maternal phylogeny involves four cytoplasmic types out of the six encountered in the Citrus genus. All lime and lemon accessions were highly heterozygous, with interspecific admixture of two, three and even the four ancestral taxa genomes. Molecular polymorphism between varieties of the same sub-group was very low. Citrus medica contributed to all limes and lemons and was the direct male parent for the main sub-groups in combination with C. micrantha or close papeda species (for C. aurata, C. excelsa, C. macrophylla and C. aurantifolia--'Mexican' lime types of Tanaka's taxa), C. reticulata(for C. limonia, C. karna and C. jambhiri varieties of Tanaka's taxa, including popular citrus rootstocks such as 'Rangpur' lime, 'Volkamer' and 'Rough' lemons), C. aurantium (for C. limetta and C. limon--yellow lemon types--varieties of Tanaka's taxa) or the C. maxima × C. reticulate hybrid (for C. limettioides--'Palestine sweet' lime types--and C. meyeri). Among triploid limes, C. latifolia accessions ('Tahiti' and 'Persian' lime types) result from the fertilization of a haploid ovule of C. limon by a diploid gamete of C. aurantifolia, while C. aurantifolia triploid accessions ('Tanepao' lime types and 'Madagascar' lemon) probably result from an interspecific backcross (a diploid ovule of C. aurantifolia fertilized by C. medica). As limes and lemons were vegetatively propagated (apomixis, horticultural practices) the intra-sub-group phenotypic diversity results from asexual variations.