Organización de la diversidad genética de los cítricos

Citrus es el género de la subfamilia Aurantioideae de mayor importancia económica. Su origen es la región sureste de Asia, en un área que incluye China, India y la península de Indochina y los archipiélagos de los alrededores. Aunque se han realizado múltiples estudios, la taxonomía del género Citrus aun no está bien definida, debido al alto nivel de diversidad morfológica encontrado en este grupo, la compatibilidad sexual entre sus especies y la apomixis de muchos genotipos. En la presente tesis doctoral se ha estudiado una amplia diversidad del género Citrus, especies relacionadas y otros taxones de la subfamilia Aurantioideae, para poder aclarar su organización y filogenia mediante el empleo de diferentes tipos de marcadores moleculares y métodos de genotipado. Más concretamente, el germoplasma de mandarino juega un papel muy importante en la mejora de variedades y patrones, pero su organización genética no está bien definida. Por lo tanto, se ha realizado un análisis en profundidad de su diversidad y organización genética. El desarrollo de marcadores moleculares de Inserción-Deleción (indel), por primera vez en cítricos, ha permitido demostrar su utilidad para estudios de diversidad y filogenia en el género Citrus. En combinación con los marcadores de tipo microsatélite (SSR), se ha cuantificado la contribución de los tres principales taxones de cítricos (C. reticulata, C. maxima and C. medica) a los genomas de las especies secundarias y cultivares modernos. También se ha definido su estructura genética a partir de los datos obtenidos en la secuenciación de 27 fragmentos de genes nucleares relacionados con la biosíntesis de compuestos que determinan la calidad de los cítricos y genes relacionados con la respuesta de la planta a estreses abióticos. El análisis de la filogenia nuclear ha permitido determinar la relación existente entre la especie C. reticulata y Fortunella, que se diferencian claramente del grupo formado por las otras dos principales especies de cítricos (C. maxima y C. medica). Este resultado está en concordancia con el origen geográfico de las especies estudiadas. A partir de este estudio, se han desarrollado marcadores moleculares de tipo SNP con un alto valor filogenético, que han sido transferidos a géneros relacionados de los cítricos. Estos marcadores han dado un resultado muy positivo en el género Citrus y serán de gran utilidad para el establecimiento de la huella genética del germoplasma en un nivel de diversidad más amplio. Se ha estudiado la organización genética dentro del germoplasma mandarino (198 genotipos de tipo mandarino pertenecientes a dos colecciones, INRA-CIRAD e IVIA), así como la introgresión de otros genomas mediante el uso de 50 y 24 marcadores de tipo SSR y indel, respectivamente, además de cuatro marcadores InDel mitocondrial (ADNmt). Se ha observado que muchos genotipos, que se creía que eran mandarinos puros, presentan introgresión de otros genomas ancestrales. Dentro del germoplasma de mandarino, se han identificado a nivel nuclear cinco grupos parentales, a partir de los cuales se originaron muchos genotipos, dando lugar a estructuras hibridas complejas. Se ha observado incluso, genotipos con un origen maternal no mandarino, determinado por los marcadores de ADNmt. La presente tesis doctoral ha aportado nueva información sobre las relaciones filogenéticas entre las especies del género Citrus, géneros cercanos, así como de las especies secundarias. Además, se han desarrollado nuevos marcadores moleculares que se complementan entre sí. Se ha establecido una nueva organización genética del germoplasma mandarino y se han caracterizado adecuadamente las dos colecciones de cítricos en estudio. Por lo tanto, todas estas contribuciones, ayudarán a los programas de mejora para la obtención de nuevas variedades de cítricos de alta calidad y permitirán optimizar la conservación y uso de los recursos genéticos existentes, así como su caracterización genética y fenotípica.García Lor, A. (2013). Organización de la diversidad genética de los cítricos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31518TESI

Identification of ovule and seed genes from Citrus clementina

[EN] Seedlessness is a highly desirable trait in fresh fruit. Citrus varieties, such as Clementine mandarin and other related species, show parthenocarpic fruit development without seeds due to self-incompatibility. In spite of that, these fruits frequently contain seeds as a result of cross-pollination by insects with compatible pollen from other citrus cultivars grown nearby. To solve this problem using a biotechnological approach we aim at the destruction of ovules and/or seeds by directing the expression of a toxic gene using the promoter of an ovule and/or seed specific gene. With the purpose of isolating this kind of genes we constructed two cDNA libraries from ovules and seeds at different developmental stages of the Clementine mandarin (Citrus clementina cv. Clemenules). A total of 1,014 ESTs from the ovule library and 1,042 ESTs from the seed library were generated, with a novelty percentage of 27% and 36% among the Spanish Citrus Functional Genomic Project (CFGP) ESTs database, respectively. Quantitative PCR analysis confirmed nearly specific expression in ovule and/or seed of two genes, TRANSPARENT TESTA16 (CcTT16) and TRANSPARENT TESTA7 (CcTT7). Expression of these two genes is restricted to early seed development, and is localized in the embryo sac and endothelium. The promoters of those genes may be useful to genetically engineer citrus species to avoid seed formation in fruits of commercial varieties.The authors thank Dr. L. Navarro and J. Juarez, from the Instituto Valenciano de Investigaciones Agrarias (Generalitat Valenciana), for the use of the Clementine orchard and Fortune pollen; Dr. J Forment and the Genomics Facility at the IBMCP for sequence analysis and maintenance of to the CFGP database; and Drs. F Tadeo and J Carbonell for critical reading of the manuscript. This work was supported by grants from the Conselleria de Agricultura, Pesca y Alimentacion (Generalitat Valenciana) and Spanish Ministerio de Ciencia y Tecnologia (research grant GEN2001-4885-C05). A.G-L. received a PhD fellowship from the Conselleria de Agricultura, Pesca y Alimentacion.García Lor, A.; Garcia Martinez, JL.; Perez Amador, MA. (2012). Identification of ovule and seed genes from Citrus clementina. Tree Genetics and Genomes. 8(2):227-235. doi:10.1007/s11295-011-0435-xS22723582Al-Shahrour F, Diaz-Uriarte R, Dopazo J (2005) Discovering molecular functions significantly related to phenotypes by combining gene expression data and biological information. Bioinformatics 21:2988–2993Al-Shahrour F, Minguez P, Tarraga J, Montaner D, Alloza E, Vaquerizas JMM, Conde L, Blaschke C, Vera J, Dopazo J (2006) BABELOMICS: a systems biology perspective in the functional annotation of genome-scale experiments. 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Plant Cell 15:2514–2531Dorcey E, Urbez C, Blazquez MA, Carbonell J, Perez-Amador MA (2009) Fertilization-dependent auxin response in ovules triggers fruit development through the modulation of gibberellin metabolism in Arabidopsis. Plant J 58:318–332Debeaujon I, Peeters AJ, Léon-Kloosterziel KM, Koornneef M (2001) The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium. Plant Cell 13:853–571Forment J, Gadea J, Huerta L, Abizanda L, Agusti J, Alamar S, Alos E, Andres F, Arribas R, Beltran JP, Berbel A, Blazquez MA, Brumos J, Canas LA, Cercos M, Colmenero-Flores JM, Conesa A, Estables B, Gandia M, Garcia-Martinez JL, Gimeno J, Gisbert A, Gomez G, Gonzalez-Candelas L, Granell A, Guerra J, Lafuente MT, Madueno F, Marcos JF, Marques MC, Martinez F, Martinez-Godoy MA, Miralles S, Moreno P, Navarro L, Pallas V, Perez-Amador MA, Perez-Valle J, Pons C, Rodrigo I, Rodriguez PL, Royo C, Serrano R, Soler G, Tadeo F, Talon M, Terol J, Trenor M, Vaello L, Vicente O, Vidal C, Zacarias L, Conejero V (2005) Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies. Plant Mol Biol 57:375–391Forment J, Gilabert F, Robles A, Conejero V, Nuez F, Blanca JM (2008) EST2uni: an open, parallel tool for automated EST analysis and database creation, with a data mining web interface and microarray expression data integration. 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Oxford University Press, Oxford, UK, pp 163–174Koltunow AM (1993) Isolation and construction of genes to control seed production in Citrus. In: Hyashi T, Omura M, Scott NS (eds) Techniques on gene diagnosis and breeding. Fruit Tree Research Station, Tsukuba, Japan, pp 101–108Koltunow AM, Soltys K, Nito N, Mcclure S (1995) Anther, ovule, seed and nucellar embryo development in Citrus sinensis cv Valencia. Can J Bot 73:1567–1582Koltunow AM, Brennan P, Bond JE, Barker SJ (1998) Evaluation of genes to reduce seed size in Arabidopsis and tobacco and their application to Citrus. Mol Breeding 4:235–251Le BH, Cheng C, Bui AQ, Wagmaister JA, Henry KF, Pelletier J, Kwong L, Belmonte M, Kirkbride R, Horvath S, Drews GN, Fischer RL, Okamuro JK, Harada JJ, Goldberg RB (2010) Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors. Proc Natl Acad Sci USA 107:8063–8070Li DD, Shi W, Deng XX (2002) Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene. Plant Cell Rep 21:153–156Marques MC, Alonso-Cantabrana H, Forment J, Arribas R, Alamar S, Conejero V, Perez-Amador MA (2009) A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus. BMC Genomics 10:428Mezzetti B, Landi L, Pandolfini T, Spena A (2004) The DefH9-iaaM auxin-synthesizing gene increases plant fecundity and fruit production in strawberry and raspberry. BMC Biotechnology 4:4Nesi N, Debeaujon I, Jond C, Stewart AJ, Jenkins GI, Caboche M, Lepiniec L (2002) The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS domain protein and is required for proper development and pigmentation of the seed coat. 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Pollen Development and Viability in Diploid and Doubled Diploid Citrus Species

Seedlessness is one of the most important agronomic traits in mandarins on the fresh fruit market. Creation of triploid plants is an important breeding strategy for development of new commercial varieties of seedless citrus. To this end, one strategy is to perform sexual hybridizations, with tetraploid genotypes as male parents. However, while seed development has been widely studied in citrus, knowledge of key steps such as microsporogenesis and microgametogenesis, is scarce, especially in polyploids. Therefore, we performed a study on the effect of ploidy level on pollen development by including diploid and tetraploid (double diploid) genotypes with different degrees of pollen performance. A comprehensive study on the pollen ontogeny of diploid and doubled diploid “Sanguinelli” blood orange and “Clemenules” clementine was performed, with focus on pollen grain germination in vitro and in planta, morphology of mature pollen grains by scanning electron microscopy (SEM), cytochemical characterization of carbohydrates by periodic acid–Shiff staining, and specific cell wall components revealed by immunolocalization. During microsporogenesis, the main difference between diploid and doubled diploid genotypes was cell area, which was larger in doubled diploid genotypes. However, after increase in size and vacuolization of microspores, but before mitosis I, doubled diploid “Clemenules” clementine showed drastic differences in shape, cell area, and starch hydrolysis, which resulted in shrinkage of pollen grains. The loss of fertility in doubled diploid “Clemenules” clementine is mainly due to lack of carbohydrate accumulation in pollen during microgametogenesis, especially starch content, which led to pollen grain abortion. All these changes make the pollen of this genotype unviable and very difficult to use as a male parent in sexual hybridization with the objective of recovering large progenies of triploid hybrids

Citrus (Rutaceae) SNP markers based on Competitive Allele-Specific PCR; transferability across the Aurantioideae subfamily.

UNLABELLED: ; PREMISE OF THE STUDY: Single nucleotide polymorphism (SNP) markers based on Competitive Allele-Specific PCR (KASPar) were developed from sequences of three Citrus species. Their transferability was tested in 63 Citrus genotypes and 19 relative genera of the subfamily Aurantioideae to estimate the potential of SNP markers, selected from a limited intrageneric discovery panel, for ongoing broader diversity analysis at the intra- and intergeneric levels and systematic germplasm bank characterization. ; METHODS AND RESULTS: Forty-two SNP markers were developed using KASPar technology. Forty-one were successfully genotyped in all of the Citrus germplasm, where intra- and interspecific polymorphisms were observed. The transferability and diversity decreased with increasing taxonomic distance. ; CONCLUSIONS: SNP markers based on the KASPar method developed from sequence data of a limited intrageneric discovery panel provide a valuable molecular resource for genetic diversity analysis of germplasm within a genus and should be useful for germplasm fingerprinting at a much broader diversity level

Alborea: A New Mid-late Mandarin Triploid Hybrid [(Citrus clementina x C. tangerina) x (C. nobilis x C. deliciosa)]

Citrus is one of the most important fruit crops worldwide, with more than 130 million tons produced in 2017. Mandarins represent 25% of this production (Food and Agriculture Organization, 2018) and are mainly destined for fresh consumption. The Mediterranean area is the most important region for mandarin exports, with 60% of the total volume, and Spain is the leading country. The fresh market demands high-quality, seedless fruit that can be harvested throughout the marketing season. Therefore, mandarin breeding programs worldwide are mainly aimed at obtaining new, seedless, easy-peeling varieties with an attractive fruit color and flavor, and with high organoleptic characteristics (Grosser et al., 2010; Navarro et al., 2015; Rapisarda et al., 2008). The mandarin varietal structure in Spain has several problems. It includes satsumas [Citrus unshiu (Mak.) Marc.], clementines (C. clementina Hort. ex Tan.), and mandarin hybrids. Satsumas are harvested from the end of August to mid November. Their pollen and ovules are not viable, and thus they are seedless. Clementine varieties are the most important group of mandarins in Spain and can be harvested from mid September until the second half of January. They are self-incompatible, but their pollen and ovules are viable and, consequently, they are able to pollinate and be pollinated with other compatible cultivars. ‘Hernandina’ clementine, our latest maturing clementine, is characterized by low fruit quality when grafted onto ‘Carrizo’ citrange [C. sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.], which is by far the predominant rootstock in our country. Fruit peel deteriorates quickly after mid January, and in practice no clementine fruit is available in the second half of January. Mandarin hybrids, such as ‘Nova’ [C. clementina × (C. paradisi Macf. × C. tangerina Hort. Ex Tan.)], ‘Fortune’ (C. clementina × ?), ‘Murcott’ (C. reticulata Blanco × C. sinensis), and ‘Ortanique’ (natural hybrid between mandarin and C. sinensis), were introduced to our citriculture to cover the demand of late-maturing mandarins by international markets. These cultivars are self-incompatible, but their pollen and ovules are viable and they cross-pollinate with clementines, producing seedy fruit in both groups of mandarins. In addition, some late mandarin hybrids, such as ‘Fortune’, ‘Nova’, and ‘Murcott’, are susceptible to Alternaria alternata fungus, which reduces production and depreciates the fruit commercially for the fresh-fruit market. This fungus has forced the top-grafting of trees of these susceptible cultivars, particularly ‘Fortune’, and during the past few decades there has been a decrease in late-mandarin plantations and an increase in clementine plantings, mainly ‘Clemenules’. This has created an imbalance between production and market demand that has caused a drastic reduction in prices for the growers such that, in many cases, they cannot even sell their products. As a consequence of excessive clementine production, a lot of growers decided to cultivate other late mandarin cultivars, especially ‘Nadorcott’ [(C. reticulata × C. sinensis) × ?], ‘Tango’ (irradiated variety from ‘Nadorcott’ mandarin), and ‘Orri’ (irradiated variety from ‘Orah’ mandarin) and, more recently, ‘Spring Sunshine’ (irradiated variety from ‘Murcott’ mandarin), which is susceptible to Alternaria. These varieties cover the period from February to the end of April and are managed by different private companies that limit the number of plants or the cultivated area, and also impose high royalties that, in many cases, are difficult for growers to pay. Under this scenario, the recovery of a new, high-quality seedless mandarin cultivar that matures from mid December until the end of January, when there are no other high-quality mandarins in the market, and that is resistant to Alternaria is a very important objective for our citriculture. In 1995, a triploid breeding program was started at the Instituto Valenciano de Investigaciones Agrarias (IVIA) with the objective of producing new mid- and late-maturing triploid cultivars resistant to A. alternata fungus through sexual hybridization, embryo rescue, and ploidy analysis by flow cytometry (Navarro et al., 2015). Triploid plants generally produce aneuploid gametes, thus leading to very low fertility of their pollen and ovules (Otto and Whitton, 2000). For this reason, citrus triploid hybrids can be considered sterile, producing seedless fruit, and do not induce seed formation in other varieties by cross-pollination, even in the presence of bees (Navarro et al., 2015). A routine strategy exploited for triploid citrus breeding is spontaneous female unreduced gamete formation in diploid × diploid crosses (Aleza et al., 2012; Cuenca et al., 2011, 2015), where triploid hybrids arise usually from the union of an unreduced megagametophyte formed through a second-division restitution mechanism with a haploid pollen (Cameron and Frost 1968; Cuenca et al., 2011; 2015; Esen and Soost 1971, 1973; Geraci et al., 1975; Luro et al., 2004). Triploid embryos are predominantly found in small seeds, which generally do not germinate in greenhouse conditions. Thus, embryo rescue from these small seeds is required to reach high germination rates (Aleza et al., 2010b). In addition, ploidy level determination by flow cytometry is also required in extensive triploid citrus breeding programs (Aleza et al., 2012). From this program, the mid- to late-maturing triploid varieties ‘Garbı’ [(C. clementina × C. tangerine) × (C. reticulata × C. sinensis)] (Aleza et al., 2010a) and ‘Safor’ [(C. clementina × C. tangerina) × (C. unshiu × C. nobilis Lour.)] (Cuenca et al., 2010) were released, with more than 600,000 plants commercialized until 2018. We describe a new triploid hybrid named ‘Alborea’ mandarin [(C. clementina × C. tangerina) × (C. nobilis × C. deliciosa Ten.)] that is resistant to Alternaria alternata and characterized by the production of high-quality, seedless fruit that can be harvested from mid-December until the end of January

Maximum-likelihood method identifies meiotic restitution mechanism from heterozygosity transmission of centromeric loci: application in citrus

Polyploidisation is a key source of diversification and speciation in plants. Most researchers consider sexual polyploidisation leading to unreduced gamete as its main origin. Unreduced gametes are useful in several crop breeding schemes. Their formation mechanism, i.e., First-Division Restitution (FDR) or Second-Division Restitution (SDR), greatly impacts the gametic and population structures and, therefore, the breeding efficiency. Previous methods to identify the underlying mechanism required the analysis of a large set of markers over large progeny. This work develops a new maximum-likelihood method to identify the unreduced gamete formation mechanism both at the population and individual levels using independent centromeric markers. Knowledge of marker-centromere distances greatly improves the statistical power of the comparison between the SDR and FDR hypotheses. Simulating data demonstrated the importance of selecting markers very close to the centromere to obtain significant conclusions at individual level. This new method was used to identify the meiotic restitution mechanism in nineteen mandarin genotypes used as female parents in triploid citrus breeding. SDR was identified for 85.3% of 543 triploid hybrids and FDR for 0.6%. No significant conclusions were obtained for 14.1% of the hybrids. At population level SDR was the predominant mechanisms for the 19 parental mandarins

A reference genetic map of C. clementina hort. ex Tan.; citrus evolution inferences from comparative mapping

Background: Most modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a 'Mediterranean' mandarin x sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine. Results: Five parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between 'Mediterranean' mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Conclusions: A reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the 'Mediterranean' mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents

Characterization of gibberellin 20-oxidases in the citrus hybrid Carrizo citrange

CcGA20ox1 is a gene encoding a GA 20-oxidase, a gibberellin (GA) biosynthetic enzyme, previously isolated from the citrus hybrid Carrizo citrange (Citrus sinensis (L.) Osbeck x Poncirus trifoliata (L.) Raf.). Southern blot analysis of genomic DNA of Carrizo citrange with CcGA20ox1 suggested the presence in the hybrid of another gene encoding another GA 20-oxidase. A cDNA clone from this new gene (CcGA20ox2) was isolated using RNA from the other parent C. sinensis. CcGA20ox2 encoded a protein of 372 amino acids that showed 67.1% identify with CcGA20ox1, and its expression product catalyzed the in vitro conversion of GA(12) to GA(9), confirming that it corresponds to another active GA20ox. Amplification of genomic DNA and isolation of genomic clones of CcGA20ox1 and CcGA20ox2 revealed that the parental sources these genes in the hybrid were P. trifolicata and C. sinensis, respectively. The sequences of CcGA20ox1 and CcGA20ox2 showed that both genes contained two introns, which are also conserved in GA20ox genes of other species like Arabidopsis thaliana L., Pisum sativum L. and Solanum lycopersicum L. Determination of transcript levels in the Carrizo citrange hybrid by quantitative real-time polymerase chain reaction showed that CcGA20ox1 and expressed mainly in internodes, leaves and seeds, and CcGA20ox2 in flower buds and flowers at anthesis, with the genes having similar transcript levels in young developing fruits.Peer reviewe

Identificación of ovule and seed genes from Citrus clementina

Seedlessness is a highly desirable trait in fresh fruit. Citrus varieties, such as Clementine mandarin and other related species, show parthenocarpic fruit development without seeds due to self-incompatibility. In spite of that, these fruits frequently contain seeds as a result of cross-pollination by insects with compatible pollen from other citrus cultivars grown nearby. To solve this problem using a biotechnological approach we aim at the destruction of ovules and/or seeds by directing the expression of a toxic gene using the promoter of an ovule and/or seed specific gene. With the purpose of isolating this kind of genes we constructed two cDNA libraries from ovules and seeds at different developmental stages of the Clementine mandarin (Citrus clementina cv. Clemenules). A total of 1,014 ESTs from the ovule library and 1,042 ESTs from the seed library were generated, with a novelty percentage of 27% and 36% among the Spanish Citrus Functional Genomic Project (CFGP) ESTs database, respectively. Quantitative PCR analysis confirmed nearly specific expression in ovule and/or seed of two genes, TRANSPARENT TESTA16 (CcTT16) and TRANSPARENT TESTA7 (CcTT7). Expression of these two genes is restricted to early seed development, and is localized in the embryo sac and endothelium. The promoters of those genes may be useful to genetically engineer citrus species to avoid seed formation in fruits of commercial varieties.This work was supported by grants from the Conselleria de Agricultura, Pesca y Alimentacion (Generalitat Valenciana) and Spanish Ministerio de Ciencia y Tecnologia (research grant GEN2001-4885-C05). A.G-L. received a PhD fellowship from the Conselleria de Agricultura, Pesca y Alimentacion.Peer reviewe

Acta Horticulturae

Citrus reticulata is considered one of the four main ancestral taxa of cultivated citrus and is widely associated with the mandarin horticultural group. However, several studies suggest introgressions of other taxa and the precise structure of the mandarin group is not known. To analyse the structuration of mandarin germplasm and the potential interspecific introgressions, 208 accessions were genotyped by the KASPar method, with sixty-seven single nucleotide polymorphism (SNP) markers mined in 27 nuclear genes and in clementine BAC-ends. These SNP markers were employed for the study of 208 genotypes comprised of 169 ` mandarin-like' genotypes and 39 genotypes belonging to ancestral taxa. Structure software was used to identify groups within the mandarin germplasm. Six mandarin groups were identified, some of them in agreement with the species defined by Tanaka. Many mandarins were located between these mandarin groups, indicating a high level of admixture between the identified parental groups. New insights in the organization of mandarin germplasm and its structure are presented in this work, which will be useful for better management of citrus germplasm collections