Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

Tapia Campos, Ernesto; Gutiérrez Espinosa, María Alejandra; Warburton, Marilyn L.; Santacruz Varela, Amalio; Villegas Monter, Ángel Characterization of mandarin ( SPP.) using morphological and AFLP markers Interciencia, vol. 30, núm. 11, noviembre, 2005, pp. 687-693 Asociación Interciencia Caracas, Venezuela

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative CHARACTERIZATION OF MANDARIN (Citrus spp.) USING MORPHOLOGICAL AND AFLP MARKERS

Ernesto Tapia Campos, María Alejandra Gutiérrez Espinosa, Marilyn L. Warburton, Amalio Santacruz Varela and Ángel Villegas Monter

SUMMARY

Sixty-three mandarin (Citrus spp.) cultivars from the collec- 86% polymorphism. Both morphological and molecular analysis tion of the Campo Citrícola Experimental Francisco Villa, showed a high degree of variation among analyzed accessions, Tamaulipas, Mexico, were evaluated using morphological and indicating an important source of genetic diversity that can be Amplified Fragment Length Polymorphism (AFLP) markers. used in future breeding programs. Although comparison of mor- Twenty quantitative and 10 qualitative morphological characters phological and molecular data using the Mantel test did not in- from leaves, flowers and were evaluated. The Mse +CAG dicate a significant correlation (r= 0,31), both techniques ap- plus Eco +ACA, and Mse + CAA plus Eco + AGG AFLP prim- peared to be complementary for mandarin characterization. ers were the best combinations and generated 109 bands with

Introduction heterogeneous of the three true selection of characters, there tion of accessions in a germ- Citrus (Moore, 2001). Tradi- are several barriers to the full plasm bank allows solving Mandarin (Citrus spp.), to- tionally, morphological charac- utilization of this variability management problems such as gether with the (C. ters have been used to identify (sterility, incompatibility, nu- to avoid duplication in the ex- grandis L.) and (C. Citrus; however, there is a cellar embryony, juvenility). In change and in the conserva- medica L.) are considered the high level of genetic variabil- fact, this genus includes some tion of the germplasm within three true Citrus species. Man- ity which can sometimes make of the most difficult species to the bank, detect mislabeled darin is the second most im- an accurate separation for each improve. accessions, certify propagated portant citrus plant worldwide. variety impossible. Although The exchange of materials material and infer the genetic The mandarin group is com- there is a large amount of for plant improvement is one variability that the collection prised of numerous species as variability within the Citrus of the most important pur- represents in order to increase well as intergeneric and inter- genus with which the breeder poses of maintaining germ- or maintain an appropriate specific hybrids which made can work, and closely related plasm collections. Correct range of genetic diversity. Ac- them the most phenotypically genera provide an even wider classification and identifica- cessions may be classified,

KEY WORDS / Citrus Germplasm / Cluster Analysis / Genetic Similarity / Molecular Markers / Received: 04/27/2005. Modified: 08/19/2005. Accepted: 09/01/2005.

Ernesto Tapia Campos. M.Sc. in María Alejandra Gutiérrez Davies, USA. Researcher, Inter- Ángel Villegas Monter. Doctor, Fruitculture, Instituto de Espinosa. Ph.D. in Horticul- national Maize and Wheat Im- Universidad de Córdova, Espa- Recursos Genéticos y Producti- tural Sciences, University of provement Center (CIMMYT), ña, Professor, IREGEP/CP, vidad, Colegio de Postgraduados Florida, USA. Professor, Mexico. e mail: Mexico. e-mail: (IREGEP/CP), México. Doctoral IREGEP/CP, Mexico. e-mail: [email protected] [email protected] Student, IREGEP/CP. Address: [email protected] Amalio Santacruz Varela. Ph.D. Km. 36.5 carretera México- Marilyn L. Warburton. B.S. and in Plant Breeding, University Texcoco. Montecillo, Edo. de M.S., University of Arizona, of Iowa, USA. Professor, México 56230, Mexico. e-mail: USA. PhD. in Molecular Genet- IREGEP/CP, Mexico. e-mail: [email protected] ics, University of California at [email protected]

NOV 2005, VOL. 30 Nº 11 0378-1844/05/11/687-07 $ 3.00/0 687 RESUMEN

Se evaluaron 63 cultivares de mandarina (Citrus spp.) pro- fismo. Tanto los marcadores morfológicos como los moleculares venientes de la colección del Campo Citrícola Experimental mostraron un alto grado de variación entre los individuos anali- Francisco Villa, Tamaulipas, México, usando marcadores morfo- zados, lo que indica una importante fuente de diversidad genéti- lógicos y AFLP (Amplified Fragment Length Polymorphism). Se ca que puede ser utilizada en futuros programas de mejoramien- usaron 20 caracteres cuantitativos y 10 cualitativos de hojas, to genético. Aunque la comparación de los datos morfológicos y flores y frutos. Las mejores combinaciones de iniciadores AFLP moleculares usando la prueba de Mantel no mostró una correla- fueron la Mse +CAG más Eco +ACA, y Mse +CAA más Eco ción significativa (r= 0.31), ambas técnicas parecen ser comple- +AGG, dando un total de 109 bandas con un 86% de polimor- mentarias para la caracterización de mandarinas.

RESUMO

Avaliaram-se 63 cultivares de mandarina (Citrus spp.) da bandas com um polimorfismo de 86%. Tanto a análise morfoló- coleção do Campo Citrícola Experimental Francisco Villa, gica como a molecular mostraram um alto nível de variação Tamaulipas, México utilizando marcadores morfológicos e AFLP indicando a existência de uma fonte importante de diversidade (Amplified Fragment Length Polymorphism). Avaliaram-se 20 genética que pode ser usada em futuros programas de melhora- caracteres quantitativos e 10 qualitativos em folhas, flores e fru- mento. Mesmo que a comparação dos dados morfológicos e tos, os quais mostraram um alto nível de variação entre os ma- moleculares utilizando a prova de Mantel indicou uma correla- teriais avaliados. As combinações Mse +CAG mais Eco +ACA, ção baixa entre estes (r=0,31), ambas as técnicas parecem ser y Mse +CAA mais Eco +AGG foram as melhores gerando 109 complementarias para a caracterização de mandarinas.

levels of diversity measured phic DNA analysis (RAPD) distributed throughout the ge- Morphological analysis and relationships between in- has been used in the identifica- nome. In crops like dividuals or populations may tion of mutants (Deng et grapevine (Cervera et al., Twenty quantitative and be established using a variety al., 1995), chimeras (Sugarawa 1998; Martínez et al., 2003), 10 qualitative characters of methods. Molecular mark- and Oowada, 1995), somatic papaya (Kim et al., 2002) and were evaluated from 10 ers may extend and comple- hybrids (Guo et al., 2000) coconut (Perera et al., 1998), leaves, 5 flowers and 5 ment characterization based and polyembryony (Ramalho AFLP has been used success- fruits from each plant on morphological or bio- et al., 2000; Andrade-Rodrí- fully in diversity assays. (Table II). The selection of chemical descriptions, provid- guez et al., 2004). Simple Se- The goals of this work morphological characters ing more accurate and de- quence Repeat (SSR) analysis were to characterize acces- was made by applying the tailed information than classi- has been used in phylogenetic sions of mandarin (Citrus IPGRI Citrus spp descriptor cal phenotypic data. Some of and linkage analysis in Citrus spp.) using morphological and (IPGRI, 2000). Morphologi- these techniques are used rou- and Poncirus (Kijas et al., AFLP markers, in order to cal character data were stan- tinely in the management of 1995), mandarin characteriza- evaluate the genetic diversity dardized using the YBAR op- germplasm collections of hor- tion (Koehler-Santos et al., of these accessions and to de- tion of the Stand program ticultural species (Karp et al., 2003) and variabil- tect redundancies inside the from the NTSYS-pc 2.1 soft- 1997). ity (Corazza-Nunes et al., collection. ware (Rohlf, 2000). Dupli- A variety of methods have 2002). Inter-Simple Sequence cate measurements for each been used to analyze genetic Repeat (ISSR) analysis has Materials and Methods specimen were averaged and diversity of Citrus cultivars. been used in Poncirus (Fang used to design a data matrix Isozyme analysis has been et al., 1997) and Citrus iden- Plant material of pairwise similarities be- used to identify nucellar and tification (Fang and Roose, tween genotypes. The Simple zygotic embryos (Torres et 1997). Inter-Retrotransposon Sixty-three mandarin acces- Matching Coefficient (SM) al., 1982) and trifoliate or- Amplified Polymorphism sions from the collection of was used to measure similar- ange cultivars (Khan and (IRAPs) and Amplified Frag- the Campo Citrícola Experi- ity, as it was the coefficient Roose, 1988; Fang et al., ment Length Polymorphism mental Francisco Villa in with the best results follow- 1997). Restriction Fragment (AFLP) have been used in Tamaulipas, Mexico, were ing a cophenetic test (Mar- Length Polymorphism (RFLP) Clementina mandarin identifi- analyzed (Table I). One to tínez et al., 2003). Principal markers have been reported to cation (Breto et al., 2001). three plants were used to rep- Components Analysis (PCA) be highly polymorphic in Cit- Amplified Fragment Length resent each accession, and a was used to depict non-hier- rus (Liou et al., 1996), and Polymorphism (AFLP; Vos et total of 152 plants were ana- archical relationships among used to assay genetic relation al., 1995) is a PCR based lyzed. The materials were the specimens. Eigenvalues among Citrus species (Green fingerprinting technique originally imported from and eigenvectors were calcu- et al., 1986). With the devel- which has been used exten- Texas in 1974 by CONA- lated by the Eigen program opment of the polymerase sively for studying genetic FRUT (National Council for using a correlation matrix as chain reaction (PCR), numer- diversity in different plant Fruit crops). The entire col- input (calculated using stan- ous molecular technologies species, since it can detect a lection comprises 262 differ- dardized morphological data) have been developed, which large number of polymor- ent citrus cultivars or citrus and 2Dplot were used to can be used for the detection, phisms in a single reaction; relatives. In the present work generate the two-dimensional characterization and evalua- and presents a good repeat- only those accessions belong- PCA plot from the software tion of the genetic diversity. ability, generating primarily ing to the mandarin group NTSYS-pc 2.1 (Rohlf, Random Amplified Polymor- dominant markers that are were analyzed. 2000).

688 NOV 2005, VOL. 30 Nº 11 TABLE I DNA isolation AFLP analysis MANDARIN VARIETIES* AND NUMBER OF PLANTS ANALYZED PER VARIETY Very young, healthy leaves AFLP analysis was based from each accession were col- on the protocol of Vos et al. Variety Nº plants lected, labeled, packed in ice (1995). Briefly, 1µg of ge- 1 Changsha mandarin 2 and stored at -80ºC at the nomic DNA was digested 2 Kansu 3 Laboratorio de Biotecnología with two pairs of restriction 3 Lou Chang Mandarin 1 de Frutales, State of México. endonucleases (MseI + EcoRI 4 Long Huang Kat Mandarin 2 DNA was extracted using the or MseI + PstI), respectively, 5 Pon Koa Mandarin 2 Saghai-Maroof et al. (1984) for 4h under conditions rec- 6 Sah Himbra Mandarin 3 7 Seh Seim Mandarin 2** protocol with modifications: ommended by the supplier. 8 Sunegusha Kata Mandarin 1 2g of leaf tissue were ground Restricted DNA fragments 9 Szuwi Kom Mandarin 2 to powder in a mortar with were ligated to previously an- 10 Furuni P.J. 137570 3 liquid N2. Nine ml of the ex- nealed adaptors and preampli- 11 Nuc. Muine Shui Chang 2 traction buffer [100mM Tris fication was carried out using 12 Tizon Mandarin 2 pH 7.7; 700mM NaCl; 50mM EcoRI+1 / Mse1+1 and 13 Karaji x s.o. (H) 2 EDTA pH 8.0; 1% CTAB PstI+1 / MseI+I primers. 14 Kinjoji P.I. 263067 3** (mixed alkyltrimethyl-ammo- Twenty four EcoRI+3 / 15 Tamurana P.I. 280540 3 nium bromide); 140mM β- Mse1+3 and PstI+3 / MseI+3 16 C. Taiwanica Mand x s.o.(H) 3 mercaptoethanol] were added primers combinations were 17 9-5 1 and the mixture was incu- used for selective amplifica- 18 O.L. Clementine Tangerine (Everchard’s) 3 bated for 60min at 65ºC. tion, the forward primer in 20 Nuc. Tang. (olson) 3 4.5ml chloroform/octanol each selective amplification 22 Mara Mandarin 3 (24:1) were added and centri- reaction was labeled with 23 Fairchild Tangerine 3 25 Frost Dancy Tangerine 1 fuged at 1500g for 10min at digoxigenin. 26 Mandarin 3 room temperature. The super- PCR products produced 27 Naranjo Cravo 3 natant was transferred to a from selective amplification 29 Honey Mandarin 3 new tube and the chloroform/ were analyzed on a 6% 30 Nova Tangerine 3 octanol step was repeated. acrylamide gel run in a 1X 31 Oceola Tangerine 3 Thirty µl of RNase (10mg/ml) TBE buffer on a Bio-Rad 32 Robinson Tangerine 1** were added and the mixture sequencing gel apparatus at 33 Nuc. Tankau Tangerine 1 was incubated at 37ºC for 100W for 3h, and then were 34 Nuc. Willowleaf Mandarin 3 45min. An equal volume of transferred to a nylon mem- 35 King Tangerine 3 isopropanol was added, mixed brane previously soaked in 36 Empress Mandarin 2 and incubated at -20ºC for an 0.5X TBE. The mem- 37 Cowhill Murcott Mandarin 3 15min. The pellet formed af- brane was dried for 15min 39 Blair Tangerine 1 ter centrifugation for 10min at at 65ºC and cross linked at 40 Bowers Tangerine 1 1500g at room temperature 12000µjoules. Dig-labeled 41 Clark Tangerine 3 42 Bruce Tangerine 3 was washed with 10µl of 5M fragments were detected us- 43 Manadarinette Mandarin 3 NaCl and 800µl of absolute ing CSPD (disodium 3-(4- 44 Mark Tangerine 1 ethanol. The DNA pellet was methoxispirol {1,2-diox- 45 Page Tangerine 2 left to dry and dissolved in etane-3,2'(5'-chloro) tricyclo 46 Miaray Mandarin 3 200µl of distilled water for [3.3.1.13.7]decan}-4-yl) phe- 47 West Tangerine 1 storage at 4ºC. nyl phosphate). 48 Avana Mandarin 3 49 Australiana citrus 2 50 Kinokuni Mandarin 3 TABLE II 51 Swen-Kat Mandarin 3 MORPHOLOGICAL TRAITS USED IN MANDARIN 52 Sunki Mandarin 3 CHARACTERIZATION 53 Tinkat Mandarin 2 55 Citris depressa (Shekwasa) 3 Code Trait Code Trait 56 Nuc. Satsuma 3 FC Flower color LEW Leaf width (cm) 57 Owari Satsuma 3 FL Flower length (mm) RLW Ratio length/with (leaf) 58 O.L. Orlando 3 LP Length of pedicel (mm) LESH Leaf shape 59 Thornton Tangelo 3 DC Diameter of caliz (mm) LESA Leaf shape of apex 60 Swanne Tangelo 2 NP Number petals FRD Fruit diameter (cm) 62 Ugli Tangelo 2 PL Petal length (mm) FRL Fruit large 63 Pearl Tangelo 3 65 Wekiwa Tangelo (Pink) 2 PW Petal width (mm) FRS Fruit shape 66 Vero Hybrid 3 STAL Stamens length (mm) FRBS Fruit basal shape 68 D.i.-4C Tangelo 3 NS Number stamens FRAS Fruit apex shape 69 Nuc. Wat Tangelo 3 STYL Style length (mm) SKT Skin thickness (mm) 70 Frava 1** RSP Ratio stamens/pistil NSEG Number of segments 71 Tarraco Tangor 3 PIL Pistil length (mm) AXS Axis saturation 72 Dweet Tangor 3 WP Wings on petiole AXF Axis form IAP Incisions of apex AXD Axis diameter (mm) * From the Campo Citrícola Experimental Francisco Villa collection. ** Accessions for which only molecular data are available. LEL Leaf length (cm) NSE Number of seeds

NOV 2005, VOL. 30 Nº 11 689 AFLP bands were scored The cophenetic correlation Group 1, contained 41 acces- Tizon and Bowers (0.89), in- based on the presence (1) or was calculated in order to sions, mainly from mandarin dicating that these pairs are absence (0) of polymorphic find the degree of association and Mediterranean groups, closely related varieties. On fragments for each primer, between the original similarity and consisted of two sub- the other hand, the Vero Hy- and used to calculate a ge- matrix and the tree matrix in groups (A and B) and three brid and Clark mandarin ac- netic similarity matrix using both morphological and AFLP accessions (Karaji P.I., Kansu cessions had the lowest simi- the Jaccard (J) coefficient, analysis. Comparison between and Australiana) which did larity values (0.10). Although which is more appropriate for both methods was performed not fall into a subgroup. there were accessions with dominant markers as it does for the accessions for which Group 2 also formed two sub- similar names (for example, not count 0/0 matches in the both morphological and AFLP groups (C and D) and con- Nucellar Satsuma and Sat- calculation. The genetic dis- data were available by calcu- sisted of 18 accessions, suma Owari), they do not ap- tance between each pair of lating the correlation between mainly hybrids from manda- pear to be related to each genotypes was calculated by the two data sets using the rins with other species such other. SIMGEND analysis of the Mantel test in NTSYS-pc. as and (sub- The cophenetic analyses NYSYS-pc software package group D) and some mandarins comparing the UPGMA clus- version 2.1 (Rohlf, 2000). Results and Discussion like Satsuma, Clementine, ter analysis and the simple Cluster analysis was per- Tamurana, Furuni, Kara, and matching similarity matrix formed on both, morphologi- Morphological analysis Taiwanica (subgroup C). demonstrated a correlation of cal and molecular, similarity Group 2 contained one outlier r= 0.82, indicating that data matrices using the unweighted The UPGMA dendrogram (Vero Hybrid) which did not in the matrix was well repre- pair group method using obtained using morphological fall into either subgroup. sented by the dendrogram. arithmetic means (UPGMA) characters (Figure 1) sepa- The average genetic simi- Accessions were clustered algorithm, from which den- rated the mandarin accessions larity among the mandarin ac- mainly based on size of flow- drograms depicting similarity into two main groups, which cessions was 0.65, with val- ers and fruits. Individuals among varieties were drawn diverged at a similarity index ues ranging from 0.10 to from Group 1 tended to have and plotted using NTSYS-pc. of 0.41. The larger group, 0.90. Accessions Swen Kat smaller flowers and fruit than and Tinkat showed a very did individuals from Group 2. high degree of similarity The classical morphological (0.90), as did accessions classification of mandarin di-

TABLE III DESCRIPTIVE STATISTICS AND EIGENVECTOR COEFFI- CIENTS OF QUANTITATIVE AND QUALITATIVE CHAR- ACTERS EVALUATED IN THE MANDARIN VARIETIES Character Mean Std. Dev. Range CP1 CP2 CP3 FC 1.0169 0.1302 -0.064 -0.1001 0.0413 FL 14.0847 3.191 7-22 0.2433 0.1188 -0.1639 LP 5.2712 2.3181 2-13 0.2475 0.0915 -0.1857 DC 5.1695 1.101 3-8 0.2635 0.1551 -0.0026 NP 4.9661 0.1825 4-5 -0.1387 0.1362 -0.1130 PL 13.9153 3.2125 7-2 0.2644 0.1491 -0.0489 PW 5.9492 1.3445 4-10 0.2544 0.0117 0.1155 STAL 10.0339 2.3339 5-17 0.2778 0.0371 -0.0685 NS 20.9322 3.3055 15-30 0.1768 0.1605 -0.0734 STYL 5.8983 1.3733 3-10 0.2571 0.1405 -0.0034 RSP 4.5254 0.8581 3-5 0.0026 -0.3616 -0.1492 PIL 9.9492 2.2851 5-17 0.2832 0.1325 -0.0196 WP 1.0339 0.1825 0.1021 -0.2371 0.0666 IAP 2.3051 0.8357 0.0607 -0.2318 0.2195 LEL 7.5324 1.3853 5.56-12.6 0.2080 0.1235 -0.0690 LEW 3.642 1.0478 2.4-8.06 0.1229 -0.0211 0.4995 RLW 2.1497 0.4411 0.8523-3.99 -0.0737 0.1274 -0.4796 LESH 2.5254 1.3816 -0.1494 0.0048 -0.1259 LESA 3.4576 1.0225 -0.0057 0.0587 0.3438 FRD 7.3411 1.2111 3.825-10.075 0.2001 0.1178 0.0944 FRL 6.5813 2.698 2.975-23.95 0.2458 0.0092 0.1211 FRS 4.0508 1.6756 -0.2310 0.2054 0.0481 FRBS 3.4237 1.07 -0.1909 0.1087 0.1929 FRAS 4.0678 0.5208 -0.1155 0.0575 0.2360 SKT 3.8139 1.3873 1.88-8.25 0.2156 -0.0829 0.1053 NSEG 10.5564 1.1135 7.5-13.25 -0.0392 0.0160 -0.1307 AXS 2.5085 0.774 -0.1561 0.3710 0.0284 Figure 1. UPGMA dendrogram (based on Simple Matching Similarity Coefficients) of 59 mandarin cultivars from the Campo Citrícola Experi- AXF 1.5085 0.5042 -0.1062 0.3633 -0.0242 mental Francisco Villa collection, generated by 20 quantitative and 10 AXD 1.3555 0.7892 0.6-6.5 -0.0866 0.3724 0.2208 qualitative morphological characters. NSE 11.5637 10.5369 000-44.75 -0.0430 0.2734 0.0853

690 NOV 2005, VOL. 30 Nº 11 variance; 28.8% in the first netic values resulted in a cor- and 8.6% in the second relation of r= 0.92, indicating (Table IV). The first PCA that data in the matrix was was most highly influenced very well represented by the by traits related with flower dendrogram. Similar to the and fruit morphology, the sec- morphological analysis, two ond PCA was most influ- main groups were distin- enced by characteristics of the guished and they diverged at axes and the third PCA was a genetic similarity coefficient most influenced by leaf char- of 0.53. Group 1 (containing acters (Table III). The projec- two subgroups, A and B) was tion of the 59 mandarin ac- composed of 42 accessions, cessions onto the graph de- mainly from mandarin and fined by the 2 first principal Mediterranean groups. Group components confirmed the 2 contained 21 accessions; same groups reflected in the subgroup C mainly integrated dendrogram (Figure 2). Group by some mandarins from the 1 showed more diversity than Satsuma group and tangelos, Group 2. Tangor and Tangelo and subgroup D composed of accessions were clearly the tangores. The subgroups grouped in the graph, along diverged at a genetic similar- with some of few other man- ity of 0.65. darin accessions from manda- The AFLP primers used in rins. Only Vero Hybrid and this work were able to dis- Kansu accessions were sepa- criminate between all manda- Figure 2. Plot of the first and second principal components, resulting rated from the rest of the ac- rin accessions analyzed, in- from a PCA of mandarin varieties assessing 30 morphological charac- cessions, which was agreed cluding those with the same ters (correlation matrix used as input). The first and second components with the subgroup generated common name. This high dis- explain 28.77 and 8.56% of the variation respectively. from the dendrogram. crimination between (presum- ably) closely related acces- vides this species into four by several authors (Domin- AFLP analysis sions is the result of the large principal groups: Satsuma gues et al., 1999; Koehler- heterogeneity in the mandarin mandarin, common mandarin, Santos et al., 2003). Cluster Level of polymorphism. An group. The level of variation Mediterranean ‘Willowleaf’ analysis results from these initial analysis was carried out in mandarins has been previ- mandarin, and natural and authors and the present one with 24 combinations of ously reported by several au- man-made hybrids (Davies were different; although it is EcoRI-MseI and PstI-MseI thors using different tech- and Albrigo, 1994). However, important to consider that primers with three nucleotide niques including isozymes these four groups were not some accessions and charac- extensions, in order to deter- (Torres et al., 1978; Torres et completely clear in the mor- ters were different in those mine the best combination of al., 1982), ISSR (Fang and phological dendrogram. In works. enzymes and selective ampli- Roose, 1997; Fang et al., Group 1, there were acces- Principal components fication primers. The Mse 1998), RFLP and RAPD (Fe- sions from at least 3 of the 4 analysis (PCA) was used to CAG plus Eco ACA, and Mse derici et al., 1998) and micro- main groups listed, while in identify multidimensional re- CAA plus Eco AGG were the satellites (Koehler-Santos et Group 2 there were mainly lationships among characters best combinations, based on al., 2003). All of them found accessions from Satsuma and for the definition of groups. the number of bands which a large heterogeneity within natural and man made hy- The first 5 principal compo- could be unambiguously this group, and all concluded brids. Characterization of nents accounted for 56.1% of scored. A total of 54 and 55 that the mandarin group is the mandarins using morphologi- the total variability and the bands were obtained, respec- most variable of the three true cal characters have been used first two for 37.3% of the tively, similar to what Vos et Citrus species (C. grandis, C. al. (1995) reported previously. medica and C. reticulata), TABLE IV Of the 109 total bands, 94 both when measured with EIGENVALUES, DIFFERENCES, AND PROPORTIONS OF variable bands were recorded molecular markers and mor- VARIABILITY FOR THE 10 PRINCIPAL COMPONENTS (86% polymorphism). phologically. Breto et al. AMONG 30 CHARACTERS FOR 59 MANDARIN VARIETIES The mean genetic similarity (2001) analyzed 24 Clemen- Component Eigenvalue Difference Proportion % Cumulative % between each pair of geno- tina mandarin accessions by types was 0.75 and ranged AFLP and found a low poly- 1 8.63038 6.06212 28.77 28.77 from 0.94 between the acces- morphism level between ac- 2 2.56825 0.43484 8.56 37.33 sions Robinson and the cessions; however, this result 3 2.13341 0.28688 7.11 44.44 Tankau, and Kinokuni and was logical because all culti- 4 1.84652 0.19539 6.16 50.60 Swen-Kat, to 0.21 between vars of Clementine mandarins 5 1.65113 0.04259 5.50 56.10 Clementine and the Tangor were derived from a single 6 1.60853 0.29856 5.36 61.46 Tarraco. UPGMA cluster plant and thus the genetic 7 1.30997 0.13452 4.37 65.83 analysis of the AFLP genetic variability level was low. 8 1.17545 0.13293 3.92 69.75 similarity matrix resulted in 9 1.04252 0.02219 3.48 73.22 the dendrogram in Figure 3. Comparison between AFLP 10 1.02032 0.20256 3.40 76.62 A comparison of the cophe- and morphology. Comparison

NOV 2005, VOL. 30 Nº 11 691 dependent, due to different Villa citrus collection were selection and evolutionary characterized using molecular factors. Although AFLP mark- markers since it was created ers can cover a high propor- in 1974. This is one of the tion of the genome because largest collections in México, of the high number of bands and includes not only acces- scored in each analysis, due sions with commercial charac- its neutral origin, there is no teristics (such as Satsuma and guarantee that such bands fall Clementina) but also acces- in coding regions of the ge- sions used as rootstocks nome involved in morphologi- (Sunki mandarin and Citrus cal and agronomic traits. amblicarpa) and as sources of Traditionally, germplasm disease resistance (Citrus has been classified on the ba- depressa). Despite the high sis of morphological and ag- diversity present in the man- ronomical traits, but recently darin group, only a small the use of molecular markers number of cultivars are used to study diversity and charac- commercially, and so the ex- terization in plants has be- ploitation of the genetic diver- come common. In this study sity in the in situ collections the characterized accessions to support breeding programs were mainly grouped accord- and other research efforts is ing to flower and fruit mor- important. The characteriza- phology, which are complex tion information of the broad and multigenic characters. genetic variation found in this Such characters are environ- work can be used in germ- mentally affected and there- plasm management, variety fore liable to subjective protection, and new efforts in evaluation. In this sense, the citrus improvement. molecular characterization is more efficient in the genera- Conclusions tion of an unbiased picture of diversity than an agro- Both morphological and nomic approach. However, molecular markers show a the agronomic characteriza- high degree of variation tion is still important in ger- among the mandarin acces- mplasm management, and de- sions analyzed. These acces- Figure 3. UPGMA dendrogram (based on Jaccard’s similarity coeffi- termination of molecular di- sions should represent an im- cients) of 63 mandarin cultivars from the Campo Citrícola Experimental versity should not be seen as portant source of genetic di- Francisco Villa collection generated using AFLP markers. replacing traditional charac- versity in citrus and can be terization but rather as a used in future breeding pro- of matrices of AFLP and Australiana accessions, which complement to it. grams; there were no redun- morphological data shows a did not fall into one of the Although the correlation dancies inside the collection. low correlation between den- subgroups in the morphologi- between the morphological Both dendrograms separate drograms (r= 0.31, P=1.0) cal dendrogram, but in the and AFLP data was low, both the mandarin accessions ana- following 500 random permu- molecular analysis they clus- methods allowed grouping the lyzed in two main groups and tations with the Mxcomp pro- tered closely to other acces- mandarin accessions analyzed four subgroups with some dif- cedure from the NTSYS pro- sions within a subgroup. in this work. Despite the fact ferences. Although the corre- gram. Despite this low corre- The low correlation be- that morphological traits were lation between morphological lation between morphological tween AFLP and morphologi- relatively less efficient for and AFLP data is low, both and molecular analysis, there cal traits had been reported in precise discrimination of techniques can be used com- were similar groups formed in other studies in European bar- closely related genotypes, the plementarily in mandarin the respective dendrograms. ley varieties (Schut et al., cost and time invested were characterization. The formation of two main 1997), synthetic hexaploid lower than for the molecular groups was consistently found wheats and their parents analysis. 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