Molecular Diversity in Pineapple Assessed by RFLP Markers
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Theor Appl Genet (2001) 102:83-90 O Springer-Verlag2001 M.F. Duval J.L. Noyer X. Perrier G. Coppens d’Eeckenbrugge P/Hamon Molecular diversity in pineapple assessed by RFLP markers Received 8 March 2000 / Accepted: 14 April 2000 Abstract Pineapple, Ananas comosus (L.) L.A.xr,is the productive barrier exists in this germplasm anb these da- third most important tropical fruit cultivated in all tropi- ta indicate the existence of gene flow, enhancing the role cal and subtropical countries. Pineapple germplasm in- of effective sexual reproduction in a species with largely cludes ali seven species of the genus Ananas and the predominant vegetative mutiplication. unique species of the related genus Pseudananas. A knowledge of its diversity structure is needed to develop Keywords Pineapple Ananas Pseudananas . new breeding programs. Restriction fragment length Genetic diversity . RFLP markers polymorphism (RFLP) was used to study molecular di- versity in a set of 301 accessions, most of which were re- cently collected. This sample was analysed using 18 ho- Introduction mologous genomic probes. Dissimilarities were calculat- ed by a Dice index and submitted to Factorial Anqlysis. Pineapple, Anaizas conzosus 6.)Merr., is the bromeliad The same data were represented as a diversity tree con- with the highest economic importance. It is cultivated in structed with the score method. Pseudananas sagenarius all tropical and subtropical countries and ranks third in displayed a high polymorphism and shares 58.7% of its production among tropical fruits. It was domesticated bands with Ananas. Within Ananas, variation appears long before its first historic mention by Christophorus continuous and was found mostly at the intraspecific lev- Columbus in 1493 (Morrison 1973). Pineapple genetic el, particularly in the wild species Ananas ananassoides resources are generally considered to include the brome- and Ananas parguazensis. As for the cultivated species, liads whose flowers and fruits are fused in a syncarp Ananas CO~OSUSappears relatively homogeneous despite (Coppens d’Eeckenbrugge et al. 1997). According to the its wide morphological variation and Ananas bracteatus, last classification by Smith and Downs (1979), which which is grown as a fence and for fruit, appears still was amended by Leal (1990), this corresponds to the much less variable. By contrast Ananas lucidus, cultivat- genera Pseudananas and Ananas, both native to South ed by the Amerindians for fiber, displays a high poly- America. The former is monospecific. Pseudananas sag- morphism. This tree displayed a loose assemblage of nu- enarius (Armda da Câmara) Camargo has been used by merous clusters separated by short distances. Most spe- the Amerindians as a fiber source, but grows generally cies were scattered in various clusters, a few of these be- wild. The latter includes seven species. In addition to ing monospecific. Some accessions which had not been Ananas comosus, Ananns bracteatus (Lindley) Schultes classified, as they shared morphological traits typical of is cultivated as a fence or for fruit and Ananas lucidus different species, re-group with one or the other, and Miller for fiber, although all three species can be found sometimes with both species in mixed clusters. No re- in a wild state. The wild species are Ananas ananasso- ides (Baker) L.B. Smith, Ananas nanus (L.B. Smith), which was previously considered as a dwarf variety of Communicated by H.F. Linskens the former (Smith 1961), Ananas pargziazertsis Camargo M.F. Duval (íXI) . J.L. Noyer . X. Perrier & L.B. Smith., and Ananas fritzinuelleri Camargo. Cirad, BP 5035,34032 Montpellier, France The placement of these taxa at the specific or generic C. d’Eeckenbrugge level has been seriously questioned (Loison-Cabot 1992; CiraMpgri, C/O CIAT, AA 6713, Cali, Colombia Duval et al. 1998). Indeed the current taxonomic key is P. Hamon mainly based on traits that depend on single genes or ~ Université Montpellier 3/IRD, BP 5045, , vary greatly with the environment. Numerous accessions 34032 Montpellier cedexl, France collected recently could not be classified because they 84 . combine typical traits of distinct species according to the tures, extracted using a Miniprep Boehringer kit and tested along botanical classification. Neither discontinuous morpho- with mechanically broken pineapple DNA labelled with d(CTP)- j 32P. A subset of 60 inserts, assumed to be IOWcopy number se- logical variation nor reproductive barriers exist in the ge- quences, was selected, labelled and tested on a subsample of the nus Alzanas, which would justify the regrouping of all plant material restricted with nine endonucleases. Twenty five in- the current Ananas species into a single species (Leal et serts revealed polymorphism. al. 1998). Isozymic polymorphism has been studied in pineap- RFLP ple to clarify classification and for phylogenetic analysis (García 1988; De Wald et al. 1992; Aradhya et al. 1994). As each accession is a clone, DNA was extracted from young Up to 37 systems have been studied but only nine could leaves of only one plant following Gawell and Jarret (1991). Pine- be used and these revealed 31 alleles at ten loci. Obvi- apple DNA probes were labelled with d(CTP)-32P applying the random primer method (Feinberg and Vogelstein 1983). RFLP ously, this is a limited number of markers in a genus pos- procedures were performed as described in Noyer et al. (1997). sessing many chromosomes (~25)(Collins and Kerns The 25 selected probes were hybridized with DNA digested with 1931). four 6-base restriction enzymes: EcoRV, Sstl, HindIII and EcoRI. WLPs are potentially more numerous and efficient in revealing polymorphism. In sorghum, RFZP markers Data analysis proved to be efficient for the racial classification of Culti- vated landraces (Deu et al. 1994; de Oliveira 1996). In Only some RFLP markers revealed a single locus, the others dis- the genus Musa, nuclear RFLPs demonstrate strong rela- played numerous bands that could not be attributed to a particular tion between A-genomes in wild clones and diploid and locus. Consequently, for each accession, bands were scored only for presence (1) or absence (O). The objective for the analysis of triploid cultivars (Jenny et al. 1999). In pineapple, a first these data was to describe the organisation of this set of accessions attempt conducted by Noyer (1991); with five heterolo- in terms of genetic diversity. A similarity-measure is calculated gous and nine homologous RFLP probes on sets of re- between each pair of accessions to descri6e at best their genetic spectively 21 and 14 Ananas accessions, displayed poor proximity. For Co-dominant markers such as RFLPs, a shared band variability with one polymorphic marker out of five in absence is not significant. Through indices based on presencdab- sence (0/1), only those based on shared presences are pertinent. the first set and two out of nine in the second set. Poly- Among them, the Dice indice was reported to give a good approxi- morphism was higher in A. ananassoides. A further mation of the genetic dissimilarity Which could be calculated study conducted with the polymorphic DNA ribosome when scoring alleles (Pemer et al. 1999). marker on DNA restricted by eight endonucleases sepa- For two accessions, i and j, diTj=2 nI1/(2 nll+nlo~~l),where nI1is the number of bands present m both accessions I and J, with rated 92 accessions into six clusters which do not match nloand nol the number of bands present for one accession and ab- up to species, with the exception of A. bracteatus (Noyer sent for the other. et al. 1997). First, a factorial analysis was carried out on the Dice dissimi- In the present work, molecular markers were devel- larity matrix. A projection of the global struture was constructed on the fist factorial plane. This representation gives a good over- oped and used €or the genetic-diversity analysis of the view of the general organization of the set, but is much less ade- genera Ananas and Pseudananas. The results are dis- quate to represent its lower levels. Consequently, the same matrix cussed in relation to the level of differentiation observed. was used to construct a diversity tree with the scoring method of (Sattath and Tversky 1977), which can be viewed as an ordinate and thus is a more robust version of the frequently used Neighbor Joining method (Saitou and Nei 1987). This representation is more Materials and methods pertinent to study the lower levels of organization, although the global structure representation may be less accurate and is a good Plant material complement to the factorial analysis. Both analyses were carried 4. out with the DARWIN 3.5 software {Perrier et al. 1999). Three Iiundred and one accessions belonging to the seven men- tioned species of the genus Ananas (also including some unclassi- fied accessions) and to the unique species of the genus Pseudana- nas were used for DNA analysis. Most of these were recently col- Results lected in Brazil, French Guiana, Venezuela and Paraguay, and maintained ex-situ in the field collections of the French Cirad- Levels of polymorphism Flhor or the Brazilian Embrapa-Mandioca e Fruticultura. The oth- er accessions came from the French collection, including acces- sions collected by Baker and Collins (1939) and obtained through Five out of the 25 selected polymorphic probes showed . exchanges with the USDA National Clonal Germplasm Reposito- complex band patterns. Two did not hybridize with the ry in Hawaii. The sample composition and origin of the material DNA of some species. All seven probes were therefore are detailed in Table 1. discarded to avoid bias. The 18 selected probes revealed a variable number of bands (from 3 for G 421B to 16 for Probe development G 104) with a total of 143 bands for the whole set, i.e. an average of 7.9 bands per probe (Table 2).