Mol Breeding (2012) 29:245–260 DOI 10.1007/s11032-010-9543-9
A genetic map of pineapple (Ananas comosus (L.) Merr.) including SCAR, CAPS, SSR and EST-SSR markers Jorge Dias Carlier • Nelson Horta Sousa • Tatiana Espı´rito Santo • Geo Coppens d’Eeckenbrugge Jose´ Manuel Leita˜o
•
Received: 2 May 2010 / Accepted: 5 December 2010 / Published online: 29 December 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Despite the paramount importance of pineapple (Ananas comosus L.) in world production and trade of tropical fruits, the genomics of this crop is still lagging behind that of other tropical fruit crops such as banana or papaya. A genetic map of pineapple was constructed using an F2 segregating population obtained from a single selfed F1 plant of a cross A. comosus var. comosus (cv. Rondon, clone BR 50) 9 A. comosus var. bracteatus (Branco do mato, clone BR 20). Multiple randomly amplified markers (RAPD, ISSR and AFLP) were brought together with SSR and EST-SSR markers identified among sequences uploaded to public databases and with sequence-specific markers (SCAR, SSR and CAPS) derived from random amplified markers. Sixty-three randomly amplified markers (RAPD, ISSR and AFLP) were selected and cloned, resulting in 71 sequences which were used to generate sequence-specific SCAR and CAPS markers. The present map includes 492 Electronic supplementary material The online version of this article (doi:10.1007/s11032-010-9543-9) contains supplementary material, which is available to authorized users. J. D. Carlier N. H. Sousa T. E. Santo J. M. Leita˜o (&) BioFIG, FCT, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal e-mail:
[email protected] G. C. d’Eeckenbrugge CIRAD, UMR 5175 CEFE, 1919 Route de Mende, 34293 Montpellier, France
DNA markers: 57 RAPD, 22 ISSR, 348 AFLP, 20 SSR, 12 EST-SSR, 25 SCARs, 8 CAPS, and the morphological trait locus ‘‘piping’’, gathered into 33 linkage groups that integrate markers inherited from both botanical varieties, four linkage groups with markers only from var. comosus and three linkage groups with markers exclusively from var. bracteatus. The relatively higher mapping efficiency of sequence-specific markers derived from randomly amplified markers (50.7%) versus SSR (31.4%) and EST-SSR (28.9%) markers is discussed. Spanning over 80% of the 2,470 cM estimated average length of the genome, the present map constitutes a useful research tool for molecular breeding and genomics projects in pineapple and other Bromeliaceae species. Keywords Pineapple Ananas comosus A. comosus var. bracteatus Genetic map Genomics SCAR CAPS SSR EST-SSR Pineapple genomics
Introduction Cultivated in most tropical and subtropical countries and in some mild climate regions (e.g. Azores Islands, Portugal), pineapple (Ananas comosus L.) ranks third in world production among tropical fruits, after banana and citrus (Botella and Smith 2008). The pineapple taxonomy was revised by Coppens d’Eeckenbrugge and Leal (2003) who simplified
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the previous classification of two genera and seven species (Smith and Downs 1979) into one genus Ananas with two species: A. comosus (L.) Merr. (diploid, 2n = 50) and A. macrodontes Morren (tetraploid, 2n = 100; formerly Pseudananas sagenarius). According to this new classification, A. comosus includes five botanical varieties: comosus, ananassoides, parguazensis, erectifolius, and bracteatus. The genetic divergence between A. macrodontes and A. comosus and the low level of genetic differentiation among the botanical varieties of A. comosus that underlie the new classification are supported by a large panoply of studies using molecular marker techniques such as isozymes (Garcı´a 1988; Aradhya et al. 1994), randomly amplified polymorphic DNA (RAPD) (Ruas et al. 2001), restriction fragment length polymorphism (RFLP) (Duval et al. 2001), amplified fragment length polymorphism (AFLP) (Kato et al. 2004; Paz et al. 2005) and cpDNA PCR–RFLP (Duval et al. 2003), as well as cytogenetic (Lin et al. 1987; Cotias-de-Oliveira et al. 2000) and floral biology data (Collins 1960; Coppens d’Eeckenbrugge et al. 1993). Genomics of tropical crops is progressing rapidly. For some species such as banana and papaya, it has already progressed to the establishment of large research consortia such as the Global Musa Genomics Consortium (http://www.musagenomics.org) or the International Papaya Genome Consortium, which in the latter case resulted in the publication of a first draft of the genome sequence (Ming et al. 2008). For other crops, e.g. cacao (Lanaud et al. 1995; Pugh et al. 2004), avocado (Sharon et al. 1997; Borrone et al. 2009), coffee (Pearl et al. 2004), taro (Quero-Garcı´a et al. 2006) and cashew (Cavalcanti and Wilkinson 2007), genetic maps have been published and, in some cases, subsequently improved. Pineapple genomics, however, is still ‘‘in its infancy’’ (Botella and Smith 2008) and very few genes have been cloned and functionally characterised (for review, see Carlier et al. 2007; Botella and Smith 2008). Nevertheless, a large number of expressed sequence tag (EST) sequences have been uploaded to genome databases over the last few years (e.g. Moyle et al. 2005, 2006) and an online pineapple bioinformatics resource, PineappleDB (www.pgel.com.au), containing over 5,600 EST and 3,383 consensus sequences has been created and microarrays based on over 9,000 pineapple cDNAs have been produced (Botella and Smith 2008).
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Regarding genome mapping, the first and very incomplete genetic maps of pineapple—A. comosus var. bracteatus and A. comosus var. comosus—were constructed by Carlier et al. (2004) on the basis of 46 F1 plants. Gathering together 491 molecular markers (RAPD, inter simple sequence repeat [ISSR] and AFLP) and the locus for the morphological trait ‘‘piping’’, the two maps were constructed simultaneously using the ‘‘two-way pseudo-testcross’’ strategy (Grattapaglia and Sederoff 1994; Hemmat et al. 1994). Preliminary maps of randomly amplified markers analysed among F1 and F2 populations have been constructed and reported in recent reviews on pineapple genomics (Carlier et al. 2007; Botella and Smith 2008). Here we present the first pineapple map completely constructed on the basis of an F2 population and composed of randomly amplified (RAPD, ISSR, AFLP) and sequence-specific (SSR, EST-SSR, CAPS and SCAR) markers. In addition to the simple sequence repeat (SSR) and EST-SSR markers identified in public databases, over sixty randomly amplified markers (RAPD, ISSR, AFLP) were cloned, sequenced and transformed into sequence characterised amplified region (SCAR) markers. Polymorphic SCAR markers were amplified among the segregating population and remapped. Monomorphic SCARs were tested with bulks of restriction enzymes for their transformation into co-dominant cleaved amplified polymorphic sequence (CAPS) markers. In some cases the marker sequences contained microsatellite motifs and the corresponding SCAR markers were denoted as SSR.
Materials and methods Plant materials A mapping population consisting of 135 F2 plants was obtained from a single selfed F1 plant of a cross A. comosus var comosus (cv. Rondon, clone BR 50) 9 A. comosus var. bracteatus (Branco do mato, clone BR 20). Crosses and selfings were performed at the CIRAD-FLHOR station in Martinique. Leaves from parental, F1 and F2 plants were received at the Laboratory of Genomics and Genetic Improvement,
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BioFIG, FCT, University of Algarve, for molecular analyses and map construction. DNA marker analyses Procedures for DNA isolation and for RAPD and AFLP analyses were previously described in Carlier et al. (2004). Primers with the respective code numbers and the procedures for analysis of ISSR markers can be found in Farinho´ et al. (2004). Procedures for transformation of randomly amplified molecular markers (RAPD, ISSR and AFLP) into SCARs, for analysis of these markers and for their conversion into CAPS have been previously described in Farinho´ et al. (2007). All the 50 pineapple sequences uploaded as microsatellites to the NCBI database by Duval et al. (www.ncbi.nlm.nih.gov) and three additional microsatellite sequences identified by Hamon et al. (1998) were retrieved for generation of SSR markers. Additionally the program SSR Taxonomy Browser [http://bioinformatics.pbcbasc.latrobe.edu.au/cgibin/ssr_ taxonomy_browser.cgi (2006)] was used for identification of microsatellite sequences among the EST and core nucleotide pineapple sequences uploaded to the NCBI database. SSR primers were designed within a length range of 16–24 nucleotides, with CG% over 40%, using FastPCR-version 4.0.13 software (Kalendar 2007) with default settings for hairpin and cross or self-dimer analysis. Microsatellite reactions were performed in a T-Gradient thermocycler (Biometra, Go¨ttingen, Germany) programmed for one initial denaturation step of 2.5 min at 94°C, 35 cycles of denaturation (1 min at 94°C), annealing (1 min at specific temperature between 55 and 65°C), and extension (1 min at 72°C), followed by a final extension of 10 min at 72°C. Amplified SSRs were first analyzed on 4% agarose gels. SSR markers whose analysis was ambiguous on these gels were further analysed on non-denaturing 8% polyacrylamide gels (19:1 mix) using a Mini-PROTEAN (Bio-Rad) apparatus. Both agarose and polyacrylamide gels were revealed by transillumination with UV light after ethidium bromide staining and recorded with a Kodak EDAS 120 system.
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Labelling of markers The suffixes Ab or Ac were added to the name of the markers with dominant inheritance according to their parental origin, var. bracteatus or var. comosus, respectively. These suffixes are absent in co-dominant markers. RAPD markers are denoted by the identifying letters of the Operon Technologies primer kit followed by the code number of the primer (01–20) and the estimated size of the marker in base pairs, e.g. OPAE15_1600_Ab stands for the 1,600-bp marker amplified by primer 15 of the kit AE inherited from var. bracteatus. ISSR markers are identified by this acronym followed by the two-digit number ascribed to the primer (Farinho´ et al. 2004) followed by the estimated size of the marker, e.g. ISSR02_1300_Ac for the 1,300-bp ISSR marker generated by primer 02 and inherited from var. comosus. AFLP markers are denoted by the two or three last nucleotides of the primers in each combination, followed by the length of the marker in bp, e.g. AAG.CTA_124_Ac stands for the 124-bp marker amplified by primers EcoRI and MseI with 30 random nucleotide sequences AAG and CTA, respectively, inherited from var. comosus. SSR and EST-SSR markers are identified, respectively, by these acronyms followed by the identification code of the respective accession in the NCBI database, e.g. EST_SSR_CO731235. SCAR markers are labeled by the prefix ‘‘Sc’’ followed by the designation of the respective original marker, e.g. Sc_ISSR03_662_Ac stands for the dominant SCAR marker, inherited from var. comosus, derived from marker ISSR03_662_Ac. CAPS markers are denoted by the acronym CAP followed by the name of the marker it originates from and the acronym of the restriction enzyme used, e.g. CAP_OPA03_332/MboI stands for the CAPS marker originated from the RAPD marker OPA03_332 whose polymorphism is revealed by the restriction enzyme MboI. Additional information regarding marker identification and labeling is provided in the text and in the caption of Fig. 1.
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Map construction and genome length estimation The JoinMap 3.0 program (Van Ooijen and Voorrips 2001) set for the Kosambi mapping function was used for map construction and for graphical representation of the linkage groups. With very few exceptions, linkage groups were constructed with a minimum LOD of 4.0 for establishment of linkage between markers and a minimum LOD of 2.0 for the order of markers within the linkage group. Markers showing slight distortion (v2a¼0:05 v2 \v2a¼0:01 ), more pronounced distortion (v2a¼0:01 v2 \v2a¼0:001 ) and very strong distortion (v2 v2a¼0:001 ) in comparison to the expected Mendelian segregation ratios (3:1 or 1:2:1) were labelled, respectively, with one, two or three asterisks. The genome lengths of the parental genotypes (var. bracteatus and var. comosus) were calculated using method 3 of Chakravarti et al. (1991). The genome length of the species (A. comosus) was calculated as the arithmetical average of the genome length of the two botanical varieties.
Results Marker segregation analysis Forty-five RAPD primers, selected for amplification among the F2 population, have generated 86 markers used for map construction. After the construction of a preliminary version of the map (not shown), 50 RAPD markers were selected for cloning, which resulted in 56 sequences used for conversion into sequence-specific markers: (a) 22 of these new markers segregated as the original RAPD marker and were re-mapped as sequence-specific markers (18 SCARs, two CAPS; two SSR); (b) seven sequences were converted into two SCAR and five CAPS that mapped as new markers apart from the location of the initial RAPD marker; (c) 25 sequences resulted in monomorphic sequence-specific markers that could not be remapped and the corresponding original RAPD markers were suffixed with a ‘‘#’’. Among the 64 markers mapped as RAPDs, 57 were included in the map and seven remained unlinked (Fig. 1). Among the 32 ISSR primers assayed in this study, 19 primers generated 30 markers useful for mapping.
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Mol Breeding (2012) 29:245–260 Fig. 1 Linkage groups 1–33 congregate markers from both c botanical varieties: bracteatus and comosus. Linkage groups 34–36 gather markers only from var. bracteatus. Linkage groups 37–40 gather markers uniquely from var. comosus. (#) Markers with one putative sequence assigned. (2#) Markers with two putative sequences assigned. (b) Sequenced markers or markers with a previously known sequence. (ib) Markers with incomplete sequence. Numbers in parentheses above linkage groups: LOD score for the establishment of linkage group followed by the LOD score for the order of markers within the group
Twelve of these ISSR markers were cloned and originated 14 sequences used to generate sequencespecific markers. Five of these new markers (four SCAR and one SSR-ISSR) segregated exactly as the original ISSR markers (one remained unlinked) while one SCAR and one SSR-ISSR were mapped as new markers in a location different from the original markers. The primers designed for one sequence did not generate any amplified product and six mapped ISSR markers were labelled with ‘‘#’’ since their sequence-derived specific markers were monomorphic and not suitable for remapping. Among the 25 markers that remained denoted as ISSR in the mapping matrix, 22 were included in the map and three remained unlinked. Because of the much higher multiplex ratio of the AFLP technique, the 46 primer combinations used generated 374 polymorphic markers—an average of approximately eight polymorphic markers per primer combination—which were analysed among the mapping population. One AFLP marker was cloned, sequenced and mapped as a SCAR marker. A total of 348 AFLP markers were included in the map while 25 remained unlinked. Altogether, the 63 cloned markers (RAPD, ISSR and AFLP) resulted in 71 sequences (Table 1) of which 37 (52%) were converted into polymorphic sequence-specific markers successfully used for mapping. Nine (24.3%) out of the sequence-specific markers mapped were new, but five of them originated from markers that originated more than one sequence. Primers were designed for 48 out of the 50 microsatellite (SSR) sequences from A. comosus var. bracteatus uploaded to the NCBI public databases by Duval et al. (www.ncbi.nlm.nih.gov) and for three additional SSR sequences identified by Hamon et al. (1998) (Electronic Supplementary Material). However
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Fig. 1 continued
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Fig. 1 continued
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Table 1 Sequenced random amplified markers Original marker
Accession number
Resulting marker
Original marker
GU726827
Sc_AC.CAA_335_Ab
AFLP
Accession number
Resulting marker
RAPD (cont.)
AC.CAA_335_Ab ISSR
OPH08_650_Ab
DQ386605
SSR_OPH08_650
OPH08_990_Ac
GU726825
Sc_OPH08_971_Ac OPL03_1235_Ab_#
ISSR03_200_Ac
GU726810
ISSR03_193_Ac_#
OPL03_1250_Ab
GU726805
ISSR03_400_Ab
GU726783
Sc_ISSR03_386_Ab
OPL03_200_Ab
GU726794
OPL03_201_Ab_#
ISSR03_690_Ac
GU726784
Sc_ISSR03_662_Ac
OPL03_450_Ab3
GU726795
OPL03_450_Ab3
ISSR06_430_Ac
EF688074
Sc_ISSR06_354_Ac
–
CAP_OPL03_442/MspI2
1
EF688075
SSR_ISSR06_369
OPL12_690_Ac
GU726772
Sc_OPL12_665_Ac
ISSR06_480_Ac
GU726809
ISSR06_499_Ac_#
OPL15_200_Ab
DQ386599
Sc_OPL15_185_Ab
ISSR09_470_Ab
GU726828
SSR_ISSR09_443_Ab
OPL15_390_Ac
GU726796
OPL15_374_Ac_#
ISSR13_610_Ac
GU726786
Sc_ISSR13_591_Ac
OPL15_510_Ab3
GU726797
OPL15_510_Ab3
ISSR14_400_Ac
GU726811
ISSR14_383_Ac_#
–
CAP_OPL15_490/Hin6I2
ISSR14_610_Ac
GU726812
ISSR14_614_Ac_#
OPM06_1200_Ac
GU726781
Sc_OPM06_1072_Ac
ISSR15_800_Ab
GU726832
ISSR15_800_Ab_2#
OPM10_600_Ab
EF688078
Sc_OPM10_583 OPM12_381_Ac_#
GU726831
–
OPM12_400_Ac
EF688083
ISSR18_500_Ab
GU726813
ISSR18_495_Ab_#
OPM12_480_Ac
GU726798
OPM12_461_Ac_#
ISSR19_580_Ab3
GU726785
ISSR19_580_Ab3
OPM12_570
GU726799
OPM12_554_Ab_#
–
Sc_ISSR19_5712
OPO18_320_Ac
EU004074
OPO18_348_Ac_#
OPP03_710_Ac
EF688082
SSR_OPP03_722
RAPD OPA02_340_Ac
GU726806
OPA02_335_Ac_#
OPQ18_450_Ac
GU726780
Sc_OPQ18_425
OPA03_350_Ab
DQ386596
CAP_OPA03_332/MboI
OPR13_1050_Ab
GU726782
Sc_OPR13_1036
OPA03_750_Ab
GU726779
Sc_OPA03_727_Ab
OPR15_400_Ac
GU726773
Sc_OPR15_385_Ac
OPAB09_500_Ac
GU726771
Sc_OPAB09_489
OPR15_850_Ab
GU726774
Sc_OPR15_841_Ab
OPAB09_800_Ac
GU726821/22
OPAB09_800_Ac_#_i
OPU06_440_Ab
GU726802
OPU06_439_Ab_#
OPAC07_600_Ab
GU726807
OPAC07_596_Ab_#
OPU12_610_Ab
GU726800
OPU12_602_Ab_#
OPAE12_750_Ab
GU726824
Sc_OPAE12_740_Ab
OPX16_880_Ac
GU726788
CAP_OPX16_848/BsuRI
OPAE14_1050_Ab
GU726778
Sc_OPAE14_1051_Ab
OPX16_900_Ab
GU726803
OPX16_903_Ab_#
OPB15_490_Ac
EF688081
OPB15_507_Ac_#
OPX18_1200_Ac
GU726775
Sc_OPX18_1186_Ac
OPC05_590_Ac
GU726791
OPC05_568_Ac_#
OPU16_1000_Ab
GU726801
OPU16_1017_Ab_#
OPC09_1800_Ab
GU726833/34
OPC09_1800_Ab*_#_i
GU726790
Sc_OPU16_515_Ab1
OPC19_650_Ab
DQ386601
OPC19_621_Ab_#
GU726776
Sc_OPU18_755_Ab
OPE03_250_Ac
GU726792
OPE03_246_Ac_#
GU726777
Sc_OPU18_765_Ab1
OPE03_430_Ab
GU726826
Sc_OPE03_422
OPE12_600_Ab
EF688076
Sc_OPE12_566_Ab
OPF19_1350_Ab
GU726808
OPF19_1300_Ab_#
OPF19_500_Ab
GU726830
OPF19_524_Ab_#
DQ386597
Sc_OPH02_349_Ab
OPH02_360_Ab 3
OPH08_1150_Ab OPH08_450_Ac
GU726829
OPU18_800_Ab OPR15_250_Ab OPD01_650_Ac OPAE14_1000_Ac
3
OPH08_1150_Ab
2
–
CAP_OPH08_1267/HinfI_Ab
GU726793
OPH08_404_Ac_#
1
New markers resulting from additional sequences
2
Markers whose segregation does not match with that of the original marker
3
Markers whose single sequence-specific marker shows a different segregation pattern
#
Markers with one putative sequence assigned
2#
Markers with two putative sequences assigned
123
GU726814
OPR15_250_Ab_2#
GU726815
–
GU726804
OPD01_627_Ac_#
GU726787
CAP_OPD01_615/MspI1
GU726816
OPAE14_1000_Ac_2#
GU726789
CAP_OPAE14_988/Sau96I1
GU726817
–
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the mapping efficiency of the SSR markers was relatively low as 15 SSRs were monomorphic, eight generated complex patterns of difficult analysis and 11, although polymorphic, were not present in the selfed F1 plant that produced the F2 population. Ultimately, only 17 out of this set of 51 SSR markers were included in the mapping matrix: 16 were mapped and one remained unlinked (Table 2). The in silico search for microsatellite sequences among the 5,649 ESTs and 92 core nucleotide sequences of A. comosus var. comosus available at the time in public databases (www.ncbi.nlm.nih.gov) allowed the identification of 592 microsatellite loci (238 di-, 320 tri-, 21 tetra- and 13 penta-nucleotide repeats). Among the 45 EST-SSR loci for which primers were designed, 12 exhibited polymorphic alleles clear and easy to score among the F2 progeny and were mapped (Table 2). Additionally, one ESTSSR was mapped as a CAPS marker. The remaining 32 EST-SSR markers were either monomorphic, displayed complex patterns of difficult interpretation or did not segregate among the F2 progeny. Eleven out of the 13 EST-SSR sequences integrated in the map (including one as CAPS marker) were identified within the EST sequences uploaded by Moyle et al. (2005), one EST-SSR marker was derived from a RING zinc finger protein mRNA sequence identified by Neuteboom et al. (2002) and another one from a polyphenol oxidase gene sequence (Zhou et al. 2003). Map construction Among the 530 markers that were included into the mapping matrix 37 (*7.0%)—one SSR, one Sc-ISSR, three ISSR, seven RAPD and 25 AFLP— remained unlinked. The new map integrates 492 DNA markers (57 RAPD, 22 ISSR, 348 AFLP, 20 SSR, 12 EST-SSR, 25 SCARs, 8 CAPS) and the morphological trait locus ‘‘piping’’, characterized by the monogenic and dominantly inherited absence of spines along the leaf margin (Collins and Kerns 1946). The 20 SSR markers were derived from 16 SSR sequences retrieved from the genomic databases, and from two RAPD and two ISSR markers. The 25 SCAR markers were derived from 20 RAPD, four ISSR and one AFLP markers. The eight CAPS markers originated from seven RAPD and one EST-SSR markers.
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The 493 loci that constitute the new map are assembled in: (a) 33 linkage groups that integrate markers inherited from both parents (458 DNA markers plus the morphological trait piping); (b) three groups that congregate 14 markers inherited from var. bracteatus and (c) four groups that gather 20 markers inherited from var. comosus. With the exception of four cases, the linkage groups were constructed using a minimal LOD threshold of 4.0 for the establishment of linkage among markers and, except in two cases, the order of the markers within each group was determined for a minimal LOD threshold of 2.0. Nevertheless, for most of the groups these minimal LOD score values were largely exceeded. In fact, 26 linkage groups were established for a LOD equal or over 5.0, whereas a LOD equal or over 2.5 was used to order 32 groups (Fig. 1). The 33 linkage groups that integrate markers inherited from both progenitors—although five of the groups are very small triplets and doublets of markers—still do not correspond to the 25 pineapple chromosomes. Altogether these linkage groups total 1549.4 cM, when the average distance between markers (3.7 cM) is added to the extremities of each group (Marques et al. 1998) their overall span increases to 1793.6 cM. Computed in the same way, the four linkage groups with markers from var. comosus and the three linkage groups with markers exclusively from var. bracteatus span over 109.9 cM and 70.4 cM, respectively. Taken as a whole, and doubly increased with the computed average distance between markers in all groups, the 40 linkage groups of the map span over 1974.2 cM. Seventy-nine (14.9%) of the markers in the matrix showed distorted segregation: 59 slight distortion, 16 more pronounced distortion and four strong distorted segregation. The percentage of distorted markers was slightly lower (14.2%) among mapped markers and clearly higher (24.3%) among the unlinked ones. The four markers that showed very strong distortion, including one SCAR and one CAPS marker, were integrated in the map where they are identified by three asterisks. The distribution of markers with distorted segregation throughout the map was not random: six linkage groups (LG1, LG3, LG4, LG8, LG11 and LG25) gather
123
123 AGATCCAAACAACGTTGAAG
CAP_OPX16_848/BsuRI
AGGAAGATCTGGACCGT GTATTTTGGGTGAGGTCG ACATGGAGCTGTTCATGC ATGGGATGTGGCATTGT
Sc_ISSR03_386_Ab
Sc_ISSR03_662_Ac
Sc_ISSR13_591_Ac
Sc_ISSR19_571
SCAR_ISSR
TGAGATTTGCCGCCA GCACACTAAAGGGATTCC TCTTTCCACCGCGCCTCATC
Sc_OPAE14_1051_Ab
Sc_OPE03_422
Sc_OPE12_566_Ab
CATCATGGGTCTTGTGAG
AGTGAGAGGGAATCCTC TCACGTGGAGGGAGAG
EST_SSR_CO731871_Ab Sc_AC.CAA_335_Ab
SCAR_AFLP
Sc_OPAE12_740_Ab
CTCCTCAGCTTCGTCGCC
EST_SSR_CO731816
CGACTACAGAGCACGA
CTTTTGGGCTATGTTGCG
EST_SSR_CO731753
Sc_OPAB09_489
CACATGTCCACGTATTGG
EST_SSR_CO731431_Ac
GCTAGGGATGTTACCAGT
GTAACACAACTACTCTACTCGGCA
EST_SSR_CO731330
Sc_OPA03_727_Ab
AGGGAAGCTTTGGAGGTGCAG
EST_SSR_CO731287
SCAR_RAPD
TTCTGCTCCGCTCTTCTTC
EST_SSR_CO730888 ATTTCGAGCCCTTGGTCG
CGCATCAGCGCCAAACGC
EST_SSR_CO730886
EST_SSR_CO731235
CTTAGGGTTGAATGGTCC CTCATCTCACTCCATACC
EST_SSR_AY149881_Ac
GTATATTACCGTGGATGCGGGAG
CAP_OPL15_490/Hin6I
EST_SSR_AY098521
TAACCTGATGACGCTTCTC
CAP_OPH08_1267/HinfI_Ab CAP_OPL03_442/MspI
EST_SSR_CO730928_Ac
EST_SSR
CTCCATCGTTTAGGGTTAC CAAGCTCCAAACCAAGC CCAGCAGCTTGATAATGTT
CAP_OPD01_615/MspI
AGTCAGCCACCCGTACA CAACCGACTACAGCCT
CAPS_RAPD
CGAGCAATGACGCTCGGG
CAP_OPA03_332/MboI
CAP_EST_CO731374/BcuI
CAPS_EST
Forward primer sequence (50 –30 )
CAP_OPAE14_988/Sau96I
Marker
Marker type
Table 2 Mapped sequence-specific markers
ACCCACCATTGTTGCAAG
ACCATGGACCTTCAAAGA
GTTGTTCCATGCAGTC
CGAGTCGATGTGGTACA
ATGTTTACCCCGGAATG
GTTCCGCAGTGCTTAC
GCACCCAAATATGCATCG
ACGTACAACACCCACCA
TGACGCTACCCACTTCA
TCAATTCTGCGGGTACT
CTTATTCACCTCGTTGCC CGTAAGCACGAAGATTG
GACGAGATTGGCGTATCCC
TGCTAAAGTACCCACCAG
GTAGCTCCACTCAGCCC
CCTCCTCCAATTAAGATCCCTCAA
TGCAATAGCGATGATAAACCCCAG
TTTATGGGGTCGCGTCGG
AATTGAGGCGATCGATGC
GGAAGCGAAAGGAGATCG
CCATTCGATATCGCCTTC
ATCTGAGACCCAAGTTCG
AGCATCAAGGGGTCCCGAGTT
CTGTGGTTATAGCTGAACTC
AGAAACTGTGACCTAACTCG
GATACTGGACCTGGTACTG AGCAGCTTACCACAAATC
AGTGCTTCTTTACGGTTG
GAAACATGGGAGCTTGAG
GGAGGAAGCGAACAAAGACGC
CACACCGAGAAAGCCCGG
Reverse primer sequence (50 –30 )
271
263
588
532
433
622
298
419
463
284
248 308
211
227
294
112
159
185
196
171
301
293
148
638
346
427 440
431
247
314
395
Expected size (bp)
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SSR_ISSR
SSR
SCAR_RAPD
Marker type
Table 2 continued
TCGCGTATTATTCAAACAGCC TCATCACCCCGCGCCTTTGC CAGTGGTGATTGAAGCCATGC TGCTGGCTCTGTGGGATG TGTAGGCATATGGTGGGTCTG GCCTCGAAAACACTGCTAGGC TCCCCCTAATCATCGGAAGCC ACCCAGCCATTGTCGTGCCTG TCATTTAGGATGCTGCATGG ACATTCCTCAGAGTGACCAGC
SSR_AJ845040 SSR_AJ845048
SSR_AJ845052
SSR_AJ845056
SSR_AJ845060
SSR_AJ845062
SSR_AJ845069
SSR_AJ845076
SSR_AJ845077
SSR_AJ845081
SSR_ISSR06_369
AGAGAGAGCTGGCTGAATG
GTTCGCAGGAGAATAGAG
TTGGAGCCGATATTATCGTCC
SSR_AJ845039
SSR_G134
TGATCATGGCGACGACCCAG
SSR_AJ845038
GTTATTGGTTTGGCCAGT
Sc_OPX18_1186_Ac
AGGTGAAGGTGGAGCTCACC
ATGCTTGTCCTTGAGCT
Sc_OPU18_765_Ab
SSR_AJ845036
CTAATCGCATCCAAATGC
Sc_OPU18_755_Ab
TCCACAGTGGGCGCAAAC
TGGATCGTTGACAAATCC
Sc_OPU16_515_Ab
GTATACCCCTCACCACCCAAG
TGGATACGATGGTCAGTC
Sc_OPR15_841_Ab
SSR_AJ845033
GACAACGAGTGACGTGG
Sc_OPR15_385_Ac
SSR_AJ845035
GACAGCCTAATCTTACTTGG
Sc_OPR13_1036
AGGCCATATCAGCCTGTG
Sc_OPL15_185_Ab
CTGGGTGATGGTACTAGA
GGTACTTTGCCTAGGTG
Sc_OPL12_665_Ac
Sc_OPQ18_425
CACAGAGAAGCTTGATGG
Sc_OPH08_971_Ac
ACATGATCGTTGTTGAGCC TCTGGCGCACATGGTGA
TCGGACGTGAGGGCGTG
Sc_OPH02_349_Ab
Sc_OPM06_1072_Ac Sc_OPM10_583
Forward primer sequence (50 –30 )
Marker
AGAGAACGGGCACAAACCT
CGTTGTAATGCAGTGAAC
CACTAATCCTTGACCCAGACC
TCTCTCATGCGCACATGC
AGTTTACAAGGCGCATAGG
GGATGATGATGGTCACCTCTG
GGTGTGTCAATTAGGCCTGAC
ATCTCTTAATCCAAGGGCCG
TTAGGTTTTCAGTGGAGAGAG
TTCACACCGAGAAAGCCCGG
ACTGAGGGGGTTCACGAG TGCCAAGCCATCCTCAGACG
ACGATCTCACAATGCTCCTCG
TCATTGTCGCGGCACCATG
GTCGCCGTTAATCGACACGTG
GCGCAATCCATAGCGCAAGTC
AAAGGACATGAGGTAGGCC
ACTAGGTGGTCACTTAGG
CCTACCACTGAGACCAG
AGGAAGCTTTGTGTAGC
GAAGCGAGTTTACTGCA
CTGTTCAATCTACCAGAACG
GGACAACGAGGATTCAACA
CACAAGAACTAGCTCAGC
ACAAGTATGGTGCCAAC
CTAGACAATTCACTCACAGG TGAGCGACAATCGTGGATGA
ATATCTCTAACACTGTAG
GGTATGAAGAAGGACATCG
AAGCAACCAACTCAACC
CGGACGTGACACACACACTGG
Reverse primer sequence (50 –30 )
192
75
214
214
368
261
308
166
306
112
207 214
142
347
312
408
149
1089
671
583
515
751
384
937
402
1017 553
144
612
518
348
Expected size (bp)
Mol Breeding (2012) 29:245–260 255
123
538
185
252
Mol Breeding (2012) 29:245–260 Expected size (bp)
256
more than 70% of the distorted markers, being more than one-third (35.7%) congregated in only two linkage groups (LG3 and LG11).
Reverse primer sequence (50 –30 )
GGATCACTAGCATACCATTC
CGCAGTTCTTAGCTGGTC
AGTCCACTAACCAACTCGCT
Forward primer sequence (50 –30 )
CACACACACACACTGTAG
TCCTGAGCTCTAACCATG
CTGATACGGCCTGCATAC
Genome coverage Based on the mapping data and using method 3 of Chakravarti et al. (1991), the genome lengths of var. bracteatus and var. comosus were estimated to be 2,126 and 2,814 cM, respectively. Accepting the arithmetical average of the genome length of the two botanical varieties, 2,470 cM, as an approximate estimation of the genome length of the species (A. comosus), the 33 linkage groups that integrate markers from both parental genotypes were calculated to cover 72.5% of the pineapple genome. However, if the other two sets of linkage groups that gather markers inherited uniquely from one botanical variety (Fig. 1) are also taken into consideration, then the total map (40 linkage groups) covers *80% (79.93%) of the pineapple genome. Based on the genetic length of the pineapple genome estimated above (2,470 cM) and average size (485 Mbp) of the haploid genomes of var. bracteatus (444 Mbp) and var. comosus (526 Mbp) calculated by Arumuganathan and Earle (1991), an approximate ratio between physical and genetic distances of 196 Kbp per cM can be calculated for the pineapple genome.
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SSR_OPP03_722
SSR_ISSR09_443_Ab
SSR_OPH08_650 SSR_RAPD
Marker type
Table 2 continued
Marker
Discussion A reference genetic map of pineapple is needed that could function as a basic framework in programs of physical mapping and genome sequencing, and as a helpful tool for fast map location of any locus of interest and for marker-assisted selection in breeding programs. Recently, we have identified (Carlier et al. 2007) two tasks that emerged as the most imperative in order for such a map to be constructed: (1) enrichment with microsatellite markers of the maps constructed to date; and (2) conversion of multiple random amplified markers selected along the map into sequence-specific markers. The construction of the present genetic map constitutes a step towards the accomplishment of these two tasks.
Mol Breeding (2012) 29:245–260
In order to accomplish the first task—to map a large number of SSR markers—an in silico search for microsatellite sequences was carried out which resulted in the identification of 53 SSR and 592 ESTSSR sequences among the pineapple core genomic and EST sequences uploaded to the public genome databases, respectively. Although primers were designed for 51 SSR and 45 EST-SSR loci, only 16 (31.4%) SSR and 13 (28.9%) EST-SSR (one converted into CAPS) markers were successfully mapped. In order to accomplish the second priority task, a set of 63 random amplified markers (50 RAPD, 12 ISSR and one AFLP) mapped into an initial map draft were selected and cloned, giving rise to 71 sequences for which primers were designed for their conversion into sequence-specific markers. Thirty-seven out of these new sequence-specific markers were successfully analysed among the F2 progeny and only one among them remained unlinked. The mapping efficiency of the new sequencespecific markers (50.7%) was clearly higher than that of the EST-SSRs (28.9%) and SSRs (31.4%). Although expected, since most of the sequenced markers were selected based on their location in a previously constructed draft map, these results must be emphasized, since they highlight the importance of frequently discredited markers such as RAPD and ISSR. Actually, these and other similar markers, despite the frequent criticisms regarding their reproducibility, can be very useful if correctly employed, particularly for the generation of sequence-specific markers (SCARs, SSRs, CAPS, etc.) either for map construction, map-based cloning, or marker-assisted selection (MAS). Another positive aspect of sequencing and remapping randomly amplified markers is that the sequences corresponding to 31 monomorphic sequence-specific markers were putatively assigned to the original RAPD and ISSR markers. Our data showed that 75.7% (28 out of 37) of the polymorphic sequences were converted into specific markers that segregate as the original markers. Considering these results, as long as polymorphisms are identified within the assigned sequences by other strategies (e.g. by direct sequencing) or using other mapping populations, the confirmation of the putatively assigned sequences is expected to occur in about the same percentage (75%) of cases.
257
The SSR and EST-SSR markers tested in the current study tend to be co-dominantly inherited and are therefore more informative than the sequence-specific markers originated from previously mapped randomly amplified markers. Thus, all polymorphic SSR markers retrieved from genomic sequences and eight of 12 EST-SSR markers segregated co-dominantly. Conversely, although more efficiently (re)mapped, the sequence-specific markers were mostly dominantly inherited, i.e. 22 dominant vs. 15 co-dominant (the latter also included three new SSR and six CAPS markers). Sequence-specific markers are usually generated in small numbers, in studies aimed at the construction of genetic maps from very restricted and specific genomic regions, often encompassing genes of interest. Nevertheless, in a few studies in which a larger number of sequence-specific markers were used for whole genome map construction, these markers exhibited relatively high variability of polymorphisms and mapping efficiency. In the present study, the mapping efficiency of the new sequence-specific markers was high (50.7%), similar to Piquemal et al. (2005) who found 41.6% of 134 SCAR primer pairs tested useful for mapping of Brassica napus, while only 26.3% of 911 SSR markers were useful. A relatively lower mapping efficiency for sequencespecific markers was found by Lee et al. (2009), who were able to include only 32.4% of 71 markers derived from bacterial artificial chromosome (BAC) ends in an integrated map of pepper, and by Celton et al. (2009) who identified as polymorphic 21.9% of the 82 SCARs used in the construction of a genetic map of apple rootstocks. The mapping efficiency (30.2% on average) of EST-SSR and SSR markers tested in the present work was lower than expected, but within the range reported for other species. It is generally considered that SSR markers are highly polymorphic. However, they exhibit wide variation in polymorphisms and mapping efficiency which, in many cases, may be low. For example, the proportion of polymorphic markers among 500 SSRs in three different mapping populations of Cucurbita sp. was, respectively, 23.2, 37.4 and 39.6% (Gong et al. 2008), and of 397 Prunus SSRs tested on peach only 20% were polymorphic (Dirlewanger et al. 2006). However, this last percentage varied according to the origin of the markers: from 27.5% among SSRs derived from
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peach genomic DNA to 10.9% for those derived from peach cDNA (EST-SSR). A very low proportion of polymorphic markers, 12.6% of 1,145 SSRs, were also observed during the construction of a peanut genetic map by Varshney et al. (2009). Paradoxically, in cucumber, two different sets of SSR markers exhibited very different levels of polymorphism, 20.0% versus 68.1%, between the same parental lines of a RIL mapping population (Ren et al. 2009). The percentage of amplified markers that exhibited segregation distortion (15%) was in the range observed during the construction of the first pineapple maps using the F1 population (Carlier et al. 2004) and in the range found for other plant species (Jenczewski et al. 1997). The putative reasons for marker segregation distortion have been discussed elsewhere (Carlier et al. 2004; Farinho´ et al. 2004). Compared to the previously constructed pineapple maps (Carlier et al. 2004), the present map constitutes a clear improvement both in terms of number and quality of markers—a large number of sequenced and co-dominant markers are included in the present map—and of genome coverage. While the pineapple maps previously constructed on the basis of the F1 population (Carlier et al. 2004) covered barely 31.6 and 57.2% of the var. comosus and var. bracteatus genomes, respectively, the present map covers about 80% of the average genome of these two botanical varieties. An indication of the high completeness of the present map is the relatively low percentage (7.0%) of markers that remained unlinked. This result contrasts soundly with the 30 and 43% unlinked markers, respectively, found during the construction of the F1-based maps of var. bracteatus and var. comosus (Carlier et al. 2004). The high number of unlinked markers upon construction of the F1 maps was most likely a consequence of the small size of the mapping population and of the limited genome coverage of the maps. A detailed analysis of the map will show that the dominantly segregating markers do not seem to be randomly inherited from both progenitors, as 269 markers arise from var. bracteatus and 185 markers from var. comosus, a statistically very significant difference (v2 = 15.54; P \ 0.0001). Taken together with some particular points in the map, such as linkage group 7 (LG7: 82.2 cM) which is almost exclusively constituted by markers arising from var. bracteatus,
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the above data indicate the existence of large genomic regions of var. bracteatus with no homologous counterpart in var. comosus. Although speculative, this working hypothesis explains the larger size of the var. bracteatus genome determined by Arumuganathan and Earle (1991) and is in line with the identified closer genetic relatedness of var. bracteatus to the tetraploid A. macrodontes (formerly P. sagenarius). In particular, the most common form of var. bracteatus, including Branco do mato, shares rare nuclear RFLP markers with A. macrodontes (Duval et al. 2001), while exhibiting a chlorotype typical of A. comosus (Duval et al. 2003). Although, these data seem to point to a hybrid origin of var. bracteatus, most probably the final answer to this hypothesis will be found only in future physical mapping and/or genome sequencing programs of pineapple. Based on this mapping work we estimated the ratio between physical and genetic distances in pineapple to be 196 Kbp per cM. Although an approximate estimate, this ratio indicates that 1 centimorgan (cM) will corresponds roughly to the physical distance spanned by two overlapping BAC clones. This result is very encouraging regarding multiple practical pineapple genomic issues. The present map composed of statistically wellsupported linkage groups spanning about 80% of the computed genome length, with 65 sequence-specific markers and 31 other sequences distributed along the genome, constitutes a useful research tool available for use in projects of MAS, map-based cloning, construction of physical maps or genome sequencing, in pineapple and other Bromeliaceae species. Acknowledgments This research was supported by the project PTDC/AGR-GPL/77398/2006: ‘‘Construction of an integrated genetic map of Pineapple’’, funded by the Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT), Portugal. Jorge Dias Carlier is the recipient of the post-doctoral grant SFRH/BPD/ 41714/2007 awarded by the FCT. Tatiana Espı´rito Santo and Nelson Horta Sousa were recipients of Research fellowships awarded by the FCT within the framework of the project PTDC/AGR-GPL/77398/2006.
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