Genetic relatedness among Aechmea species and hybrids inferred from AFLP markers and pedigree data

Scientia Horticulturae 139 (2012) 39–45

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Genetic relatedness among Aechmea species and hybrids inferred from AFLP markers and pedigree data Fei Zhang ∗ , Weiyong Wang, Yaying Ge, Xiaolan Shen, Danqing Tian, Jianxin Liu, Xiaojing Liu, Xinyin Yu, Zhi Zhang Flower Research and Development Centre, Zhejiang Academy of Agricultural Sciences, Hangzhou 311202, China

a r t i c l e

i n f o

Article history: Received 6 December 2011 Received in revised form 29 February 2012 Accepted 1 March 2012 Keywords: Aechmea Bromeliads Genetic relatedness AFLP Pedigree

a b s t r a c t Bromeliads are among the most economically important potted ornamentals, however little is available about genetic variability among bromeliad cultivars or hybrids for breeding purposes. In this study, genetic relatedness among cultivars and selected hybrids of Aechmea bromeliad was investigated using amplified fragment length polymorphism (AFLP) markers and pedigree data. Eight AFLP primer combinations produced 1498 DNA fragments, of which 1465 (97.8%) were polymorphic. The AFLP-based genetic similarity ranged from 0.20 to 0.61 and averaged 0.33, while the corresponding pedigree-based coefficient of coancestry varied from 0 to 0.5, with an average of 0.07. This reveals large variability and genetic diversity among the investigated Aechmea bromeliads. The AFLP-based cluster analysis grouped the Aechmea bromeliads well according to their breeding years and ancestors, whereas pedigree-based cluster analysis divided the Aechmea bromeliads corresponding only to their ancestors. Additionally, the AFLPbased genetic similarity followed a normal distribution, while pedigree-based coefficient of coancestry displayed a skewed distribution. A significant though moderate correlation (r = 0.35, P < 0.001) and a significant linear relationship with low coefficient of determination (R2 = 0.12) were observed between the two measures. This indicates that AFLP is more effective in quantifying genetic relatedness among Aechmea bromeliads. The present study provides useful implications for the efficient conservation and utilization of bromeliad germplasm. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Ornamental bromeliads, originated in tropical and subtropical zone of Central and South America, are among the most commercially important ornamentals and occupy a valuable part in flower industry worldwide. Ornamental bromeliad was first introduced in China in 1980s and now becomes one of the most important potted flowers in China. Presently, some modern breeding methods (i.e. transgenic technique) emerged in many famous ornamentals, such as rose (Ma et al., 2008; Yasmin and Debener, 2010), lily (Shahin et al., 2011; Yamagishi et al., 2010) and chrysanthemum (Lv et al., 2011). However, the conventional hybridization and selection is still the most efficient method of breeding new varieties for bromeliad. As the Florida Committee of Bromeliads Society (http://www.fcbc.org) recorded, more than 90% of the ornamental bromeliad varieties are interspecific hybrids, underscoring to some extent the importance of novel bromeliad hybrids for market demands. Through intensive breeding programs, many novel materials derived from

∗ Corresponding author. Tel.: +86 571 82704035; fax: +86 571 82721053. E-mail address: [email protected] (F. Zhang). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2012.03.002

inter-specific and generic hybridizations make new bromeliad varieties possible. Nevertheless, the germplasm introgression through outcrossing leads to the complex genetic backgrounds of bromeliads and consequently brings great difficulty in assessing their genetic relationships. Until now, several methods have been used to determine genetic variability of crops. The inexpensive approach based on morphological and biochemical traits has been often applied in inferring genetic relatedness, however this method has limitations and many times does not reflect the real diversity of germplasm, because environmental factors and the developmental stage of plant usually influence their expression (Lima et al., 2002). The pedigree-based coefficient of coancestry indirectly measures the degree of genetic relatedness by estimating the probability that a random allele from one genotype is identical by descent to a random allele of another genotype at the same locus (Weir et al., 2006). However, the accuracy of coefficient of coancestry depends on the availability of reliable and detailed pedigree records, and assumptions made when calculating coefficient of coancestry regarding the relatedness of ancestors, selection pressure, and genetic drift are generally not met (Graner et al., 1994). Nevertheless, pedigree information offers crop breeders a simple and inexpensive way to estimate genetic relatedness among breeding materials (Chen et al., 2009;

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F. Zhang et al. / Scientia Horticulturae 139 (2012) 39–45

Priolli et al., 2010). In contrast to the former two methods, molecular markers based on DNA polymorphism are more informative, independent of environmental conditions and unlimited in number (Agarwal et al., 2008). Compared to other DNA-based markers (i.e. RAPD, ISSR, SSR and SRAP), however, AFLP has notable advantages, namely reproducibility, high levels of polymorphism that can be detected in a single reaction, genome-wide distribution of markers, and no need of prior DNA sequence information (Vos et al., 1995). Thus far, AFLP has been successfully used in assessing genetic relationships of many species, including some ornamental crops such as Phalaenopsis (Chang et al., 2009; Gawenda et al., 2011), Gladiolus (Ranjan et al., 2010) and Hippohano (Raina et al., 2011). This gives a good reference for the present study on genetic relatedness among ornamental bromeliads of Aechmea. Aechmea constitutes an important part in bromeliad industry due to their diversified flower color and type, and stronger cold resistance than other ornamental bromeliads of Neoregelia, Guzmania and Vriesea. Therefore, the excellent gene pools in Aechmea are quite useful for breeding novel varieties of ornamental bromeliads. Despite the ornamental and economic importance of Aechmea, however, the genetic potential of Aechmea has not been fully exploited except some assessments of genetic diversity within several wild species of Aechmea (Izquierdo and Pinero, 2001; Sousa et al., 2009). Hence, the purpose of the present study was to (1) estimate the level of genetic variability among 27 Aechmea bromeliad cultivars and hybrids using AFLP and pedigree data, and (2) determine the correlation between estimates of AFLP-based genetic similarity and pedigree-based coefficient of coancestry in Aechmea bromeliads. This is the first report on genetic variability among Aechmea bromeliad cultivars and hybrids. 2. Materials and methods 2.1. Plant materials and DNA isolation A total of 27 accessions of Aechmea cultivars and hybrids were involved in this study (Table 1). The A-coded accessions were cultivars obtained from various breeders and the P-coded accessions were selected hybrids derived from the crosses between A-coded cultivars. All the accessions were maintained at the Ornamental Bromeliad Germplasm Nursery, Zhejiang Academy of Agricultural Sciences. Young leaves were collected from each accession, frozen with liquid nitrogen and grinded into powder. Genomic DNA was isolated following a CTAB-based procedure (Murray and Thompson, 1980). DNA concentration was estimated in comparison with known concentrations of Lamda DNA in 0.8% agarose gel. 2.2. AFLP analysis AFLP analyses were performed as described by Vos et al. (1995) with some modifications. The pre-amplification products were diluted 20-fold and were used as a template for the selective amplification. Selective amplification was conducted by using fluorescently labeled EcoRI or Pstl primers. A total of 48 selective primers of EcoRI + 3/Pstl + 3 were initially applied to screen polymorphisms using the DNA of four randomly chosen accessions, consequently, eight polymorphic primer combinations were chosen to genotype the whole accessions. Amplification was conducted on a Gene Amp PCR System 9600 (Perkin Elmer, USA). PCR products were mixed with loading buffer and loaded on 4% polyacrylamide gels after heat denaturation. The products were fractionated on PRISM 377 sequencer (ABI) using electrophoresis. The internal 70–500 size standard ROX 500 (ABI) was run with the samples to estimate the size of fragments. All AFLP fragments

were scored dominantly and recorded in a 0/1-matrix for the peak absence/presence along with their sizes. The binary scores were manually compared with the electropherograms to re-confirm the presence or absence of peaks. 2.3. AFLP-based cluster and principal coordinated analysis Based on the 0/1-matrix, a cluster analysis was conducted using unweighted pair group method with arithmetic mean (UPGMA) based on the Dice index (Nei and Li, 1979). This analysis was performed using the FreeTree software package (Hampl et al., 2001). Bootstrap values based on 1000 re-samplings were used to estimate the reliability of the clusters. The principal coordinated analysis (PCoA) was conducted with the same program using the DCENTER and ELGEN procedures of NTSYS-pc 2.2 (Rohlf, 2005). This multivariate approach was chosen to complement the cluster analysis information, because cluster analysis is more sensitive to closely related individuals, whereas PCoA is more informative regarding distances among major groups. 2.4. Pedigree analysis The pedigree information for all Aechmea bromeliad cultivars was obtained from cultivar descriptions, breeding records, personal communication with breeders, and the Florida Council of Bromeliads Society (http://fcbc.org). Coefficient of coancestry was computed for all pair-wise combination of Aechmea bromeliads and corresponded to the probability that a randomly selected allele at a particular locus of one genotype is identical by descent to a randomly selected allele at the same locus of another genotype. The pedigree data were used to calculate coefficient of coancestry by the method as Weir et al. (2006). In general, the assumptions suggested by Cox et al. (1985) were followed, and coefficient of coancestry was considered 0 among the remote ancestors without known pedigree. Pair-wise coefficient of coancestry values were used to generate the coefficient of coancestry matrix. The TRANSF subroutine of NTSYS-pc 2.2 was used to transform this matrix into a distance (1-coefficient of coancestry) matrix. Clusters of Aechmea bromeliads were accomplished using the SAHN subroutine of NTSYS-pc 2.2 with UPGMA method. 2.5. Matrix comparison Plots of frequency distribution for the AFLP-based genetic similarity matrix and the pedigree-based coefficient of coancestry matrix were structured using SPSS software. The two matrices were compared by the MAXCOMP subroutine of NTSYS-pc. The normalized Mantel statistic Z test (Mantel, 1967) was used to determine the level of association and the REGRESSION subroutine of NTSYSpc 2.2 was used to plot the linear relationship between the two matrices. 3. Results 3.1. AFLP analysis A total of 48 AFLP primers were screened with four random Aechmea broleliads, and eight primer combinations producing reproducible and informative patterns were selected to genotype the 27 accessions of Aechmea bromeliads. The number of polymorphic fragments for each primer combination varied from 168 to 192 with an average of 187.3 per primer combination (Table 2). The eight AFLP primer combinations generated a total of 1498 fragments ranging in size from 70 bp to 500 bp, of which 1465 (97.8%) were polymorphic, suggesting high diversity in the Aechmea bromeliads.

F. Zhang et al. / Scientia Horticulturae 139 (2012) 39–45

41

Table 1 List of pedigree data for the 27 accessions of Aechmea bromeliads. Code

Name

Pedigree data

Cultivar/selected hybrid

A1 A3 A27 A30 A37 A40 A41 A43 A50 A64 A66 A71 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15

Primera Frost Melanocrater Yellow Foster’s favorite 2216 2137 3342 Sockeye 4215 Red ribbons Suenos A40 × A27 A40 × A27 A40 × A27 A37 × A64 A66 × A50 A66 × A50 A66 × A50 A41 × A40 A41 × A40 A41 × A40 A43 × A30 A43 × A30 A43 × A30 A64 × A71 A64 × A71

A. fasciata A. fasciata A. caudata, A. organensis A. caudata A. racinae, A. victoriana var. discolor A. distichantha var. glaziovii A. distichantha var. schlumbergeri A. distichantha, A. organensis A. gamosepala A. recurvata A. racinae, A. victoriana var. discolor A. recurvata var. benrathii, A. gamosepala A. distichantha var. glaziovii, A. caudata, A. organensis A. distichantha var. glaziovii, A. caudata, A. organensis A. distichantha var. glaziovii, A. caudata, A. organensis A. racinae, A. victoriana var. discolor, A. recurvata A. racinae, A. victoriana var. discolor, A. gamosepala A. racinae, A. victoriana var. discolor, A. gamosepala A. racinae, A. victoriana var. discolor, A. gamosepala A. distichantha var. schlumbergeri, A. distichantha var. glaziovii A. distichantha var. schlumbergeri, A. distichantha var. glaziovii A. distichantha var. schlumbergeri, A. distichantha var. glaziovii A. distichantha, A. organensis, A. caudata A. distichantha, A. organensis, A. caudata A. distichantha, A. organensis, A. caudata A. recurvata, A. recurvata var. benrathii, A. gamosepala A. recurvata, A. recurvata var. benrathii, A. gamosepala

Cultivar Cultivar Cultivar Cultivar Cultivar Cultivar Cultivar Cultivar Cultivar Cultivar Cultivar Cultivar Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid Selected hybrid

P8 P2 100

39

P1

28

P3

43

i

70

P9

57

P10 P13

100

62

P11

74

P12 P4

93

ii

98

I

P7 100 64

P5 P6 A71

100

iii

P15 A43

100

a i 58

II

P14

82

ii

61

iii 45 50

iv 0.1

90 100

96

A41 A50

98

b

A40

A64 A37

94

A66

100

A1 A3 A27 A30

Fig. 1. Dendrogram of 27 accessions of Aechmea bromeliads generated by UPGMA cluster analysis using AFLP-based genetic similarity estimates (Dice index) using FreeTree program. Numbers on the branches are bootstrap values obtained from 1000 re-samplings.

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F. Zhang et al. / Scientia Horticulturae 139 (2012) 39–45

Table 2 The number of scored AFLP loci examined using eight primer combinations as well as percentage of polymorphic loci per primer combination among 27 accessions of Aechmea species and hybrids. Primer combination Number of total loci E-AAC/M-CAG E-AAC/M-CTA E-AAC/M-CTC E-AAG/M-CAA E-AAG/M-CTT E-AGG/M-CAG E-AGG/M-CTA E-AGG/M-CTC Total Average

Number of polymorphic loci

% of polymorphic loci

196 184 175 191 187 192 181 192

189 181 168 187 185 192 176 187

96.4 98.4 96.0 97.9 98.9 100.0 97.2 97.4

1498 187.3

1465 183.1

97.8 –

The AFLP-based genetic similarity among pair-wise Aechmea bromeliads ranged from 0.20 to 0.61, with a mean of 0.33. A dendrogram based on AFLP-based genetic distance was shown in Fig. 1. In this dendrogram, the Aechmea broleliads were clustered into two separate major groups (I and II). Group I contained all the Pcoded hybrids and could be divided into three subgroups (i, ii, and iii). Subgroup i included nine accessions derived from three different crosses with an ancestor (Aechmea distichantha or its variant). Subgroup ii included four accessions derived from two different crosses with an ancestor, A37. The three accessions together with their ancestor (A71) made up subgroup iii. The A-coded accessions excluding A71 constituted group II. Similarly, group II could be divided into four subgroups (i, ii, iii, and iv), and each subgroup also clustered accessions that shared same ancestors. Bootstrap values ranged from 28% to 100%; however, most of them (85%) were larger than 50% (Fig. 1). Associations among the Aechmea bromeliads were also examined with PCoA (Fig. 2). In general, the results of PCoA corresponded well to that from the UPGMA cluster analysis. The principal coordinates (PC1 and PC2) encompassed 18.0% and 17.1% of the total variations, respectively. The A-coded cultivars apparently clustered in the center, and most of the P-coded hybrids were generally around the A-coded cultivars.

0.2

P6

P11

P9

0.1 P7

PC 2 (17.1%)

P15 A66

-0.0 A27 P10

P2 A43 P3

A64 P8

A50 P13

P1 A40 A71 A3 A1 P14 A37 P4 P12

A30

A41

-0.1

-0.3 -0.2

P5

-0.1

0.0

PC 1 1 (18.0%)

0.1

0.3

Fig. 2. Two-dimensional principal co-ordinate analysis based on the AFLP-based genetic similarity estimates (Dice index) for the 27 accessions of Aechmea bromeliads. PC1 and PC2 represent the first and second principal co-ordinate, respectively.

3.2. Pedigree analysis For pedigree analysis, the ancestry of Aechmea accessions was traced through three generations. The pedigree-based coefficient of coancestry ranged from 0 to 0.5, with a mean of 0.07. Based on coefficient of coancestry, the UPGMA dendrogram clustered the Aechmea bromeliads into five major groups (Fig. 3). Group I contains two cultivars (A1 and A3) of Aechmea fasciata. Group II included eight accessions sharing an ancestor (Aechmea caudate). Group III consisted of six accessions that shared an ancestor, A. distichantha. Group IV contained six accessions that had an ancestor, Aechmea racinae. The selected hybrids P14 and P15, along with their cross parent (A64 and A71) and grandparent (A50), made up group V. Therefore, pedigree-based coefficient of coancestry also identified the Aechmea bromeliads according to different crosses or ancestors. 3.3. Comparison between genetic similarity and coefficient of coancestry The frequency distribution of AFLP-based genetic similarity and pedigree-based coefficient of coancestry was shown in Fig. 4. Generally the AFLP-based genetic similarity values followed a normal distribution, whereas the coefficient of coancestry distributed towards low values and followed a skewed distribution. A highly significant though moderate correlation (r = 0.35, P < 0.001) was detected between AFLP-based genetic similarity and pedigreebased coefficient of coancestry. The AFLP-based genetic similarity estimates for the possible pairs of related Aechmea bromeliads were plotted against their corresponding coefficient of coancestry values (Fig. 5). There was a significant linear relationship between the two measures; nevertheless, the coefficient of determination was rather low (R2 = 0.12). 4. Discussion A successful breeding program depends on the complete knowledge and understanding of the genetic diversity of the available germplasm. Considerable interest has focused on the taxonomy and molecular ecology of wild species of Bromeliaceae, including Tillandsia (González-Astorga et al., 2004), Vriesea (Palma-Silva et al., 2009), Alcantarea (Barbabá et al., 2009), Puya (Sgorbati et al., 2004), Pitcairnia (Boisselier-Dubayle et al., 2010), Encholirium (Cavallari et al., 2006), Quesnelia (Almeida et al., 2009), Lymania (Sousa et al., 2007), and Aechmea (Faria et al., 2004; Izquierdo and Pinero, 2001; Sousa et al., 2009). However, this gives few references to breeding program of ornamental bromeliads and little information on the genetic variability among ornamental bromeliad cultivars or hybrids was available. To fill this gap, genetic relatedness among Aechmea cultivars and hybrids were first investigated by AFLP markers and pedigree data in present study. In this study, eight AFLP primer combinations produced a total of 1465 (97.8%) polymorphic loci in Aechmea bromeliad cultivars and hybrids. Recently, Xiang et al. (2003) observed 83% polymorphic AFLP markers in Dendrobium orchid hybrids, and Chang et al. (2009) reported that 95% of AFLP markers showed polymorphism in Phalaenopsis orchids. The informative AFLP markers herein were higher than that identified in orchids. This is likely due to the intensive germplasm introgressions through inter-specific crossing among Aechmea species. The AFLP-based genetic similarity ranging from 0.20 to 0.61, together with the large number of identified polymorphic markers, demonstrated the abundant diversity in Aechmea bromeliads. Thus there exists a huge potential for breeding novel interspecific bromeliad hybrids in Aechmea species. Gawenda et al. (2011) reported that some relatively new Phalaenoposis hybrids clustered together. In the present study, the

F. Zhang et al. / Scientia Horticulturae 139 (2012) 39–45

43 A1 A3

I

A27 A30 P1 P2

II

P3 P11 P12 P13 A40 A41 P8 P9 P10

III

A43 A37 A66 P4 P5

IV

P6 P7 A50 A71 A64

V

P14 P15

1.00

0.88

0.75

0.63

Coefficient

0.50

Fig. 3. Dendrogram of 27 accessions of Aechmea bromeliads generated by UPGMA cluster analysis based on the matrix of coefficient of coancestry.

A-coded Aechmea bromeliad cultivars were bred around 2000, and the P-coded Aechmea hybrids were recently selected from the crosses between A-coded bromeliad cultivars. The AFLP-based dendrogram clustered the P- and A-coded Aechmea bromeliads into two separate major groups, suggesting that the two separate clusters corresponded well to their breeding years. Furthermore, the subdivisions within each major group were highly correlated with whether the accessions were originated from the same ancestors. This suggests that AFLP-based genetic similarity can accurately identify Aechmea cultivars and recently selected hybrids, reflecting the real genetic relationships among the Aechmea bromeliads. The pedigree-based coefficient of coancestry, however, divided the Aechmea bromeliads into different clusters generally corresponding to their ancestors and often mixed cultivars and hybrids. Even though, the AFLP- and pedigree-based clusters were somewhat similar. This is attributed to the explicit pedigree data for the Aechmea bromeliads investigated herein. PCoA showed that some

A

70

Aechmea hybrids, i.e. P5, P6, P9 and P10, were far away from the Acoded cultivars. This suggests that interspecific crosses broaden the genetic basis of Aechmea bromeliads, indicative of the possibility to breed excellent interspecific hybrids with Aechmea species. Many studies have demonstrated significant correlations (P < 0.001) between marker-based genetic similarity and pedigreebased coefficient of coancestry. Lima et al. (2002) described a moderate correlation (r = 0.42, P < 0.001) between AFLP-based genetic similarity and coefficient of coancestry in sugarcane. A moderate correlation ∼0.3 were observed between marker-based genetic similarity and pedigree-based coefficient of coancestry in many crops such as winter triticale (Tams et al., 2004, 2005) and soybean (Priolli et al., 2010). Lower correlations (r < 0.2) were detected in soybean (Bonato et al., 2006) and barley (Soleimani et al., 2007), while higher correlation (r > 0.7) were also found in Phalaenopsis orchids (Chang et al., 2009) between the two measurements. Here, the correlation between AFLP-based genetic

B

300

60 50 200 40 30 100 20 10 0

0 .20

.25

.30

.35

.40

.45

.50

AFLP-based genetic similarity

.55

.60

0.00

.13

.25

.38

.50

Coefficient of coancestry

Fig. 4. Frequency distributions of (A) AFLP based genetic similarity estimates and (B) coefficients of coancestry for possible pairs of 27 accessions of Aechmea broleliads.

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F. Zhang et al. / Scientia Horticulturae 139 (2012) 39–45

Zhejiang Province (2009C12095), the No-profit Technology Research Program of Zhejiang Province (2000C22014), the Key Scientific and Technological Innovation Team Project of Zhejiang Province (2011R50034), Hangzhou Seeds and Seedlings of Special Project of Zhejiang Province (20110332H15), the Science and Technology Innovation Ability Promoting Project of Zhejiang Academy of Agricultural Sciences (2011R25Y01D01).

.7

.6

.5

.4

References

.3

y = 0.25x + 0.31 r = 0.35

.2

.1 -.1

0.0

.1

.2

.3

.4

.5

.6

Coefficient of coancestry Fig. 5. Plot of AFLP-based genetic similarity estimates (Dice index) and coefficient of co-ancestry for possible pairs of Aechmea bromeliads. Pearson’s correlation r is 0.35.

similarity and pedigree-based coefficient of coancestry was moderate (r = 0.35, P < 0.001) for Aechmea bromeliads. As described by the former authors, the significant differences in the correlation should be accounted for by selection pressure or genetic drift, as each gene pool is derived from a few major ancestors with different gene frequencies. In the present study, the AFLP-based genetic similarity values followed a normal distribution, whereas the pedigree-based coefficient of coancestry displayed a skewed distribution. Additionally, a significant linear relationship was observed between the two measurements, but the coefficient of determination was rather low (R2 = 0.12). This indicates that the pedigree-based coefficient of coancestry only explains a small part of the variations observed for AFLP-based genetic similarity. Therefore, AFLP is more effective in quantifying the genetic diversity among Aechmea bromeliads. As has been further confirmed herein, nevertheless, the complete pedigree data will continue to be a useful and inexpensive way in assessing genetic relationship among crops (Sun et al., 2003; Weir et al., 2006; Chang et al., 2009). As we know, there are huge varieties for bromeliads and most of them are hybrids with complex genetic backgrounds of two or more species. With extensive exchange of germplasm among breeders, information such as origin, name and pedigree data of bromeliad cultivars are easily confused or lost, as is the case in China. The present study verified the reliability of both DNA genotyping and the known pedigrees in assessing genetic relatedness among Aechmea bromeliad cultivars and hybrids, but more effective the former. This gives a hint that DNA fingerprinting could be credibly relied on in estimating genetic relationships among bromeliads without known pedigrees. Results from the present study help to select appropriate parents for future cross breeding for Aechmea bromeliads and provide some useful information for the efficient conservation and utilization of genetic resources of other bromeliads. Acknowledgments This research was financially supported by the National Natural Science Foundation of China (Grant No. 31101570), Zhejiang Provincial Natural Science Foundation of China (Grant No. Y3110271), the Key Science Technology Specific Projects of

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