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Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers
Scientia Horticulturae 123 (2010) 562–567
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Short communication
Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers Pragya Ranjan a,*, K.V. Bhat b, R.L. Misra a, S.K. Singh a, J.K. Ranjan a a b
Discipline of Horticulture, Indian Agricultural Research Institute, New Delhi, 110012, India National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources, New Delhi, 110012, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2009 Received in revised form 31 October 2009 Accepted 19 November 2009
Gladiolus is one of the important commercial flowers with a large number of cultivars. However, genetic relationships among its genotypes have not been reported. This study analyzed genetic relatedness of 54 gladiolus cultivars using amplified fragment length polymorphism (AFLP) markers. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for AFLP reactions. Fluorescence-labeled amplification products were subjected to electrophoresis and then analyzed using an automated sequencer. A dendrogram was constructed by the unweighted pair group method using the arithmetic average (UPGMA). The number of AFLP fragments generated per primer set ranged from 10 to 151 with fragment sizes varying from 50 to 450 bp. A total of 660 AFLP fragments were detected, of which 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. All cultivars were clearly differentiated by their AFLP profiles. The AFLP data were compared with previously obtained RAPD data and combined to generate a common dendrogram. The first cluster was dominated with indigenously bred cultivars while the second was dominated with exotic cultivars. This shows that most of the exotic cultivars as well as indigenous cultivars are closely related with each other. However, two indigenous cultivars viz., Pusa Suhagin and Pusa Archana share genetic similarity with exotic cultivars. Among the genotypes selected for the investigation, Pusa Gunjan was identified as the most distinct genotype. The AFLP markers developed will help future Gladiolus cultivar identification, germplasm conservation and new cultivar development. The assessed genetic relationships among gladiolus cultivars may enhance the efficiency of breeding program by selecting desirable parents with reduced breeding cycle. ß 2009 Elsevier B.V. All rights reserved.
Keywords: AFLP markers Bulbous ornamental Genetic relationship Gladiolus Iridaceae
1. Introduction Gladiolus spp. is an important bulbous ornamental prized for the beauty of its spikes as well as longer vase-life. The genus Gladiolus is a member of family Iridaceae and sub-family Ixioideae (Goldblatt, 1991). The name gladiolus is derived from the Latin word ‘gladius’, meaning sword for the shape of its leaves. An ancient name for gladiolus was xiphium, from the Greek word ‘xiphos’, also meaning sword. Commonly gladiolus is known as ‘Sword lily’. Tournefort christened Gladiolus as a genus, which was accepted by Linnaeus in botanical literature (Lewis et al., 1972). Due to magnificent inflorescence with wide array of colours, gladioli are very much
Abbreviations: CTAB, cetyltrimethylammonium bromide; ISSR, inter simple sequence repeat; PCR, polymerase chain reaction. * Corresponding author at: Central Institute of Temperate Horticulture-Regional Station, Mukteshwar, Distt: Nainital, Uttarakhand, 263138, India. Tel.: +91 5942 286027; fax: +91 5942 286027. E-mail address: [email protected] (P. Ranjan). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.11.013
popular among growers as cut-flower. The flower spikes are used in vase or value-added in bouquet. Gladioli are also quite suitable as herbaceous border, bedding and potted plant. South Africa, particularly the Cape of Good Hope, is considered to be the centre of diversity for this genus. The current number of species in the genus is 255 (Goldblatt and Manning, 1998) and all the species are herbaceous perennial in nature. The modern cultivars of G. grandiflora are believed to be originated from a number of wild species viz., G. cruentus, G. natalensis, G. oppositiflorus, G. papilio and G. saundersii (Barnard, 1972; Imanishi, 1989). The modern cultivars are derived from interspecific crosses among several species. Hence, wide variation is exhibited by gladiolus cultivars for their growth habit, size, shape and colour of spikes, and florets. The assessment of natural genetic variation is important not only for ethical and aesthetic reasons but also to ensure that genetic resources may be used even more efficiently and sustainably in agriculture and other industries. Thus, there is a need to assess the variation that already exist and how it can be conserved and utilized effectively.
P. Ranjan et al. / Scientia Horticulturae 123 (2010) 562–567
Although gladiolus is being grown as a popular cut-flower worldwide, neither the US National Plant Germplasm System (NPGS) nor the Biodiversity International formerly known as International Board for Plant Genetic Resources (IBPGR) had been actively involved in conservation of gladiolus. Even the Ornamental Plant Germplasm Centre (OPGC) established in 1999 at Ohio State University has not assigned gladiolus as priority genera for conservation and maintenance. Looking into the importance of this crop, efforts are needed for systematic characterization of this crop. Proper characterization helps in unambiguous discrimination between accessions, identification of economically important plant resources, detection of redundancies and in monitoring genetic changes appearing during maintenance. Estimates of diversity are useful to guide collection missions, monitoring genetic erosion, establish core subset and to develop on-farm conservation strategies. Molecular marker techniques also have significant utility in plant breeding programs through assisting plant variety protection as well as distinctness, uniformity and stability testing processes (Heckenberger et al., 2006) which is highly applicable to gladiolus in which the number of newly constituted varieties is continuously increasing. Moreover, proper characterization and assessment of genetic relationships among diverse accessions allow breeders to select desirable genes from different sources and to accumulate those genes in one cultivar. Thus, estimates of genetic relatedness between cultivars may enhance the efficiency of effective breeding program. Application of molecular techniques for the study of genetic relationships or cultivar identification in gladiolus has perhaps not been reported yet. Amplified fragment length polymorphism (AFLP), a more robust and reliable technique (Vos et al., 1995) has been used as an efficient technique to assess genetic diversity and relationships in a number of crops including some bulbous ornamentals such as Aglaonema (Chen et al., 2004); Alstroemeria (Han et al., 2000); Caladium venten spp. (Loh et al., 1999, 2000) and Hemerocallis spp. (Tomkins et al., 2001). Among the molecular markers deployed, AFLPs have several advantages, including high levels of reproducibility and a high information content (Mueller and Wolfenbarger, 1999; Hodkinson et al., 2002). Moreover, when fluorescence-labeled oligonucleotides are employed in combination with automated capillary electrophoresis, it gives a much higher resolution than other AFLP detection systems. Besides these, fluorescence-labeled fragments have distinct advantages as these can be scored using computerized analysis rather than visual analysis (Carr et al., 2003). The main objectives of this study were to use of fluorescence based AFLP technique to estimate genetic relationships among 54 gladiolus cultivars. Probably, no report is available in gladiolus regarding estimation of genetic relationship using any molecular marker techniques. Therefore, as a first report of its kind on gladiolus cultivars, this study strives to generate information that is crucial in guiding improvement and conservation program of gladiolus.
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Table 1 List of 54 Gladiolus cultivars and a duplicate sample of Pusa Shweta used in this study and their parentages. Sr. no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.
Cultivar
Parentage
Friendship
(Maid of Orleans Picardy) (Gloaming Seedling) Unknown Unknown Unknown Unknown Crusador Statuette Diamond Leona Unknown Eloise Rosemarie Pfitzer Creamy Green American Beauty Parfait Domino Unknown Sylvia seedling Big Time Plum Pudding Marjorie Nadean R S Mayne Unknown Unknown Unknown Unknown Apricot Glow Emile Aubrun Unknown Jester sport Salome Maolete Jr Prom Lucky Star Unknown White Oak Oscar Unknown Unknown Pastorale unknown Unknown Ruffled Ebony Ace of spades Unknown Old South Statuette Unknown Comm Koehl Moorish King Beauty’s Blush Rose Charm Picardy Early Rose Unknown Unknown Open pollinated seedling of Ratna’s Butterfly Open pollinated seedling of White Oak Sylvia Partica Ave Christian Jane Creamy Green American Beauty Cygnet Little Fawn Red Bantam Flaura Belle Seedling of (Wind Song Pink Frost) Seedling of (Wind Song Pink Frost) Spontaneous mutant of Dhanvantari Red soft glow open Mayur Heady Wine Berlew Heady Wine Viola open Snow Princess Viola White Oak Oscar
Yellow Jame Creamy Green Light Purple Morello Ripples Priscilla Apricot Queen Anglia Pusa Archana Gold Dust Punjab Dawn Pusa Suhagin Smoky Lady The Barton Christian Jane Errato Royal Supreme Ave Picardy Summer Rose Jester Gold Peter Pears Pusa Dhanvantari Snow Princess Pusa Shagun Plumtart Chantiler Viola Bonaire Regency Australian Fair Little Fawn Bis–Bis Melody Apple Blossom Rose Delight Pusa Chandani George Mazure Pusa Bindiya Pusa Sarang Neelam Pusa Mohini Pusa Lohit Pusa Chirag Pusa Rangmahal Pusa Shweta 1 Pusa Shweta 2 Pusa Swarnima Pusa Urvashi Pusa Shringarika Pusa Urmi Pusa Swapnil Pusa Gulaal Pusa Gunjan
2. Materials and methods 2.1. Plant material A total of 54 (33 exotic and 21 indigenous) commercial varieties of gladiolus (Table 1) along with one duplicated sample were analyzed in this study. Some of them are developed at the Division of Floriculture and Landscaping, IARI, New Delhi and rest are maintained as procured from different countries vis-a`-vis indigenous sources. 2.2. Fluorescent-AFLP analysis Young, fresh and healthy leaves of 55 samples were used for DNA extraction using CTAB method as per the protocol of Sanghai-
Maroof et al. (1984) with minor modifications. AFLP analysis was conducted using the Plant Mapping kit for regular plant genomes (500–6000 Mb) AFLPTM (PE Applied Biosystems, USA). Total DNA (500 ng) was digested with 1 ml of a mixture of EcoRI/Mse I (1.25 units ml 1) at 37 8C overnight and ligated to EcoRI/Mse I adapters with 1.5 ml (1 unit ml 1) of T4 DNA ligase at 25 8C for at least 6 h. The adaptor ligated DNA was amplified and subjected to PCR amplification in a thermocycler (Perkin Elmer 9600, PE Applied Biosystems, USA). EcoRI primers of AFLP kit were got labeled with fluorescent dyes of different colours (5-FAM, JOE and NED), which have an added advantage of multiplexing three primers in one reaction. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for further
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analysis. After selective amplification, electrophoresis (ABI Prism 310 Automated Sequencer) was performed at a constant voltage (14.9 kV) and current (10 mA) at a temperature of 60 8C. The data collection software collects light intensities using software selectable filters and stores them as electronic signals for processing. These signals are in the form of peaks. Each peak is equivalent to one band and peak height represents intensity of the band. The ‘‘Genescan’’ and ‘‘Genotyper’’ software package (Applied Biosystems, USA) was used for processing and scoring of the results. The similarity coefficient matrix (Jacard, 1908) was subjected to cluster analysis by unweighted pair group method of arithmetic average (UPGMA) analysis and dendrogram was generated with the help of NTSYS-pc software version 1.70 (Exeter Software, New York, USA). 3. Results and discussion 3.1. DNA profiles of gladiolus genotypes The nine primer pairs generated clear-cut AFLP profiles for 54 gladiolus cultivars and AFLP fragments ranged from 50 to 450 bp with a total of 660 scorable fragments. The duplicate cultivar ‘Pusa Shweta’ showed identical AFLP patterns with each primer set. The number of bands generated per primer ranged from 10 (E-AGG/MCTA) to 151 (E-ACT/M-CAT) with a mean of 73.33 bands per primer. Out of the 660 bands generated, 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. The levels of polymorphisms detected in the gladiolus germplasm under investigation indicate that the amount of genetic variability present in this genome is relatively high. The number of peaks scored per primer pair ranged from 10 to 151 (Table 2). Electropherogram of Gladiolus cultivars Gold
Dust, Punjab Dawn, Pusa Suhagin, Smoky Lady and The Barton are presented in Fig. 1. The average number of amplification products per primer per cultivar ranged from 4.89 (E-AGG/M-CTA) to 88.33 (E-ACT/M-CAT). 3.2. Cluster analysis and genetic relationships among cultivars Based on Jaccard’s similarity matrix and UPGMA clustering a wide range of degree of relatedness between cultivars was reported in this study; some are closely related while others were found quite distinct from each other. The greatest similarity was found between Pusa Lohit and Pusa Swarnima (Jaccard coefficient = 0.788), while Pusa Gunjan was found to be the most distinct genotype. The dendrogram based on UPGMA clustering (Fig. 2) resulted in the identification of seven major clusters. Cultivars Pusa Gunjan and Little Fawn were not confined to any of the clusters and were present as out groups. Pusa Gunjan was found to be the most distinct genotype. Cluster I was the largest and dominated with indigenous cultivars. Among these cultivars, Pusa Urmi is a cross of Berlew Heady Wine, while Shringarika has been developed by hybridizing Mayur and Heady Wine. It shows that they share a common male parentage, i.e. Heady Wine. Thus, their presence in the same sub-cluster could be obvious. However, rest of the cultivars did not share common parentages but clustered together. It might be possible that their parents have got greater degree of similarity in their genetic constitution. Friendship and Rose Delight were closely related and their relatedness could be proved by their parental relationship. Friendship had been developed through hybridization of (Maid of Orlean Picardy) (Gloaming Seedling), while Rose Delight has originated as a result of cross Picardy Early Rose, i.e. one of the parent is common. However, rest
Table 2 AFLP primer combinations, primer sequences, total number of bands generated by each primer set, number of polymorphic bands detected, percentages of polymorphic bands, effective multiplex ratio, diversity index, marker index and primer resolving power of each primer set used in the study of AFLP profiles of Gladiolus cultivars. Sr. no.
Primer pair
Total no. of amplicons
No. of polymorphic amplification products
No. of amplicons/ primer/cv.
% polymorphism
Effective multiplex ratio (EMR)
Diversity index (DI)
Marker index (MI)
Primer resolving power (Rp)
1 2 3 4 5 6 7 8 9
M-CTA/E-AGG M-CTA/E-AGC M-CTA/E-ACT M-CAT/E-ACG M-CTG/E-ACA M-CAT/E-ACC M-CAT/E-ACT M-CTG/E-AGC M-CTG/E-AAG
10 12 50 64 59 70 151 120 124
8 12 50 64 59 70 151 120 124
4.89 7.44 33.68 27.23 19.56 29.05 88.33 53.40 54.53
80 100 100 100 100 100 100 100 100
6.40 12.00 50.00 64.00 59.00 70.00 151.00 120.00 124.00
0.51 0.38 0.33 0.57 0.67 0.58 0.42 0.55 0.56
3.27 4.56 16.32 36.77 39.44 40.95 62.67 66.60 69.47
0.77 3.79 19.19 24.04 15.89 28.53 60.81 65.65 53.82
Fig. 1. Electropherogram of Gladiolus cultivars Gold Dust, Punjab Dawn, Pusa Suhagin, Smoky Lady and The Barton with primer pair E-ACT/M-CAT analyzed using GENOTYPER software. Shaded areas represent polymorphic bands between the cultivars.
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Fig. 2. Dendrgram of 54 Gladiolus cultivars and a duplicate sample of Pusa Shweta resulting from UPGMA cluster analysis based on Jaccard estimates of similarity obtained from 658 number of polymorphic AFLP bands.
of the cultivars did not share common parentages but clustered together. Considering the highly heterozygous nature of the crop, this might be possible. A large number of exotic cultivars clustered to form the II major cluster. However, two indigenous cultivars viz., Pusa Archana and Pusa Suhagin were confined to that cluster. The parentages of these cultivars are quite different but of exotic origin. Pusa Archana has been developed through hybridization of Creamy Green American Beauty whereas Pusa Suhagin is a seedling of Sylvia. Their presence in this particular cluster could be justified by assuming chance similarity in their parentages at genotypic level. Cultivar Errato was found to be the most distinct cultivar in this cluster.
The third cluster was formed by four Pusa cultivars viz., Pusa Lohit, Pusa Swarnima, Pusa Dhanvantari and Pusa Shagun. Pusa Lohit is a cross of Creamy Green American Beauty while Pusa Shagun is developed through White oak Oscar. Pusa Lohit and Shagun are also morphologically related. They have got dark red large sized florets. Pusa Dhanwantari had been developed by Jr Prom Lucky Star, while Pusa Swarnima is a spontaneous mutant of Pusa Dhanwantari. Thus, clustering of Pusa Swarnima and Dhanwantari could be well justified. Clusters IV, V, VI and VII are formed by Pusa Bindiya, Yellow Jame and Australian Fair, Chantiler and Melody, Creamy Green and Snow Princess and Smoky Lady and Viola, respectively. However, Little Fawn and Pusa Gunjan were recognized as two different outgroups. Sufficient pedigree
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information for gladiolus cultivars used in this study was not available in order to conduct comparative analysis based on similarities of AFLP profiles to pedigree data. It has been reported that genetic diversity estimates have compared well with pedigree information in other genera (Jones et al., 1997; Barrett et al., 1998). Schut et al. (1997) compared AFLP markers to pedigree information and morphological traits, and found a moderate correlation. In general, genetic relationships determined through use of the AFLP technique were consistent with previous studies (Perera et al., 1998; Lima et al., 2002; Carr et al., 2003). 3.3. AFLP versus RAPD The clustering of gladiolus cultivars on the basis of AFLP analysis was compared to the previously obtained clustering of the same genotypes on the basis of RAPD data (P. Ranjan, IARI, New Delhi, India, unpublished research). Agreements as well as discrepancies have been observed. In both analyses, Pusa Archana and Pusa Suhagin are placed in the cluster dominated with exotic cultivars. This suggests that these cultivars while close to each other are quite distinct from all other indigenous bred cultivars in their genetic constitution. Also, cultivar pairs The Barton and
Christian Jane; Apricot Queen and Royal Supreme and Pusa Lohit and Pusa Swarnima were found to cluster together in both the dendrograms suggesting their genetic relatedness. The genetic relatedness of indigenous cultivars viz., Pusa Mohini, Pusa Shringarika, Pusa Urvashi, Pusa Urmi, Pusa Sarang, Pusa Swapnil and Pusa Chandani was supported by both RAPD and AFLP analyses through their presence in the same cluster. In the same way, Morello, Priscilla, Apricot Queen, Royal Supreme, Ave, Errato, Gold Dust, Light Purple, Ripples and Anglia were clustered together in both the dendrograms. Pusa Gunjan was found to be the most distinct genotype. However, in RAPD analysis, it was found close to Pusa Chirag. The parentages of Pusa Gunjan and Pusa Chirag are quite different. Pusa Gunjan is a cross of White Oak and Oscar, while Pusa Chirag has been developed by crossing Cygnet Little Fawn. This might be the reason of their diversity in AFLP analysis. Although the parentages of Pusa Gunjan are similar to Pusa Shagun (White Oak Oscar), they were not related with each other through AFLP as well as RAPD analysis. This might be due to the highly heterozygous nature of their parentages. Depending of the type of sequence variation detected with each marker system, a different estimate of the relationships may be obtained (Powell et al., 1996).
Fig. 3. Dendrgram of 54 Gladiolus cultivars and a duplicate sample of Pusa Shweta resulting from a UPGMA cluster analysis based on Jaccard estimates of similarity obtained from combining RAPD and AFLP data.
P. Ranjan et al. / Scientia Horticulturae 123 (2010) 562–567
That is why the genetic relationships between the cultivars of gladiolus estimated by RAPD and AFLP analysis revealed some differences. Differences in the genetic relationships through RAPD and AFLP markers were earlier demonstrated by Wen et al. (2004) in Rosa roxburghii. The dominant nature of RAPD markers, and the limitations of the RAPD technique to detect polymorphism in case of heterozygosity (Parker et al., 1998), may account for the inconsistencies between the analyses. Since the independent dendrograms indicated different clustering patterns, the data of RAPD and AFLP were combined to generate a common UPGMA dendrogram (Fig. 3). The difference in clustering was indicative of the independence of markers studied with these two methods. The UPGMA dendrogram generated by combining RAPD and AFLP markers revealed that the cultivars under study could be divided into four major clusters. Five cultivars viz., Snow Princess, Australian Fair, Little fawn, Viola and Pusa Gunjan were present as separate outgroups. Pusa Gunjan was recognized as the most distinct genotype as in the case of AFLP based dendrogram. Pusa Archana was found closely related with Gold Dust. Although the parentages of Pusa Archana (Creamy Green American Beauty) and Gold Dust (Parfait Domino) are quite different, they were present in the same sub-cluster. This might be due to chance similarity in their genotypes. 3.4. Implications for Gladiolus breeding Clear separation of all 54 gladiolus cultivars proves the sensitivity of AFLP markers for cultivar identification. This sensitivity is further illustrated by the fact that two cultivars selected from the same cross could be distinguished. For example, Pusa Lohit and Pusa Archana are the progenies of Creamy Green and American Beauty. Due to the parental heterozygosities, F1 is actually a segregating generation. Selection can thus be initiated in the F1 generation and desirable plants could be reproduced by vegetative propagation. The AFLP analysis could also detect mutational changes in old genotypes. Pusa Swarnima is a mutant of Pusa Dhanwantari which was isolated based on morphological variations. Jaccard’s similarity coefficient between these two genotypes was 0.688. Among the genotypes under investigation, Pusa Gunjan was the most distinct genotype with superior commercial characters. Thus, this genotype can be efficiently used in effective breeding program and also to study the inheritance pattern of different economical traits. Furthermore, breeders can use this genotype for broadening of genetic base in gladiolus using different breeding methods. The AFLP profiles and clusters identified can be used as a basis for comparison of other gladiolus cultivars. The genetic similarity among cultivars established can help future gladiolus germplasm identification, conservation and new cultivar development. Acknowledgement The Director, National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources, New Delhi, India is thanked for providing the facilities required for molecular work.
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Short communication
Genetic relationships of gladiolus cultivars inferred from fluorescence based AFLP markers Pragya Ranjan a,*, K.V. Bhat b, R.L. Misra a, S.K. Singh a, J.K. Ranjan a a b
Discipline of Horticulture, Indian Agricultural Research Institute, New Delhi, 110012, India National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources, New Delhi, 110012, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2009 Received in revised form 31 October 2009 Accepted 19 November 2009
Gladiolus is one of the important commercial flowers with a large number of cultivars. However, genetic relationships among its genotypes have not been reported. This study analyzed genetic relatedness of 54 gladiolus cultivars using amplified fragment length polymorphism (AFLP) markers. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for AFLP reactions. Fluorescence-labeled amplification products were subjected to electrophoresis and then analyzed using an automated sequencer. A dendrogram was constructed by the unweighted pair group method using the arithmetic average (UPGMA). The number of AFLP fragments generated per primer set ranged from 10 to 151 with fragment sizes varying from 50 to 450 bp. A total of 660 AFLP fragments were detected, of which 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. All cultivars were clearly differentiated by their AFLP profiles. The AFLP data were compared with previously obtained RAPD data and combined to generate a common dendrogram. The first cluster was dominated with indigenously bred cultivars while the second was dominated with exotic cultivars. This shows that most of the exotic cultivars as well as indigenous cultivars are closely related with each other. However, two indigenous cultivars viz., Pusa Suhagin and Pusa Archana share genetic similarity with exotic cultivars. Among the genotypes selected for the investigation, Pusa Gunjan was identified as the most distinct genotype. The AFLP markers developed will help future Gladiolus cultivar identification, germplasm conservation and new cultivar development. The assessed genetic relationships among gladiolus cultivars may enhance the efficiency of breeding program by selecting desirable parents with reduced breeding cycle. ß 2009 Elsevier B.V. All rights reserved.
Keywords: AFLP markers Bulbous ornamental Genetic relationship Gladiolus Iridaceae
1. Introduction Gladiolus spp. is an important bulbous ornamental prized for the beauty of its spikes as well as longer vase-life. The genus Gladiolus is a member of family Iridaceae and sub-family Ixioideae (Goldblatt, 1991). The name gladiolus is derived from the Latin word ‘gladius’, meaning sword for the shape of its leaves. An ancient name for gladiolus was xiphium, from the Greek word ‘xiphos’, also meaning sword. Commonly gladiolus is known as ‘Sword lily’. Tournefort christened Gladiolus as a genus, which was accepted by Linnaeus in botanical literature (Lewis et al., 1972). Due to magnificent inflorescence with wide array of colours, gladioli are very much
Abbreviations: CTAB, cetyltrimethylammonium bromide; ISSR, inter simple sequence repeat; PCR, polymerase chain reaction. * Corresponding author at: Central Institute of Temperate Horticulture-Regional Station, Mukteshwar, Distt: Nainital, Uttarakhand, 263138, India. Tel.: +91 5942 286027; fax: +91 5942 286027. E-mail address: [email protected] (P. Ranjan). 0304-4238/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2009.11.013
popular among growers as cut-flower. The flower spikes are used in vase or value-added in bouquet. Gladioli are also quite suitable as herbaceous border, bedding and potted plant. South Africa, particularly the Cape of Good Hope, is considered to be the centre of diversity for this genus. The current number of species in the genus is 255 (Goldblatt and Manning, 1998) and all the species are herbaceous perennial in nature. The modern cultivars of G. grandiflora are believed to be originated from a number of wild species viz., G. cruentus, G. natalensis, G. oppositiflorus, G. papilio and G. saundersii (Barnard, 1972; Imanishi, 1989). The modern cultivars are derived from interspecific crosses among several species. Hence, wide variation is exhibited by gladiolus cultivars for their growth habit, size, shape and colour of spikes, and florets. The assessment of natural genetic variation is important not only for ethical and aesthetic reasons but also to ensure that genetic resources may be used even more efficiently and sustainably in agriculture and other industries. Thus, there is a need to assess the variation that already exist and how it can be conserved and utilized effectively.
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Although gladiolus is being grown as a popular cut-flower worldwide, neither the US National Plant Germplasm System (NPGS) nor the Biodiversity International formerly known as International Board for Plant Genetic Resources (IBPGR) had been actively involved in conservation of gladiolus. Even the Ornamental Plant Germplasm Centre (OPGC) established in 1999 at Ohio State University has not assigned gladiolus as priority genera for conservation and maintenance. Looking into the importance of this crop, efforts are needed for systematic characterization of this crop. Proper characterization helps in unambiguous discrimination between accessions, identification of economically important plant resources, detection of redundancies and in monitoring genetic changes appearing during maintenance. Estimates of diversity are useful to guide collection missions, monitoring genetic erosion, establish core subset and to develop on-farm conservation strategies. Molecular marker techniques also have significant utility in plant breeding programs through assisting plant variety protection as well as distinctness, uniformity and stability testing processes (Heckenberger et al., 2006) which is highly applicable to gladiolus in which the number of newly constituted varieties is continuously increasing. Moreover, proper characterization and assessment of genetic relationships among diverse accessions allow breeders to select desirable genes from different sources and to accumulate those genes in one cultivar. Thus, estimates of genetic relatedness between cultivars may enhance the efficiency of effective breeding program. Application of molecular techniques for the study of genetic relationships or cultivar identification in gladiolus has perhaps not been reported yet. Amplified fragment length polymorphism (AFLP), a more robust and reliable technique (Vos et al., 1995) has been used as an efficient technique to assess genetic diversity and relationships in a number of crops including some bulbous ornamentals such as Aglaonema (Chen et al., 2004); Alstroemeria (Han et al., 2000); Caladium venten spp. (Loh et al., 1999, 2000) and Hemerocallis spp. (Tomkins et al., 2001). Among the molecular markers deployed, AFLPs have several advantages, including high levels of reproducibility and a high information content (Mueller and Wolfenbarger, 1999; Hodkinson et al., 2002). Moreover, when fluorescence-labeled oligonucleotides are employed in combination with automated capillary electrophoresis, it gives a much higher resolution than other AFLP detection systems. Besides these, fluorescence-labeled fragments have distinct advantages as these can be scored using computerized analysis rather than visual analysis (Carr et al., 2003). The main objectives of this study were to use of fluorescence based AFLP technique to estimate genetic relationships among 54 gladiolus cultivars. Probably, no report is available in gladiolus regarding estimation of genetic relationship using any molecular marker techniques. Therefore, as a first report of its kind on gladiolus cultivars, this study strives to generate information that is crucial in guiding improvement and conservation program of gladiolus.
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Table 1 List of 54 Gladiolus cultivars and a duplicate sample of Pusa Shweta used in this study and their parentages. Sr. no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.
Cultivar
Parentage
Friendship
(Maid of Orleans Picardy) (Gloaming Seedling) Unknown Unknown Unknown Unknown Crusador Statuette Diamond Leona Unknown Eloise Rosemarie Pfitzer Creamy Green American Beauty Parfait Domino Unknown Sylvia seedling Big Time Plum Pudding Marjorie Nadean R S Mayne Unknown Unknown Unknown Unknown Apricot Glow Emile Aubrun Unknown Jester sport Salome Maolete Jr Prom Lucky Star Unknown White Oak Oscar Unknown Unknown Pastorale unknown Unknown Ruffled Ebony Ace of spades Unknown Old South Statuette Unknown Comm Koehl Moorish King Beauty’s Blush Rose Charm Picardy Early Rose Unknown Unknown Open pollinated seedling of Ratna’s Butterfly Open pollinated seedling of White Oak Sylvia Partica Ave Christian Jane Creamy Green American Beauty Cygnet Little Fawn Red Bantam Flaura Belle Seedling of (Wind Song Pink Frost) Seedling of (Wind Song Pink Frost) Spontaneous mutant of Dhanvantari Red soft glow open Mayur Heady Wine Berlew Heady Wine Viola open Snow Princess Viola White Oak Oscar
Yellow Jame Creamy Green Light Purple Morello Ripples Priscilla Apricot Queen Anglia Pusa Archana Gold Dust Punjab Dawn Pusa Suhagin Smoky Lady The Barton Christian Jane Errato Royal Supreme Ave Picardy Summer Rose Jester Gold Peter Pears Pusa Dhanvantari Snow Princess Pusa Shagun Plumtart Chantiler Viola Bonaire Regency Australian Fair Little Fawn Bis–Bis Melody Apple Blossom Rose Delight Pusa Chandani George Mazure Pusa Bindiya Pusa Sarang Neelam Pusa Mohini Pusa Lohit Pusa Chirag Pusa Rangmahal Pusa Shweta 1 Pusa Shweta 2 Pusa Swarnima Pusa Urvashi Pusa Shringarika Pusa Urmi Pusa Swapnil Pusa Gulaal Pusa Gunjan
2. Materials and methods 2.1. Plant material A total of 54 (33 exotic and 21 indigenous) commercial varieties of gladiolus (Table 1) along with one duplicated sample were analyzed in this study. Some of them are developed at the Division of Floriculture and Landscaping, IARI, New Delhi and rest are maintained as procured from different countries vis-a`-vis indigenous sources. 2.2. Fluorescent-AFLP analysis Young, fresh and healthy leaves of 55 samples were used for DNA extraction using CTAB method as per the protocol of Sanghai-
Maroof et al. (1984) with minor modifications. AFLP analysis was conducted using the Plant Mapping kit for regular plant genomes (500–6000 Mb) AFLPTM (PE Applied Biosystems, USA). Total DNA (500 ng) was digested with 1 ml of a mixture of EcoRI/Mse I (1.25 units ml 1) at 37 8C overnight and ligated to EcoRI/Mse I adapters with 1.5 ml (1 unit ml 1) of T4 DNA ligase at 25 8C for at least 6 h. The adaptor ligated DNA was amplified and subjected to PCR amplification in a thermocycler (Perkin Elmer 9600, PE Applied Biosystems, USA). EcoRI primers of AFLP kit were got labeled with fluorescent dyes of different colours (5-FAM, JOE and NED), which have an added advantage of multiplexing three primers in one reaction. A total of 24 AFLP primer pairs with three samples were initially screened, from which 9 primer sets that showed clear scorable and highly polymorphic bands were selected for further
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analysis. After selective amplification, electrophoresis (ABI Prism 310 Automated Sequencer) was performed at a constant voltage (14.9 kV) and current (10 mA) at a temperature of 60 8C. The data collection software collects light intensities using software selectable filters and stores them as electronic signals for processing. These signals are in the form of peaks. Each peak is equivalent to one band and peak height represents intensity of the band. The ‘‘Genescan’’ and ‘‘Genotyper’’ software package (Applied Biosystems, USA) was used for processing and scoring of the results. The similarity coefficient matrix (Jacard, 1908) was subjected to cluster analysis by unweighted pair group method of arithmetic average (UPGMA) analysis and dendrogram was generated with the help of NTSYS-pc software version 1.70 (Exeter Software, New York, USA). 3. Results and discussion 3.1. DNA profiles of gladiolus genotypes The nine primer pairs generated clear-cut AFLP profiles for 54 gladiolus cultivars and AFLP fragments ranged from 50 to 450 bp with a total of 660 scorable fragments. The duplicate cultivar ‘Pusa Shweta’ showed identical AFLP patterns with each primer set. The number of bands generated per primer ranged from 10 (E-AGG/MCTA) to 151 (E-ACT/M-CAT) with a mean of 73.33 bands per primer. Out of the 660 bands generated, 658 (99.70%) were polymorphic. All the primers except E-AGG/M-CTA displayed 100% polymorphism. The levels of polymorphisms detected in the gladiolus germplasm under investigation indicate that the amount of genetic variability present in this genome is relatively high. The number of peaks scored per primer pair ranged from 10 to 151 (Table 2). Electropherogram of Gladiolus cultivars Gold
Dust, Punjab Dawn, Pusa Suhagin, Smoky Lady and The Barton are presented in Fig. 1. The average number of amplification products per primer per cultivar ranged from 4.89 (E-AGG/M-CTA) to 88.33 (E-ACT/M-CAT). 3.2. Cluster analysis and genetic relationships among cultivars Based on Jaccard’s similarity matrix and UPGMA clustering a wide range of degree of relatedness between cultivars was reported in this study; some are closely related while others were found quite distinct from each other. The greatest similarity was found between Pusa Lohit and Pusa Swarnima (Jaccard coefficient = 0.788), while Pusa Gunjan was found to be the most distinct genotype. The dendrogram based on UPGMA clustering (Fig. 2) resulted in the identification of seven major clusters. Cultivars Pusa Gunjan and Little Fawn were not confined to any of the clusters and were present as out groups. Pusa Gunjan was found to be the most distinct genotype. Cluster I was the largest and dominated with indigenous cultivars. Among these cultivars, Pusa Urmi is a cross of Berlew Heady Wine, while Shringarika has been developed by hybridizing Mayur and Heady Wine. It shows that they share a common male parentage, i.e. Heady Wine. Thus, their presence in the same sub-cluster could be obvious. However, rest of the cultivars did not share common parentages but clustered together. It might be possible that their parents have got greater degree of similarity in their genetic constitution. Friendship and Rose Delight were closely related and their relatedness could be proved by their parental relationship. Friendship had been developed through hybridization of (Maid of Orlean Picardy) (Gloaming Seedling), while Rose Delight has originated as a result of cross Picardy Early Rose, i.e. one of the parent is common. However, rest
Table 2 AFLP primer combinations, primer sequences, total number of bands generated by each primer set, number of polymorphic bands detected, percentages of polymorphic bands, effective multiplex ratio, diversity index, marker index and primer resolving power of each primer set used in the study of AFLP profiles of Gladiolus cultivars. Sr. no.
Primer pair
Total no. of amplicons
No. of polymorphic amplification products
No. of amplicons/ primer/cv.
% polymorphism
Effective multiplex ratio (EMR)
Diversity index (DI)
Marker index (MI)
Primer resolving power (Rp)
1 2 3 4 5 6 7 8 9
M-CTA/E-AGG M-CTA/E-AGC M-CTA/E-ACT M-CAT/E-ACG M-CTG/E-ACA M-CAT/E-ACC M-CAT/E-ACT M-CTG/E-AGC M-CTG/E-AAG
10 12 50 64 59 70 151 120 124
8 12 50 64 59 70 151 120 124
4.89 7.44 33.68 27.23 19.56 29.05 88.33 53.40 54.53
80 100 100 100 100 100 100 100 100
6.40 12.00 50.00 64.00 59.00 70.00 151.00 120.00 124.00
0.51 0.38 0.33 0.57 0.67 0.58 0.42 0.55 0.56
3.27 4.56 16.32 36.77 39.44 40.95 62.67 66.60 69.47
0.77 3.79 19.19 24.04 15.89 28.53 60.81 65.65 53.82
Fig. 1. Electropherogram of Gladiolus cultivars Gold Dust, Punjab Dawn, Pusa Suhagin, Smoky Lady and The Barton with primer pair E-ACT/M-CAT analyzed using GENOTYPER software. Shaded areas represent polymorphic bands between the cultivars.
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Fig. 2. Dendrgram of 54 Gladiolus cultivars and a duplicate sample of Pusa Shweta resulting from UPGMA cluster analysis based on Jaccard estimates of similarity obtained from 658 number of polymorphic AFLP bands.
of the cultivars did not share common parentages but clustered together. Considering the highly heterozygous nature of the crop, this might be possible. A large number of exotic cultivars clustered to form the II major cluster. However, two indigenous cultivars viz., Pusa Archana and Pusa Suhagin were confined to that cluster. The parentages of these cultivars are quite different but of exotic origin. Pusa Archana has been developed through hybridization of Creamy Green American Beauty whereas Pusa Suhagin is a seedling of Sylvia. Their presence in this particular cluster could be justified by assuming chance similarity in their parentages at genotypic level. Cultivar Errato was found to be the most distinct cultivar in this cluster.
The third cluster was formed by four Pusa cultivars viz., Pusa Lohit, Pusa Swarnima, Pusa Dhanvantari and Pusa Shagun. Pusa Lohit is a cross of Creamy Green American Beauty while Pusa Shagun is developed through White oak Oscar. Pusa Lohit and Shagun are also morphologically related. They have got dark red large sized florets. Pusa Dhanwantari had been developed by Jr Prom Lucky Star, while Pusa Swarnima is a spontaneous mutant of Pusa Dhanwantari. Thus, clustering of Pusa Swarnima and Dhanwantari could be well justified. Clusters IV, V, VI and VII are formed by Pusa Bindiya, Yellow Jame and Australian Fair, Chantiler and Melody, Creamy Green and Snow Princess and Smoky Lady and Viola, respectively. However, Little Fawn and Pusa Gunjan were recognized as two different outgroups. Sufficient pedigree
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information for gladiolus cultivars used in this study was not available in order to conduct comparative analysis based on similarities of AFLP profiles to pedigree data. It has been reported that genetic diversity estimates have compared well with pedigree information in other genera (Jones et al., 1997; Barrett et al., 1998). Schut et al. (1997) compared AFLP markers to pedigree information and morphological traits, and found a moderate correlation. In general, genetic relationships determined through use of the AFLP technique were consistent with previous studies (Perera et al., 1998; Lima et al., 2002; Carr et al., 2003). 3.3. AFLP versus RAPD The clustering of gladiolus cultivars on the basis of AFLP analysis was compared to the previously obtained clustering of the same genotypes on the basis of RAPD data (P. Ranjan, IARI, New Delhi, India, unpublished research). Agreements as well as discrepancies have been observed. In both analyses, Pusa Archana and Pusa Suhagin are placed in the cluster dominated with exotic cultivars. This suggests that these cultivars while close to each other are quite distinct from all other indigenous bred cultivars in their genetic constitution. Also, cultivar pairs The Barton and
Christian Jane; Apricot Queen and Royal Supreme and Pusa Lohit and Pusa Swarnima were found to cluster together in both the dendrograms suggesting their genetic relatedness. The genetic relatedness of indigenous cultivars viz., Pusa Mohini, Pusa Shringarika, Pusa Urvashi, Pusa Urmi, Pusa Sarang, Pusa Swapnil and Pusa Chandani was supported by both RAPD and AFLP analyses through their presence in the same cluster. In the same way, Morello, Priscilla, Apricot Queen, Royal Supreme, Ave, Errato, Gold Dust, Light Purple, Ripples and Anglia were clustered together in both the dendrograms. Pusa Gunjan was found to be the most distinct genotype. However, in RAPD analysis, it was found close to Pusa Chirag. The parentages of Pusa Gunjan and Pusa Chirag are quite different. Pusa Gunjan is a cross of White Oak and Oscar, while Pusa Chirag has been developed by crossing Cygnet Little Fawn. This might be the reason of their diversity in AFLP analysis. Although the parentages of Pusa Gunjan are similar to Pusa Shagun (White Oak Oscar), they were not related with each other through AFLP as well as RAPD analysis. This might be due to the highly heterozygous nature of their parentages. Depending of the type of sequence variation detected with each marker system, a different estimate of the relationships may be obtained (Powell et al., 1996).
Fig. 3. Dendrgram of 54 Gladiolus cultivars and a duplicate sample of Pusa Shweta resulting from a UPGMA cluster analysis based on Jaccard estimates of similarity obtained from combining RAPD and AFLP data.
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That is why the genetic relationships between the cultivars of gladiolus estimated by RAPD and AFLP analysis revealed some differences. Differences in the genetic relationships through RAPD and AFLP markers were earlier demonstrated by Wen et al. (2004) in Rosa roxburghii. The dominant nature of RAPD markers, and the limitations of the RAPD technique to detect polymorphism in case of heterozygosity (Parker et al., 1998), may account for the inconsistencies between the analyses. Since the independent dendrograms indicated different clustering patterns, the data of RAPD and AFLP were combined to generate a common UPGMA dendrogram (Fig. 3). The difference in clustering was indicative of the independence of markers studied with these two methods. The UPGMA dendrogram generated by combining RAPD and AFLP markers revealed that the cultivars under study could be divided into four major clusters. Five cultivars viz., Snow Princess, Australian Fair, Little fawn, Viola and Pusa Gunjan were present as separate outgroups. Pusa Gunjan was recognized as the most distinct genotype as in the case of AFLP based dendrogram. Pusa Archana was found closely related with Gold Dust. Although the parentages of Pusa Archana (Creamy Green American Beauty) and Gold Dust (Parfait Domino) are quite different, they were present in the same sub-cluster. This might be due to chance similarity in their genotypes. 3.4. Implications for Gladiolus breeding Clear separation of all 54 gladiolus cultivars proves the sensitivity of AFLP markers for cultivar identification. This sensitivity is further illustrated by the fact that two cultivars selected from the same cross could be distinguished. For example, Pusa Lohit and Pusa Archana are the progenies of Creamy Green and American Beauty. Due to the parental heterozygosities, F1 is actually a segregating generation. Selection can thus be initiated in the F1 generation and desirable plants could be reproduced by vegetative propagation. The AFLP analysis could also detect mutational changes in old genotypes. Pusa Swarnima is a mutant of Pusa Dhanwantari which was isolated based on morphological variations. Jaccard’s similarity coefficient between these two genotypes was 0.688. Among the genotypes under investigation, Pusa Gunjan was the most distinct genotype with superior commercial characters. Thus, this genotype can be efficiently used in effective breeding program and also to study the inheritance pattern of different economical traits. Furthermore, breeders can use this genotype for broadening of genetic base in gladiolus using different breeding methods. The AFLP profiles and clusters identified can be used as a basis for comparison of other gladiolus cultivars. The genetic similarity among cultivars established can help future gladiolus germplasm identification, conservation and new cultivar development. Acknowledgement The Director, National Research Centre on DNA Fingerprinting, National Bureau of Plant Genetic Resources, New Delhi, India is thanked for providing the facilities required for molecular work.
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