Saccharum(=Erianthus) native to North America is an untapped germplasm for genetic improvement of ..... distances were estimated using the modified Roger's.
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Genetic Resources and Crop Evolution 44: 235–240, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.
Genetic diversity of North American and Old World Saccharum assessed by RAPD analysis David M. Burner1 , Yong-Bao Pan1 & Robert D. Webster2 1
Sugarcane Research Unit, USDA-ARS, P.O. Box 470, Houma, LA 70361, USA; 2 South Central Family Farms Research, USDA-ARS, Rt. 2, P.O. Box 144-A, Booneville, AR 72927, USA Received 28 June 1996; accepted in revised form 4 October 1996
Key words: Erianthus, genetic distance, genomic RAPD-PCR, germplasm evaluation, sugarcane, taxonomy
Summary Saccharum (= Erianthus) native to North America is an untapped germplasm for genetic improvement of sugarcane (Saccharum spp. hybrids). There are five species and two varieties native to North America: S. alopecuroideum, S. baldwinii, S. brevibarbe vars. brevibarbe and contortum, S. coarctatum, and S. giganteum. There are three cytotypes of S. giganteum (2n = 30, 60, 90), and they overlap in gross morphology. Our objectives were to compare genetic diversity of North American and Old World members of Saccharum. Bulked DNA for five North American species, three Old World Erianthus spp. sect. Ripidium clones, and five sugarcane cultivars was tested by PCR with 13 RAPD primers. A total of 283 repeatable RAPD bands was scored for the nine taxa. Genetic distance coefficients ranged from 0.365 to 0.767 indicating substantial diversity among taxa. Taxa were assigned to one of three cluster groups: 1) S. baldwinii, S. brevibarbe var. contortum, S. coarctatum, and S. giganteum 2n = 90; 2) S. giganteum 2n = 30 and 2n = 60, S. alopecuroideum, and sugarcane cultivars; and 3) Old World Erianthus spp. The RAPD analysis indicated that sugarcane was genetically more similar to North American Saccharum than it was to Old World Erianthus. This was unexpected given that North American Saccharum is geographically, cytologically, morphologically, and possibly reproductively isolated from Old World Erianthus and sugarcane. The data support the taxonomic separation of cytotypes of S. giganteum.
Introduction Sugarcane (a trispecific hybrid of Saccharum officinarum L., S. robustum Brand. & Jesw. ex Grassl, and S. spontaneum L.), a sugar crop with world-wide importance, is a grass (Gramineae) of the tribe Andropogoneae. Sugarcane is thought to have originated in Asia. East Indonesia/New Guinea is a particularly rich source of Saccharum germplasm (Berding & Roach, 1987), and New Guinea is probably the center of diversity (Daniels & Roach, 1987) of S. officinarum (noble sugarcane). Southern Louisiana (lat 30 N) represents the near northern limit of commercial sugarcane production. Erianthus sect. Ripidium, Miscanthus sect. Diandra, Narenga, Saccharum, and Sclerostachya are so closely related that Mukherjee (1957) referred to
them as the “sugarcane complex”. Harlan and de Wet (1975) questioned whether the taxonomy of Saccharum may be overly divided, thereby exaggerating apparent wide hybridization. These taxa continue to present challenges to breeders and taxonomists because of the genetic and morphological complexity: species range from 2n = 20 [e.g., Erianthus rufipilus (Steud.) Griseb.] to 2n = about 200 (e.g., S. robustum) with many numbers in between (Burner, 1991; Sreenivasan et al., 1987). Ploidy level(s) are poorly understood, but evidence from in situ hybridization (D’Hont et al., 1995) indicates that x = 10 for Erianthus arundinaceus (Retz.) Bhar. (2n = 60) and S. officinarum (2n = 80). Saccharum spontaneum is autopolyploid with x = 8 (Da Silva et al., 1993). Celarier (1956) postulated that Erianthus is typical of the Andropogoneae with x = 5.
236 Numerous collection expeditions have provided germplasm for use in developing improved varieties and cultivars (Berding & Roach, 1987). Cultivars are exclusively hybrid derivatives whose genotypes (clones) are vegetatively propagated. Species of Erianthus native to North America, collected mainly from the southeastern U.S., were taxonomically reevaluated and moved from Erianthus to Saccharum (Webster & Shaw, 1995). The species are S. alopecuroideum L. Nutt. (2n = 30), S. baldwinii Spreng. (2n = 30), S. brevibarbe (Michx.) Pers. var. brevibarbe (Michx.) Pers. (2n = 60), S. brevibarbe var. contortum (Nutt.) R. Webster (2n = 60), S. coarctatum Fern. R. Webster (2n = 60), and S. giganteum (Walt.) Pers. (2n = 30, 60, and 90). Chromosome counts were by Burner and Webster (1994). The random amplified polymorphic DNA (RAPD) technique has been used to assess the genetic diversity of germplasm within grass genera such as Brachypodium (Catalan et al., 1995), Eleusine (Hilu, 1995), Lolium (Sweeney & Danneberger, 1994), Oryza (Ko et al., 1994; Virk et al., 1995; Yu & Nguyen, 1994), Sorghum (Tao et al., 1993), and Triticum (Joshi & Nguyen, 1993). A small number of RAPD primers may be used to identify sugarcane cultivars (Taylor et al., 1995). Our objective was to use RAPD analysis to determine the genetic diversity between North American and Old World sugarcane taxa.
Materials and method Plant materials: Live plants of North American Saccharum species were established in the greenhouse from true seed (Table 1). Young leaves were harvested for DNA isolation from five sugarcane cultivar clones and three Old World Erianthus sect. Ripidium clones from vegetatively propagated clones grown in the greenhouse or field. Clones NG 77-214 and US 57-60-2 appear to be E. rufipilus based on floral morphology and chromosome number. Clone US 57-11-2 appeared to be E. arundinaceus, not Eccoilopus longisetosus Anderss. as previously reported (AlJanabi et al., 1994), based on floral morphology and chromosome number (Burner, 1991; Gill & Grassl, 1986). Young leaves were harvested for DNA isolation from about three random seedlings from each of three accessions of S. alopecuroideum, S. baldwinii, S. brevibarbe var. contortum, S. coarctatum, and S. giganteum (2n = 30, 60, and 90).
Table 1. Sugarcane taxa and clones tested in RAPD study. Taxon/clone
Chromosome number (2n)a
Sugarcane cultivars (Saccharum spp. hybrids) CP 70-321 110 CP 74-383 104, 108–109 CP 79-332 112, 114 LCP 82-89 109–116 LCP 86-454 ND Old World Erianthus spp.b NG 77-214 E. rufipilus 20 US 57-11-2 E. arundinaceus 60 US 57-60-2 E. rufipilus 20 North American Saccharum spp. S. alopecuroideum 30 S. baldwinii 30 S. brevibarbe var. contortum 60 S. coarctatum 60 S. giganteum 30 S. giganteum 60 S. giganteum 90
Plants sampled (no.) 5
3
60 8 7 9 9 9 9 9
a
Chromosome counts from Burner (1991), Burner & Legendre (1994), Burner & Webster (1994), and unpublished. b Tentative identifications.
DNA isolation: The PuregeneR kit (Gentra Systems, Minneapolis, MN)1 was used to extract DNA from leaves (about 40 mg f.w.) of seedlings or shoots. The DNA concentration of each sample was determined spectrophotometrically and was electrophoresed on a 0.8% agarose gel to confirm quality. An aliquot was diluted to about 25 ng DNA l 1 with sterile, 18 mohm water. Some samples were brown colored, apparently due to phenolic compounds that inhibited the RAPD reaction, and were discarded. Equivolume samples were bulked by taxon. Primer selection: Thirty-two 9-, 10-, 11-, 17-, and 20-mer primers obtained either from Integrated DNA Technologies, Inc. (Coralville, IA) or Operon Technologies, Inc. (Alameda, CA) were available for testing. Each primer was diluted with water and stored at 20 C. Thirteen primers that yielded scorable, apparently polymorphic bands were repeated for the study (Table 2).
237 Table 2. RAPD primers tested for polymorphism, their sequence, and numbers of bands for sugarcane taxa.
!3)
Primer
Sequence (50
262 K19T OPA-01 OPA-04 OPA-07 OPA-08 OPA-10 OPA-11 OPA-16 OPA-17 OPA-18 OPA-19 OPA-20 Total
CGCCCCCAGT AGTTCAGGC CAGGCCCTCC AATCGGGCTG GAAACGGGTG GTGACGTAGG GTGATCGCAG CAATCGCCGT AGCCAGCGAA GACCGCTTGT AGGTGACCGT CAAACGTCGG GTTGCGATCC
a Total
0
Polymorph.a
No. bands ALOb BAL
BRE
COA
GIG30
GIG60
GIG90
OWE
CVS
Total
12 4 3 6 4 3 5 11 9 12 4 12 4 89
5 2 0 0 2 2 0 2 6 4 2 1 1 27
5 2 1 1 3 3 0 2 6 4 1 3 3 34
4 2 2 1 4 2 0 3 5 4 1 3 1 32
3 1 2 0 3 2 2 4 6 4 3 3 0 33
4 2 2 0 4 1 1 3 6 4 3 1 1 32
4 1 2 3 4 0 1 4 5 4 1 3 1 33
6 3 3 3 1 0 2 1 4 3 0 6 2 34
3 2 0 1 2 0 2 2 2 6 0 5 1 26
39 17 13 10 26 11 8 24 44 37 12 29 13 283
5 2 1 1 3 1 0 3 4 4 1 4 3 32
number of polymorphic bands across taxa. Only intensely stained bands were scored. S. alopecuroideum, BAL S. baldwinii, BRE S. brevibarbe var. contortum, COA S. coarctatum, GIG30 S. giganteum (2n 30), GIG60 S. giganteum (2n 60), GIG90 S. giganteum (2n 90), OWE Old World Erianthus spp., and CVS sugarcane cultivars (Saccharum spp. hybrids). b ALO
=
=
=
= =
=
=
PCR and agarose gel electrophoresis: Amplifications of DNA were conducted under standardized conditions in a 25 l reaction volume containing 25 ng template DNA; 50 mM KCl; 10 mM Tris-HCl (pH 8.3); 1.5 mM mgCl2 ; 0.2 mM each of dATP, dCTP, dGTP, and dTTP; 2 U Stoffel fragment; and 0.4 M primer. Reactions were conducted in a PTC-100TM thermocycler (MJ Research Inc., Watertown, MA) with the following steps: 5 min at 95 C, 30 cycles of (40 s at 93 C, 30 s at 40 C, 1 min at 72 C), and 5 min at 72 C. Reaction products were mixed with 5 l of 6X loading dye (0.25% bromophenol blue, 0.25% xylene cyanole, and 40% sucrose, w/v) and spun briefly in a centrifuge before loading. Five l were loaded in each lane of a 1.5% agarose gel made with 0.5X TBE and 0.5 g ml 1 ethidium bromide. Samples were electrophoresed at 6 V cm 1 for 2.5 h, and photographed under UV. The PCR analysis was conducted twice. Data analysis: Replicates of bulked samples amplified with a particular primer were compared in a single gel. The molecular weight of intense bands present in both replicates was determined in reference to a molecular weight marker (Cat. no. P9577, Sigma Chemical Co., St. Louis, MO). Relationships among taxa were calculated by PC SAS procedure CLUSTER (SAS, 1989) using Ward’s (1963) minimum variance method. Results from the cluster analysis were plotted using
=
=
=
=
=
the TREE procedure of PC SAS (SAS, 1989). Genetic distances were estimated using the modified Roger’s distance equation (Rogers, 1972).
Results and discussion There were 89 different polymorphic bands amplified across taxa and primers, or an average of about 7 bands per primer (Table 2). A total of 283 repeatable RAPD bands was scored for the nine taxa. As expected, numbers of bands varied with primer but averaged 22 per primer across taxa. Primers produced 0.9 bands per taxon (primer OPA-10) to 4.9 bands per taxon (primer OPA-16). Primer 262 and OPA-17 yielded 4.3 bands per taxon. Primer 262 has been reported to yield numerous RAPD bands for a number of diverse plant species (Fritsch et al., 1993; McDonald et al., 1994). Cluster analysis of the banding data suggested that S. baldwinii, S. brevibarbe var. contortum, S. coarctatum, and S. giganteum 2n = 90 were assigned to one cluster (Figure 1). The mean distance for this cluster was 0.562. The 2n = 30 and 2n = 60 cytotypes of S. giganteum, S. alopecuroideum, and sugarcane cultivars were assigned to another cluster with a mean distance of 0.589. Old World Erianthus sect. Ripidium was assigned to a separate cluster with a mean distance of 0.748. Thus, sugarcane and North American Sac-
238
Figure 1. Genetic diversity among sugarcane taxa.
charum were more genetically similar than sugarcane and Old World Erianthus. This supports the taxonomic reassignment of North American members to Saccharum from Erianthus (Webster & Shaw, 1995). Pair-wise distance coefficients for the nine taxa ranged from 0.365 to 0.767 (Table 3). Old World Erianthus had highest distance coefficients among the taxa. It was interesting that genetic distances among cytotypes of S. giganteum were comparable to interspecific distances, and that the 2n = 90 cytotype clustered apart from the 2n = 30 and 2n = 60 cytotypes (Figure 1). These data suggested that cytotypes of S. giganteum might logically be assigned to separate subspecies or varieties. Coefficients were similar in magnitude to those reported for 13 cultivars of Oryza sativa L. (Yu & Nguyen, 1994), and for 10 populations of the North American shrub Chrysothamnus nauseosus ssp. hololeucus Pallas (Britt.) (Gang & Weber, 1995). Geographical groups of the shrub had distinctive banding patterns, suggesting that RAPD data could be used in phylogenic studies of that species (Gang & Weber, 1995). Cluster analysis of the North American species based on chromosome number, pollen area, and pollen
volume suggested four cluster groups: 1) S. giganteum 2n = 60, 2) S. giganteum 2n = 90, 3) S. brevibarbe and S. coarctatum, and 4) the 2n = 30 species or cytotypes of S. alopecuroideum, S. baldwinii, and S. giganteum (Burner & Webster, 1994). Considered together, the studies confirm that S. giganteum (2n = 30 and 60) are logically distinct from S. brevibarbe and S. coarctatum. The data also support previous reports of genetic divergence between Old World Erianthus sect. Ripidium and sugarcane based on total genomic DNA (Lu et al., 1994) and extranuclear genomes (Al-Janabi et al., 1994; D’Hont et al., 1993 and 1995; Sobral et al., 1994). The North American species have not been placed within the evolutionary pathway of S. officinarum (Roach & Daniels, 1987), which is logical given their geographic isolation. The breeding potential of this germplasm has not been determined, but it possesses adaptability to temperate climate and, possibly, cold tolerance traits that may be absent from elite cultivars. Old World Erianthus sect. Ripidium, on the other hand, is considered closely related to Saccharum based on geographic distribution and chromosome homology (Roach & Daniels, 1987). Indeed, sugarcane is frequently crossed with Old World Erianthus sect. Ripidi-
239 Table 3. Distance matrix of North American and Old World taxa of Saccharum estimated by Rogers’ modified distance equation (Rogers, 1972). Taxaa
ALO
BAL
BRE
COA
GIG30
GIG60
GIG90
OWE
CVS
ALO BAL BRE COA GIG30 GIG60 GIG90 OWE CVS
0
0.587b 0
0.483 0.365 0
0.568 0.422 0.394 0
0.577 0.658 0.587 0.527 0
0.506 0.577 0.537 0.516 0.408 0
0.615 0.568 0.568 0.483 0.577 0.548 0
0.723 0.730 0.730 0.760 0.767 0.760 0.767 0
0.587 0.632 0.650 0.667 0.691 0.683 0.675 0.745 0
=
=
a The following abbreviations are used for species: ALO S. alopecuroideum, BAL S. baldwinii, BRE S. brevibarbe var. contortum, COA S. coarctatum, GIG30 S. giganteum 2n 30, GIG60 S. giganteum 2n 60, GIG90 S. giganteum 2n 90, OWE Old World Erianthus spp., and CVS sugarcane cultivars (Saccharum spp. hybrids). b Values range from 0 (no diversity) to 1.0 (no similarity).
=
=
=
=
=
um because Old World Erianthus is an excellent source of resistance to sugarcane mosaic virus (Grisham et al., 1992) and sugarcane smut, Ustilago scitaminea Syd. & P. Syd. (Burner et al., 1993). However, some North American accessions are susceptible to the H strain of sugarcane mosaic virus (unpublished), while others are susceptible to tangle top fungus, Myriogenospora atramentosa (Berk. & Curt.) Diehl (C.W. Bacon, 1996, personal communication). Successful crosses between elite sugarcane x S. brevibarbe var. contortum and elite sugarcane x S. giganteum 2n = 90 were reported by Burner and Webster (1994) based on gross plant morphology. Subsequent DNA-based tests failed to verify the plants were hybrid (unpublished). Several additional cross attempts also failed to yield hybrids. Thus, preliminary data suggest that the North American species may be less crosscompatible with sugarcane than their Old World Erianthus counterparts. This could reflect geographic isolation, cytogenetic diversity (the 2n = 90 cytotype has not been reported in Old World Erianthus), and morphological diversity (New World and Old World taxa have two and three stamens per floret, respectively). Cytotaxonomic studies are in progress to further clarify the distinctions between cytotypes of S. giganteum. Breeding studies are underway to produce interspecific hybrids and determine the value of North American germplasm in sugarcane improvement.
= =
=
=
=
Acknowledgements This research was partially supported by a grant from the American Sugar Cane League. Y.-B. Pan was supported by a USDA-ARS post-doctoral research fellowship.
Notes 1
Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
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