Crop Science 43:1516-1522 (2003)
© 2003 Crop Science Society of America
PLANT GENETIC RESOURCES
Genetic Diversity in Diploid Cultivars of Rhodesgrass Determined on the Basis of Amplified Fragment Length Polymorphism Markers
Benjamin Ewa Ubia,
Roland Köllikerb,
Masahiro Fujimoric and
Toshinori Komatsu*,c
a Department of Crop Science, Faculty of Agriculture, University of Calabar, P.M.B. 1115-Calabar, Nigeria
b Federal Research Station for Agroecology and Agriculture, FAL-Reckenholz, 8046 Zurich, Switzerland
c Department of Plant Breeding, National Grassland Research Institute, Nishinasuno, Tochigi 329-2793, Japan
* Corresponding author (tkomatsu{at}affrc.go.jp)
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ABSTRACT
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Rhodesgrass (Chloris gayana Kunth) is a highly variable, perennial forage grass widely cultivated in all tropical and subtropical regions of the world. Despite its economic importance, there is a lack of information on the genetic diversity within and among rhodesgrass cultivars, which are all based on genetic resources initially introduced from East and South Africa. The objective of this study was to assess genetic diversity within and among 13 cultivars of diploid rhodesgrass and to determine whether recent breeding efforts have resulted in cultivars distinct from the African source populations. For each cultivar, 15 individuals were examined for 237 amplified fragment length polymorphism (AFLP) markers generated from three EcoRI/MseI primer pairs. Partition of the variation revealed that the major proportion of the total genetic variation occurred within cultivars and with only 12 to 13% attributed to geographical origin or breeding history. Cluster analysis revealed three distinct groups among the cultivars investigated. Group one consisted mainly of recent Japanese cultivars and their African source population, group two contained mostly African cultivars, and group three contained one African cultivar. However, the genetic diversity within recent Japanese cultivars was comparable to the diversity within old African cultivars and there was no evidence of a reduced genetic base because of breeding efforts.
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INTRODUCTION
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RHODESGRASS is an important forage grass which originated from East Africa and has been widely cultivated in the tropical and subtropical regions of the world. It is a highly variable, allogamous, perennial species consisting of many natural ecotypes which differ greatly from each other with respect to a range of morphological, agronomic and nutritional quality traits (Bogdan, 1969; Hutton, 1961; Nakagawa et al., 1987). Rhodesgrass consists of both diploid (2n = 2x = 20) and autotetraploid (2n = 4x = 40) forms which do not readily cross (Hutton, 1961; Nakagawa et al., 1987). However, cultivars within each ploidy group are interfertile and hybridize freely (Nakagawa et al., 1987).
Rhodesgrass is becoming increasingly popular because of its high seed production potential, the ease with which it can be established, and its ability to withstand dry conditions, soil salinity, and light frost (Pérez et al., 1999; Skerman and Riveros, 1990). The species is characterized by increased germination rate and improved seedling vigor when compared with other tropical forage grasses such as guinea grass (Panicum maximum Jacq.) (Nakagawa and Momonoki, 2000). To meet growers' needs, many cultivars improved for traits such as nutritive value, yield potential, and persistence have been developed mainly in Africa (Boonman, 1978), Australia (Lambert and Graham, 1996), and Japan (Nakagawa et al., 1993). All breeding efforts were based on genetic resources initially introduced from East and South Africa (Duke, 1978).
Despite its economic importance, little research has been undertaken on genetic characterization of rhodesgrass germplasm and cultivars. Information about genetic diversity and germplasm characterization is important for any breeding program. In particular, it is often useful to identify diverse parental combinations to create segregating progenies with genetic variability that would provide further gain from selection.
Molecular markers offer an efficient tool to investigate genetic diversity in plant populations. They have been successfully used to differentiate cultivars of outcrossing species, where the high level of diversity among individual plants can obscure among cultivar variability (Huff et al., 1993; Kölliker et al., 1998a; Xu et al., 1994). Among the various marker systems available, AFLP (Vos et al., 1995) markers are particularly useful since they allow genetic diversity to be assessed at a large number of loci without prior sequence knowledge, and data are obtained quickly and reproducibly (Barrett and Kidwell, 1998; Sanchez et al., 1999; Tohme et al., 1996). Markers (AFLP) have been successfully used to characterize cultivars and accessions of outbreeding forage species including perennial ryegrass (Lolium perenne L.) (Guthridge et al., 2001; Roldán-Ruiz et al., 2000) and white clover (Trifolium repens L.) (Kölliker et al., 2001).
In rhodesgrass, random amplified polymorphic DNA (RAPD) markers allowed a clear separation of cultivars assessed for salinity tolerance (Pérez et al., 1999) and AFLP markers demonstrated significant genetic variation among diploid as well as tetraploid individuals and cultivars (Ubi et al., 2000, 2001). Still, there is a general lack of information on genetic variability within and among the most important rhodesgrass cultivars and on the influence of breeding efforts on the genetic structure of rhodesgrass germplasm.
Analysis of molecular variance (AMOVA) partitions genetic variation among groups, populations, and individuals, and may provide additional information on the genetic structure of species and germplasm collections (Excoffier et al., 1992; Huff, 1997; Kölliker et al., 1998b; Mengoni et al., 2000). The objectives of this study were to assess genetic variability within and among populations of diploid rhodesgrass and to determine whether recent breeding efforts in Japan resulted in cultivars distinct from the African source cultivars.
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MATERIALS AND METHODS
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Plant Material
Thirteen diploid cultivars of rhodesgrass from the collection maintained at the National Grassland Research Institute and the Kyushu National Agricultural Experiment Station, Japan, were used for this study (Table 1). The cultivars were selected to include some of the most important rhodesgrass cultivars, local African ecotypes, and recent Japanese cultivars. They can be classified into two groups with respect to their origin (i.e., Africa and Japan), but were initially all derived from germplasm introduced from Africa (Duke, 1978). A more detailed description of cultivars can be found in Bogdan (1969), Jones et al. (1995), Nakagawa et al. (1993), and Skerman and Riveros (1990). The term cultivar will be used subsequently for all populations analyzed.
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Table 1. Origin and breeding history of rhodesgrass (Chloris gayana Kunth.) cultivars used for investigation of genetic diversity.
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Seeds of each cultivar were germinated in trays until they attained two or three leaves. Each individual seedling was transplanted into a single pot containing horticultural-compost growing medium in the greenhouse of the National Grassland Research Institute. Plants were watered as necessary and fertilized with fertilizer (N:P:K 17:17:17) at two weeks after transplanting. At 4 wk after transplanting, approximately 3 g of young leaf tissue was harvested and stored at –80°C until DNA isolation. The AFLP analysis of cultivars was performed on 15 randomly selected individual plants from each of the 13 cultivars.
AFLP Analysis
DNA was isolated according to a modified cetyltrimethylammonium bromide (CTAB) extraction procedure of Rogers and Bendich (1988). The DNA concentration was determined spectrophotometrically. Aliquots were then run on a 0.8% (w/v) agarose gel along with undigested lambda DNA standards to confirm the determined concentration and check for DNA quality. Approximately 500 ng of genomic DNA was diluted to a final volume of 5.5 µL with purified water (Milli-Q Labo, Millipore Corp., Bedford, MA). AFLP analysis was performed according to the AFLP plant mapping protocol of Applied Biosystems (Vos et al., 1995) with some minor modifications (Ubi et al., 2000). Three EcoRI and MseI selective primer pairs were used (Table 2).
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Table 2. AFLP primer combinations used for selective amplification and percentage of polymorphic markers detected within each of 13 rhodesgrass cultivars.
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Data Analysis
All individuals were scored using a binary code (1/0) for presence or absence of AFLP bands and the data were entered into a binary matrix. Only bands that could be unequivocally scored for presence or absence across all individuals were considered for further analysis. Genetic distance among individuals was based on squared Euclidean distance (Sneath and Sokal, 1973) using the formula
where k is the number of bands and xki and xkj are the frequencies of occurrence (0 or 1) of the kth band in individual i and individual j respectively. Analysis of molecular variance (WINAMOVA 1.55 software) (Excoffier et al., 1992) based on Euclidean distance, as described for RAPD markers by Huff (1997), was used to calculate variance components within and between cultivars and groups based on geographical origin or breeding history. Unweighted pair-group method based on arithmetic means (UPGMA) clustering of cultivars was based on modified coancestry coefficients (Reynolds et al., 1983) derived from AMOVA. The genetic distance between any two cultivars was represented by its modified coancestry value, derived from AMOVA. Bartlett's test for population heteroscedasticity was used to determine the significance of differences in within cultivar diversity. Phenograms were constructed by means of UPGMA of the NTSYS-pc software package (Version 2.10; Rohlf, 2002) and modified coancestry coefficients. Reliability of the clustering was tested by computing Mantel test statistics for the comparison of the similarity matrix and the cophenetic matrix (Rohlf, 2002). Principle coordinate analysis was performed on Euclidean distances among individual plants using NTSYS-pc. The initial binary data matrix was subjected to stepwise discriminant analysis to identify a subset of AFLP markers which were the best discriminating factors among the 13 rhodesgrass cultivars. Subsequently, canonical discriminant functions were calculated by means of the previously identified subset of AFLP markers. Discriminant analyses were performed by the procedures PROC STEPDISC (P = 0.15 for adding and retaining variables) and PROC CANDISC of the SAS v. 8.0 statistical package (SAS Institute, 1999).
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RESULTS
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Distribution of AFLP Markers
A total of 237 easily scorable bands were generated from three selective primer pairs. Only 17% of these markers were found in less than 20 individual plants, while the majority, 56%, were present in more than 100 plants (data not shown). Eighty-four percent of the 237 markers were polymorphic between two or more individual plants. The percentage of markers which were polymorphic within individual cultivars ranged from 30.9 for the cultivar Tochirakukei to 57.7 for Nagabokukei (Table 2). Two unique bands were found exclusively in some individuals of the cultivar Katambora, while six bands were found unique to three cultivars, Katambora, Gunsons, and Hatsunatsu. However, none of these bands were present in more than four individuals of each of these cultivars. Unique bands present in all the individuals of a particular cultivar but not present in any other individual of another cultivar (fixed marker difference) were not observed in this study, so characterization was based on marker frequency differences among cultivars.
Partition of Genetic Variation
Analysis of molecular variance was performed twice, using either geographic origin (Africa vs. Japan) or breeding history (cultivars based on Fords-Katambora vs. remaining cultivars) as grouping criteria. In both instances, AMOVA demonstrated highly significant (P < 0.001) genetic variance within cultivars as well as among cultivars (Table 3). The variance within cultivars accounted for 82% of the total variance when groups were based on breeding history and for 83% when groups were based on geographic origin, while the among population variance contributed only 16 and 18% (Table 3) for breeding history and origin, respectively. The variance component for variation among groups was negative for geographic origin, indicating the absence of genetic structure on this level (Schneider et al., 2000). The variance component for variance among groups based on breeding history was considerably larger (2%), but still not significant (Table 3).
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Table 3. Analysis of molecular variance (AMOVA) for 13 rhodesgrass cultivars based on 237 AFLP markers and 195 individual plants.
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Diversity within Cultivars
Genetic diversity within cultivars, expressed as AMOVA mean square deviations, was positively correlated (r = 0.95; P < 0.001) with the percentage of polymorphic markers detected per cultivar (Tables 2 and 4). The Bartlett's test for population heteroscedasticity was highly significant (Bp = 3.73; P < 0.001) indicating significantly different levels of variability within specific cultivars. The least variable cultivar examined was Tochirakukei, while Nagabokukei was the most variable (Tables 2 and 4).
Diversity between Cultivars
Genetic diversity, expressed as coancestry coefficients, among 13 diploid rhodesgrass cultivars ranged from 3.0 for the comparison of Nagabokukei and Fords-Katambora to 67.8 for the Katambora/Tochirakukei comparison (Table 5). Coancestry coefficients between Katambora and other cultivars ranged from 25 to 67.8 and were generally higher than coefficients between any other two cultivars. Clustering (UPGMA) based on coancestry coefficients clearly separated the 13 cultivars into three clusters (Fig. 1). Cluster one contained four Japanese cultivars as well as the African cultivars Fords-Katambora and Morocco common, and cluster two contained the remaining African cultivars as well as the Japanese cultivar Tochirakukei. The third cluster exclusively consisted of the old African cultivar Katambora, which was clearly separated from all other cultivars. Clustering of all 195 individual plants based on Euclidean distances clearly grouped all plants of Katambora in a distinct cluster but did not result in a clear separation of the remaining cultivars. Principle coordinate analysis based on Euclidean distances did not allow a clear distinction of the 13 cultivars but resulted in a moderate separation of individual plants according to the three clusters obtained by cluster analysis (Fig. 2). Stepwise discriminant analysis identified a subset of 84 AFLP markers as the best discriminating factors among the 13 cultivars. Canonical discriminant analysis based on these 84 AFLP markers resulted in a clear separation of the cultivars Katambora, Nzoia, Asatsuyu, Hatsunatsu and Morocco common (Fig. 3). The grouping of cultivars obtained by canonical discriminant analysis largely reflected the results obtained by cluster analysis (Fig. 1 and 3).
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Table 5. Genetic distance among 13 cultivars of diploid rhodesgrass based on 237 AFLP markers and 15 individual plants per cultivar.
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Fig. 1. UPGMA clustering of 13 rhodesgrass cultivars based on coancestry coefficients (Reynolds et al., 1983) derived from 237 AFLP markers and 15 individual plants per populations. Letters in parentheses refer to the geographic origin of the cultivars (A = Africa, J = Japan; Table 1).
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Fig. 2. Principle coordinate analysis of 195 rhodesgrass plants from 13 cultivars based on Euclidean distance derived from 237 AFLP markers.
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Fig. 3. Canonical discriminant analysis of 195 rhodesgrass plants from 13 cultivars based on 84 AFLP markers which differentiated best among the 13 cultivars as determined by stepwise discriminant analysis. White, gray, and black symbols mark plants from clusters I, II, and III, respectively (Fig. 2).
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DISCUSSION
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The high level of intra- and intercultivar diversity detected in this study could be largely due to the strong outcrossing mechanism in this species which is likely to increase the degree of polymorphism (Brummer et al., 1991; Caetano-Anolles, 1998). Although some few unique bands were found in some individuals of a particular cultivar, AFLP markers present in all individuals of one cultivar but absent in all the other cultivars were not detected. Such a lack of fixed marker differences has been reported in various species based on different marker systems (Brummer et al., 1991; Huff, 1997; Kölliker et al., 1998a; Mengoni et al., 2000). However, all 13 rhodesgrass cultivars were successfully separated on the basis of differences in marker frequencies.
AFLP analysis revealed considerable variation within each cultivar examined in this study. This observation suggests that these cultivars have potential for providing further gain from selection within a breeding program. Although small, nonsignificant differences exist between intracultivar diversity based on AMOVA mean square deviation and the percentage of polymorphic markers detected in each cultivar, the two measures of within cultivar genetic diversity estimates were almost identical for all cultivars. The highest level of intracultivar variability was exhibited by Nagabokukei, a cultivar derived from multiplications of several lines of Fords-Katambora. Although, the difference to Fords-Katambora, Kumabokukei and Morocco was small, it was not significant according to Bartlett's test for population heteroscedasticity (P > 0.05). On the other hand, the very low level of intracultivar variability in Tochirakukei is not surprising since this oldest Japanese cultivar was established from a limited number of selected clones.
In the present study, the partition of genetic variation by AMOVA showed that a substantial part of the variation (82–83%) in this set of rhodesgrass cultivars is attributable to within-cultivar variation, which was about eight times larger than the variability observed among cultivars (Table 3). This is a common observation in outbreeding species and similar values of within cultivar diversity have been found in other forage grasses such as perennial ryegrass, meadow fescue (Festuca pratensis Huds.) and cocksfoot (Dactylis glomerata L.) (Huff, 1997; Kölliker et al., 1998a,b).
Although geographical origin (Japanese vs. African cultivars) and breeding history (cultivars derived from Fords-Katambora vs. remaining cultivars) did not significantly contribute to the variation among cultivars according to AMOVA, cluster analysis showed a distinct influence of these factors. Cluster I (Fig. 1) contained the old African cultivars Morocco common and Fords-Katambora as well as four Japanese cultivars which were all derived from the latter African source population, while cluster II consisted almost exclusively of African cultivars. Principle coordinate analysis based on individual plants indicated a similar, although not as clear, separation of the cultivars, while canonical discriminant analysis almost completely reflected the grouping of cultivars obtained by cluster analysis.
The clear separation of Katambora as observed in the present study on the basis of AFLP markers was also reflected in morphological and physiological traits such as growth habit, time of flowering and yield potential, where large differences were observed between Katambora and the other 12 cultivars (Komatsu, 2000, unpublished data). Moreover, Katambora was also found to be genetically distinct from other diploid cultivars based on RAPD markers (Pérez et al., 1999). The two Japanese cultivars Nagabokukei and Kumabokukei were uniquely based on clones of Fords-Katambora, while Asatsuyu is a synthetic cultivar based on clones of Fords-Katambora and Gunson. The results of the present AFLP analysis confirm this breeding history. Tochirakukei, which appears in cluster two with most African cultivars (Fig. 1), is the oldest Japanese cultivar and shares similar characteristics with Pioneer (Komatsu, 2000, unpublished data). On the basis of morphological traits, Nakagawa et al. (1987) found Pioneer to be more similar to Gunsons than to Katambora or Fords-Katambora. This, together with the close clustering based on AFLP markers, is a strong indication that Tochirakukei was initially bred from Pioneer, an old cultivar which was widely cultivated in Japan and other parts of the world.
In conclusion, a general restriction of genetic diversity was not found in any of the cultivars investigated because of recent breeding efforts. Although rhodesgrass cultivation has an old history in Japan, the species has not undergone intensive selection and intensive breeding efforts started as recently as 1979. This may explain the close relationship of the Japanese cultivars and the African source populations. However, this study clearly demonstrates the existence of three distinct rhodesgrass genepools among the cultivars investigated. This information may assist plant breeders in their decisions of what germplasm to include in their breeding program. To broaden the existing breeding germplasm, it may be useful to include cultivars or ecotypes from outside the Fords-Katambora complex. On the other hand, the genetic diversity within modern Japanese cultivars was comparable to the genetic diversity in old African cultivars. Therefore, no negative effects due to self-incompatibility and inbreeding expression are to be expected by further moderate selection within existing cultivars.
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NOTES
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This research is part of a project supported by the Japan International Science and Technology Exchange Center (JISTEC) through the award of the Science and Technology Agency Postdoctoral Fellowship to B. Ubi and was performed at the NGRI, Nishinasuno.
Received for publication June 10, 2002.
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TOP
ABSTRACT
INTRODUCTION
