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Genetic analyses of Chinese Cynodon accessions by flow cytometry and AFLP markers.

GRASSES belonging to the genus Cynodon L. C. Rich. occur in relative abundance in the southern half of China and are sparsely distributed in northern warm temperate humid regions (Anonymous, 1990; Abulaiti et al., 1998). Three Cynodon taxa, C. arcuatus J.S. Presl ex C.B. Presl, C. dactylon (L.) Pers. var. dactylon, and C. dactylon var. biflorus Merino, are listed in Chinese Floral Acta (Anonymous, 1990). The taxonomic revision of Cynodon by J.R. Harlan and colleagues included nine species and 10 varieties, but did not include C. dactylon var. biflorus (Harlan et al., 1970a; de Wet and Harlan, 1970). Harlan et al. (1970a) provided a description of C. arcuatus and indicated that it was easily distinguished and genetically isolated from other Cynodon species. C. dactylon var. dactylon is the most widely distributed and genetically variable of all Cynodon species (Harlan and de Wet, 1969; Harlan et al., 1970a). It is found on all continents and larger ocean islands between about 45[degrees] N and S latitudes. Its uses as livestock fodder, turf, and soil stabilization and remediation make it the most economically important of the Cynodon species (Burton, 1947; Harlan, 1970; Harlan et al., 1970b; Taliaferro, 1995, 2003).

Although the widespread occurrence of Cynodon in China is well documented, there is little information on kinds and magnitudes of genetic variation within the indigenous Chinese Cynodon germplasm pool. In 1974, G.W. Burton collected an unusually dark green C. dactylon plant from a Shanghai lawn that was later determined to be a hexaploid (2n = 6x = 54 chromosomes) and released as 'Tifton 10' (Hanna et al., 1990). In addition to Tiftonl0, only a few hexaploid Cynodon plants have been reported (Hurcombe, 1947; Moffett and Hurcombe, 1949; Powell et al., 1968; Felder, 1967; Malik and Tripathi, 1968; Johnston, 1975). The basic chromosome number of Cynodon is nine and the predominant cytotypes are diploid (2n = 2x = 18) and tetraploid (2n = 4x = 36) (Forbes and Burton, 1963; Harlan et al., 1970c; de Wet and Harlan, 1971; de Silva and Snaydon, 1995). Triploid (2n = 3x = 27) Cynodon plants from intra- and interspecific natural and artificial hybridizations are relatively common. Rare pentaploid plants have been reported from interspecific artificial hybridization (Johnston, 1975; Burton et al., 1993).

The PCR-based DNA fingerprinting techniques based on the analysis of information-rich nucleic acid molecules have been used in studies of genetic diversity, relatedness, phylogeny, and in identifying off-types of cultivars in Cynodon (Caetano-Anolles, 1998a). A number of researchers have employed randomly amplified polymorphic DNA (RAPD) (Roodt et al., 2002) and DNA amplified fingerprinting (DAF) procedures (Caetano-Anolles et al., 1995; Caetano-Anolles et al., 1997; Ho et al., 1997; Assefa et al., 1998; Anderson et al., 2001). Recently, AFLP (Vos et al., 1995) has been used in Cynodon to differentiate bermudagrass genotypes (Zhang et al., 1999), to detect the genetic diversity among forage bermudagrass cultivars (Karaca et al., 2002), and to quantify the genetic variation of C. transvaalensis and its relatedness to hexaploid C. dactylon (Wu et al., 2005).

The objectives of this study were to (i) determine the ploidy of 132 Cynodon accessions collected in 11 provinces of China, and (ii) quantify the genetic variation and relatedness of the accessions within and among different ploidy levels based on AFLP markers.

MATERIALS AND METHODS

Plant Materials

Plant materials consisted of 136 Cynodon clonal accessions including 132 accessions collected in China and four U.S. commercial cultivars with known chromosome number and nuclear DNA content (Table 1). The Chinese Cynodon accessions were collected from 11 provinces ranging from tropical Hainan Island to the temperate climatic region around Beijing (Fig. 1). All indigenous Chinese accessions were determined to be C. dactylon based on morphological characteristics, as described by Harlan et al. (1970a) and Clayton et al. (2005). Distinguishing characteristics of C. dactylon include racemes arranged in one whorl on inflorescences and subequal glumes at least 3/4 the length of spikelets.

[FIGURE 1 OMITTED]

The plants were grown in a greenhouse at the Agronomy Research Station, Oklahoma State University, Stillwater, OK, in 15-cm-diam. pots containing Metro-Mix 250 growing medium (Scotts-Sierra Horticultural Products Co., Marysville, OH). They were watered daily and fertilized biweekly with M77 Peat-rite Special water-soluble fertilizer (Scotts-Sierra Horticultural Products Co., Marysville, OH). All plants were actively growing and healthy at the time of the sampling.

Flow Cytometry

Flow cytometry procedures described by Taliaferro et al. (1997b) were used to measure nuclear DNA content on the 136 accessions. Flow cytometry analyses of prepared materials were conducted on a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA) with an argon laser emitting at 488 nm for excitation of propidium iodide at the Flow Cytometry Laboratory of Oklahoma State University. The mean nuclear DNA content of each plant sample, measured in picograms, was based on 5000 scanned nuclei. Channel catfish (Ictalurus punctatus) blood cells with a nuclear DNA content of 5.67 pg/ 2C were used as internal standards (Taliaferro et al., 1997b). For every plant sample, at least three measurements (replicates) were obtained in three different days. Sample DNA content was calculated by dividing the mean value of sample channel by the mean value of internal standard channel, then multiplying the result by the DNA content of the internal standard. The ploidy levels of the Chinese accessions were inferred by comparing sample DNA content to the previously reported range of respective ploidy levels (Taliaferro et al., 1997b; Arumuganathan et al., 1999).

Cytology

Somatic chromosome number was determined for all accessions having DNA content values outside the reported range for respective ploidy levels (Taliaferro et al., 1997b; Arumuganathan et al., 1999). Additionally, somatic chromosome number was determined for selected accessions having DNA content within the reported range to confirm the accuracy of the flow cytometry data. Somatic chromosome counts were determined using conventional squashes of shoot tip somatic cells under a light microscope equipped with a digital camera system. The somatic cell squashes were prepared from shoot apical meristem tissues as described by Powell (1968) with minor modifications. Briefly, actively growing shoot tips were stripped, and the stripped shoot tips were cut longitudinally to expose the apical meristem. As a pretreatment to shorten chromosomes, the sprit shoot tips were placed into a saturated solution of monobromonaphthalene for 2 h at ambient room temperature. Then the pretreated shoot tips were fixed in 3:1 ethanol-acetic acid solution for 24 h at room temperature. The fixed shoot tips were washed in flowing deionized water for 10 min, then digested in cytolase (PCL5, DSM Food Specialties, Charlotte, NC) solution at 37[degrees]C for 40 to 70 min. Chromosomes were stained with acetocarmine.

AFLP und Marker Data Analysis

In AFLP analysis, 119 Chinese Cynodon accessions plus Tifway, Tifgreen, and Tifton 10 (see Table 1 and its footnotes) were used, while the other 13 Chinese accessions were not included because of a space limitation in gel electrophoresis. DNA samples were isolated from fresh leaf tissues of the Cynodon plants with DNeasy plant mini kit from QIAGEN, Inc. (Valencia, CA). The AFLP analysis was performed as described by Vos et al. (1995) with a few minor modifications (Bai et al., 1999). The AFLP procedure details followed Wu et al. (2005).

The AFLP electrophoresis bands were visually scored twice as present (1), absent (0), or ambiguous (9) for each accession. Data of polymorphic bands were compiled for each Cynodon accession in a data matrix and analyzed using NTSYS-pc (Numerical Taxonomy System) v. 2.0 (Exeter Software, Setauket, NY). Genetic similarity coefficients of pair-wise comparisons among the Cynodon accessions were computed as simple matching coefficients within the SIMQUAL module. Polymorphic information content (PIC) indicating the ability to distinguish between genotypes for each primer combination was calculated as expected heterozygosity for polymorphic bands following Powell et al. (1996). Cluster analysis was performed according to the unweighted pair group mean algorithm (UPGMA) within the SAHN module of the NTSYS-pc program. Bootstrap analysis (250 replications) of the data matrix was performed to test the clusters robustness with FreeTree program according to the program manual (Hampl et al., 2001). A principal coordinate analysis to construct a two-dimensional array of eigenvectors was performed using the DCENTER module of the NTSYS-pc program. Comparisons of the average GSCs among three ploidy levels (4x, 5x, and 6x) of the Chinese Cynodon accessions were performed using the program MANTEL-STRUCT with an asymptotic solution (Miller, 1999).

RESULTS

Ploidy Determination

The nuclear DNA contents of the 132 Chinese Cynodon accessions and four commercial cultivars are presented in Table 1. The standard deviations of DNA content measurements ranged from 0.01 to 0.08 pg/2C [nucleus.sup.-1], demonstrating that flow cytometry was very precise. These results are consistent with previous reports regarding the precision of flow cytometry in Cynodon plants (Taliaferro et al., 1997b; Arumuganathan et al., 1999). The DNA contents of Tifton 10, Tifway, Tifgreen, and Uganda, measured along with the Chinese Cynodon accessions to verify the results, were consistent with the results reported by Taliaferro et al. (1997b) and Arumuganathan et al. (1999).

Nuclear genome size among the 132 Chinese accessions ranged from 1.55 to 3.15 pg/2C [nucleus.sup.-1] (Table 1). On the basis of previously reported data (Taliaferro et al., 1997b; Arumuganathan et al., 1999), diploid, triploid, tetraploid, and hexaploid cytotypes were indicated by respective nuclear genome sizes of 1.03 to 1.14, 1.37 to 1.62, 1.95 to 2.36, and 2.64 to 2.93 pg DNA [2C.sup.-1] per nucleus. On the basis of these ranges, four accessions were classified as triploid, 116 accessions as tetraploid, and one accession as hexaploid (see inferred ploidy in Table 1).

Eleven accessions had genome sizes outside the previously reported ranges. Somatic chromosome counts determined for these 11 accessions indicated two triploid (A12256 and A12260), three pentaploids (A12348, A12352, and A12365), and six hexaploids (A12317, A12318, A12319, A12320, A12356, and A12358) (Table 1 and Fig. 2). Somatic chromosome counts for A12272, A12282, A12257, and A12360 confirmed that the three inferred ploidy levels based on nuclear DNA contents were correct (Table 1).

AFLP Genetic Diversity and Relatedness

Thirteen AFLP selective amplification primer combinations produced a total of 763 bands among the 122 Cynodon genotypes, with an average of 58.7 [+ or -] 11.4 bands per primer combination (Table 2). Of the 763 bands scored, 466 (61.1%) were polymorphic, with an average of 35.9 [+ or -] 12.4 polymorphic bands per primer combination. The primer combinations eAGT/mCAC and eGCT/mCAG amplified the largest (61) and smallest (35) numbers of total bands and polymorphic bands per gel, respectively (Table 2). The average PIC of the 13 primer combinations was 0.21, ranging from 0.15 (eACT/mCAG and eACT/mGAC) to 0.29 (eAGT/ mCAT) (Table 2).

The genetic diversity was relatively high among the accessions in this study. The GSCs among the 122 accessions ranged from 0.55 to 0.99. The lowest GSC (0.55) was between Tifgreen and 12351, and Tifgreen and 12261. Accessions 12351 and 12261 were from China. The highest GSC was 0.99, detected between accessions 12279 and 12280. The two accessions were collected from very close sites in Aba, Sichuan Province of China.

Cluster analysis based on the GSC separated the 122 Cynodon accessions into five distinct major groups having bootstrap values of 81% or more: A, B, C, D, and E (Fig. 3). The variation patterns and groupings of the Cynodon accessions appear to be associated with their ploidy level and geographic origin. Cluster A contains Tifgreen and two triploid accessions (Fig. 3). The GSC among the three accessions ranged from 0.97 to 0.99, indicating that they are genetically very similar. Cluster B consists of Tifway, 12260, 12272, and 12282, with GSC ranging from 0.97 to 0.99 and averaging 0.98 [+ or -] 0.01. Cluster C contains only accessions 12261 and 12257. Interestingly, accession 12261 is a triploid collected from Neijiang, Sichuan. Accession 12257 is a tetraploid from Tongjiaxi, Chongqing. The genetic similarity of pair-wise comparison between the two accessions is 0.92.

[FIGURE 3 OMITTED]

Cluster D contains 109 accessions making it the largest group. The cluster contains the majority of the tetraploid cytotypes and all of the hexaploid cytotypes of the Chinese accessions (Fig. 3). The GSC in the cluster averaged 0.82 [+ or -] 0.05, and ranged from 0.69 to 0.99. The eight hexaploid cytotypes were separated into three subgroups. One subgroup contained 12317, 12318, 12320, 12356, and 12358, with a bootstrap value of 100%. Accessions 12317 and 12318 were collected from Shanghai, and 12356 and 12358 from Zhejiang and Jiangsu, respectively. Field notes indicated that accession 12320 was a fine-textured plant from Hongya, Sichuan, but the plant used in this study had a relatively coarse texture, suggesting that it was not the true 12320 accession. Mechanical mixture of the two accessions may have occurred because the original nursery plots of the two accessions were adjacent to each other. The GSC between 12318 and 12320 was 0.99, further suggesting that the accession identified as 12320 in this study was a contaminant. The other three hexaploid genotypes (Tifton10, 12319, and 12360) clustered into two subgroups. Tifton10 and 12319 were from Shanghai, and 12360 from Jiangsu. In cluster D, 101 of the tetraploid genotypes scatter into various subgroupings (Fig. 3). Collectively, the accessions from the same geographic locations or nearby regions tended to have higher genetic similarity and to cluster into the same subgroups or neighboring subgroups. As shown in Fig. 3, most accessions that originated in the western provinces, such as Sichuan, Chongqing, and Yunnan, were included in the same subgroupings or in neighboring subgroupings, and most accessions from eastern and southern provinces, including Shanghai, Jiangsu, Zhejiang, Fujian, Guangdong, and Hainan, tended to group into the same or adjacent groupings. However, seven accessions (12364, 12357, 12361, 12363, 12354, 12367, and 12359) from eastern and southern regions formed subgroupings with accessions from west regions, while two accessions (12344 and 12258) from western regions formed subgroupings closer to groupings containing predominantly eastern and southern origin accessions.

Cluster E contains one tetraploid and three pentaploid accessions (Fig. 3). Two pentaploid accessions 12348 and 12352 and the tetraploid accession 12351 were collected from Xinlong, Wanquanhe, and Haikou in Hainan Island. Another pentaploid accession 12365 originated in Minghou, Fujian. Among the pentaploid genotypes, GSCs averaged 0.96 [+ or -] 0.01, ranging from 0.95 to 0.98, indicating very low genetic diversity among the pentaploid accessions.

Principal coordinate analysis clearly indicated that Tifgreen and Tifway, and the accessions clustering with them, were widely separated from the Chinese accessions (Fig. 4). Both Tifgreen and Tifway are interspecific [F.sub.1] hybrids from tetraploid C. dactylon by diploid C. transvaalensis crosses with parents of African origin. Among the Chinese accessions, the pentaploid genotypes separated from tetraploid and hexaploid accessions (Fig. 4). However, the hexaploid accessions were grouped closer to or together with the tetraploid accessions from closer geographic regions. Mantel tests of the average GSCs (Table 3) among the ploidy levels further demonstrated that the genetic distances among the tetraploidy, pentaploidy, and hexaploidy are significant (P < 0.01). Within ploidy levels, the range of GSCs and the average GSC of tetraploids were smaller than those of the hexaploids and pentaploids, showing that the Chinese tetraploid Cynodon contained much wider genetic diversity compared with the pentaploid and hexaploid cytotypes.

[FIGURE 4 OMITTED]

DISCUSSION

Among the 132 accessions used in this study, the 116 tetraploid Cynodon accessions (87.9%) originated from 11 provincial regions, clearly indicating this to be the predominant cytotype over the geographic expanse represented by the collection. In three western provinces, 96 of 101 accessions were tetraploids. In the other eight eastern and southern provincial regions, 20 of the 31 accessions were tetraploids. The wide distribution of the tetraploid cytotype in China is consistent with previous reports (Harlan and de Wet, 1969; Harlan et al., 1970a) that tetraploid C. dactylon var. dactylon is truly cosmopolitan. Although the tetraploid accessions were collected from two distinct geographic regions in China (Fig. 1), discrete genetic differentiation within a region is not observed and genetic overlap existed between the two separate regions indicated by AFLP markers in the dendrogram (Fig. 3) and principal coordinate plot (Fig. 4). This might be explained by the open pollination behavior of Cynodon plants. The predominant mode of sexual reproduction in C. dactylon is outcrossing due to cross-pollination and self-incompatibility (Burton, 1947, 1965; Taliaferro and Lamle, 1997a). Cross-pollination results in gene flow between natural populations, which probably prevents formation of distinctly differentiated genetic groups. We believe that genetic variation and differentiation patterns of the Chinese tetraploid Cynodon germplasm pool in the main distribution regions is of a gradual mode for tetraploid forms without apparent genetic barriers.

The pentaploid and hexaploid accessions are considered to belong to the taxon C. dactylon as described by Harlan and de Wet (1969) and Harlan et al. (1970a). The pentaploid and hexaploid accessions are included in C. dactylon based on similarity of morphological features compared with tetraploid accessions. The descriptions of C. dactylon var. dactylon by Harlan and de Wet (1969) and Harlan et al. (1970a) indicated the species contained only the tetraploid cytotype. Our AFLP marker data basically supported the morphological classification. A dendrogram from the cluster analysis (Fig. 3) showed that hexaploid and pentaploid accessions formed clusters or subclusters in mixture with or close to tetraploid accessions that originated in the same geographic region as the pentaploids and hexaploids. The spread of tetraploid, pentaploid, and hexaploid forms in a two-dimensional plot of principal coordinate analysis (Fig. 4), in which principal coordinates 1 (PC1) and 2 (PC2) accounted for 10.4% and 7.2% of total variation, respectively, is basically consistent with the dendrogram (Fig. 3). The close genetic relatedness of the three different cytotypes indicates they have a recent common ancestry. Hexaploidy and pentaploidy are rare in Cynodon. Powell et al. (1968) reported a hexaploid clone from a cross of tetraploid x diploid parents, and speculated that doubling of chromosome number at an early zygote stage probably accounted for its occurrence. Another hexaploid plant was identified in the progeny of a self-pollinated plant of tetraploid C. dactylon (Felder, 1967). Felder hypothesized that the autohexaploid plant resulted from the union of an unreduced female gamete and a reduced male gamete. Accordingly, the origin of the Chinese hexaploid forms most likely resulted from the union of reduced and unreduced gametes of tetraploid parents. The pentaploid forms probably originated from the union of one normal gamete from a tetraploid parent with another normal gamete from a hexaploid parent.

This is the first report of pentaploid Cynodon plants occurring naturally. The pentaploids were collected in subtropical environments, two from Hainan Island and the third from Fujian province. We are aware of only two previous reports of pentaploid Cynodon plants. Johnston (1975) found three pentaploid plants among progeny from a hexaploid plant (derived from interspecific hybridization of C. barberi and C. dactylon) crossed with a tetraploid C. dactylon plant. Burton et al. (1993) reported 'Tifton 85', an [F.sub.1] interspecific hybrid from a C. dactylon x C. nlemfuensis cross, to be a pentaploid.

Among the six triploids, five were morphologically similar to hybrids from C. dactylon var. dactylon by C. transvaalensis crosses because of short and slender racemes, two to four per inflorescence, fine leaves and stolons, low growing and very dense sod, while one (A12261) was close to C. dactylon var. dactylon. The five triploids were probably introduced plants based on their morphological and genetic similarity to Tifgreen and Tifway and their dissimilarity to the Chinese tetraploid accessions. Turf type triploid Cynodon cultivars developed in the USA have been introduced and widely used in China (Wu et al., 2001). The morphological traits of accessions 12256 and 12369 grown in greenhouse and field plots were very similar, but different from Tifgreen in leaf length and sod density (data not reported). Accessions 12256 and 12369 probably were cultivars introduced to China and may be mutational variants of Tifgreen or 'Tifdwarf'. Caetano-Anolles (1998b) reported the genetic instability of Tifgreen and Tifdwarf detected by DAF and ASAP (arbitrary signatures from amplification profiles) analysis. Off-types of Tifgreen were phenotypically distinct from the wild type in leaf texture, length, and color, stolon internode length, and other morphological characteristics, but genetically close to each other (Caetano-Anolles, 1998b). Accessions 12260, 12272, and 12282 were morphologically uniform, but distinct from Tifway in foliage color and developmental characteristics (data not shown), particularly in the late part of a growing season. Tifway tended to have darker green color and fewer inflorescences than the other three accessions in the fall (data not reported). Caetano-Anolles et al. (1997) indicated that Tifway was genetically stable; thus, it is less likely that the three Chinese accessions are mutational variants of Tifway.

CONCLUSIONS

Tetraploid C. dactylon is prevalent among Cynodon germplasm indigenous to the 11 provinces of China represented in the collection studied. Southeast China may represent a unique geographic area relative to the frequency of occurrence of hexaploid and pentaploid Cynodon dactylon cytotypes. Genetic differentiation among the three cytotypes is distinct based on AFLP markers. Though a more comprehensive collection is needed to elucidate genetic variation within Cynodon indigenous to China, this study demonstrates that substantial genetic variation exists within Chinese C. dactylon germplasm that might contribute to genetic improvement of the species.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Mr. T. Colberg of Oklahoma State University Flow Cytometry Laboratory for his technical support in the investigation. We thank several anonymous reviewers for their constructive comments and useful suggestions.

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Zhang, L.H., P. Ozias-Akins, G. Kochert, S. Kresovich, R. Dean, and W. Hanna. 1999. Differentiation of bermudagrass (Cynodon spp.) genotypes by AFLP analyses. Theor. Appl. Genet. 98:895-902.

Abbreviations: AFLP, amplified fragment length polymorphism; DAF, DNA amplified fingerprinting; GSC, genetic similarity coefficient; PIC, polymorphic information content; UPGMA, unweighted pair group mean algorithm.

Y. Q. Wu, * C. M. Taliaferro, G. H. Bai, D. L. Martin, J. A. Anderson, M. P. Anderson, and R. M. Edwards

Y.Q. Wu, USDA-ARS, Plant Science Research Lab., 1301 N. Western Rd., Stillwater, OK 74075-2714; C.M. Taliaferro, M.P. Anderson, and R.M. Edwards, Dep. of Plant and Soil Sci., Oklahoma State Univ., Stillwater, OK 74078; D.L. Martin and J.A. Anderson, Dep. of Horticulture and Landscape Architecture, Oklahoma State Univ., Stillwater, OK 74078; G.H. Bai, Plant Science and Entomology Research Unit, USDA-ARS and Dep. of Agronomy, Kansas State Univ., Manhattan, KS 66506. This research was accomplished at Oklahoma State University. Received 16 Aug. 2005. * Corresponding author (yanqi.wu@okstate.edu).
Table 1. Identification, source, nuclear DNA content, and
inferred ploidy of 132 Chinese Cynodon accessions and four
commercial Cynodon cultivars.

  Identification       Origin or        DNA content         Inferred
                       reference      mean [+ or -] SD     ploidy (2n)

                                           pg/2C

A12253                Sichuan        2.15 [+ or -] 0.07   36
A12254                Sichuan        2.05 [+ or -] 0.06   36
A12255                Sichuan        2.04 [+ or -] 0.03   36
A12256                Shanghai       1.64 [+ or -] 0.03   27 ([dagger])
A12257                Chongqing      1.96 [+ or -] 0.07   36
A12258                Chongqing      2.09 [+ or -] 0.04   36
A12259                Sichuan        2.01 [+ or -] 0.04   36
A12260                Sichuan        1.65 [+ or -] 0.01   27 ([dagger])
A12261                Sichuan        1.55 [+ or -] 0.01   27
A12262                Yunnan         2.09 [+ or -] 0.03   36
A12263                Yunnan         2.03 [+ or -] 0.05   36
A12264                Sichuan        2.07 [+ or -] 0.01   36
A12265                Sichuan        2.02 [+ or -] 0.02   36
A12266                Sichuan        2.17 [+ or -] 0.04   36
A12267                Sichuan        2.10 [+ or -] 0.01   36
A12268                Sichuan        2.07 [+ or -] 0.04   36
A12269                Sichuan        2.05 [+ or -] 0.02   36
A12270                Sichuan        2.03 [+ or -] 0.04   36
A12271                Sichuan        2.05 [+ or -] 0.03   36
A12272                Sichuan        1.61 [+ or -] 0.01   27 ([dagger])
A12273                Sichuan        2.09 [+ or -] 0.06   36
A12274                Sichuan        2.11 [+ or -] 0.01   36
A12275                Sichuan        1.99 [+ or -] 0.04   36
A12276                Sichuan        2.22 [+ or -] 0.05   36
A12277                Sichuan        2.02 [+ or -] 0.02   36
Al2278                Sichuan        2.01 [+ or -] 0.07   36
A12279                Sichuan        2.06 [+ or -] 0.04   36
A12280                Sichuan        2.03 [+ or -] 0.05   36
A12281                Sichuan        2.20 [+ or -] 0.03   36
A12282                Sichuan        1.55 [+ or -] 0.03   27 ([dagger])
A12283                Sichuan        2.15 [+ or -] 0.04   36
A12284                Sichuan        2.12 [+ or -] 0.04   36
A12285                Sichuan        2.08 [+ or -] 0.07   36
A12286                Sichuan        2.03 [+ or -] 0.05   36
A12287                Sichuan        2.11 [+ or -] 0.05   36
A12288                Sichuan        2.00 [+ or -] 0.06   36
A12289                Sichuan        2.10 [+ or -] 0.04   36
A12290                Sichuan        2.08 [+ or -] 0.06   36
A12291                Sichuan        2.11 [+ or -] 0.05   36
A12292                Chongqing      2.04 [+ or -] 0.06   36
A12293                Chongqing      2.10 [+ or -] 0.05   36
A12294                Sichuan        2.12 [+ or -] 0.05   36
A12295                Sichuan        2.11 [+ or -] 0.04   36
A12337                Sichuan        2.05 [+ or -] 0.05   36
A12338                Sichuan        2.10 [+ or -] 0.05   36
A12339                Sichuan        2.30 [+ or -] 0.03   36
A12340                Sichuan        2.14 [+ or -] 0.06   36
A12341                Sichuan        2.01 [+ or -] 0.05   36
A12342                Sichuan        2.06 [+ or -] 0.06   36
A12343                Sichuan        2.17 [+ or -] 0.07   36
A12344                Sichuan        2.05 [+ or -] 0.02   36
A12345                Sichuan        2.12 [+ or -] 0.02   36
A12346                Sichuan        2.12 [+ or -] 0.01   36
A12347                Sichuan        2.12 [+ or -] 0.03   36
A12348                Hainan         2.45 [+ or -] 0.04   45 ([dagger])
A12349                Hainan         2.16 [+ or -] 0.07   36
A12350                Guangdong      2.14 [+ or -] 0.07   36
A12351                Hainan         2.08 [+ or -] 0.02   36
A12352                Hainan         2.37 [+ or -] 0.02   45 ([dagger])
A12353                Guangdong      2.04 [+ or -] 0.05   36
A12354                Guangdong      2.03 [+ or -] 0.03   36
A12355                Zhejiang       2.13 [+ or -] 0.06   36
A12356                Zhejiang       3.13 [+ or -] 0.05   54 ([dagger])
A12357                Jiangsu        2.02 [+ or -] 0.07   36
A12358                Jiangsu        2.98 [+ or -] 0.05   54 ([dagger])
A12359                Jiangsu        2.02 [+ or -] 0.06   36
A12360                Jiangsu        2.90 [+ or -] 0.08   54 ([dagger])
A12361                Jiangsu        2.01 [+ or -] 0.07   36
A12296                Sichuan        2.13 [+ or -] 0.05   36
A12297                Sichuan        2.08 [+ or -] 0.02   36
A12298                Chongqing      2.06 [+ or -] 0.06   36
A12299                Chongqing      2.22 [+ or -] 0.05   36
A12300                Sichuan        2.04 [+ or -] 0.07   36
A12301                Sichuan        2.05 [+ or -] 0.04   36
A12302                Sichuan        2.02 [+ or -] 0.05   36
A12303                Sichuan        2.04 [+ or -] 0.04   36
A12304                Sichuan        2.09 [+ or -] 0.05   36
A12305                Sichuan        2.08 [+ or -] 0.04   36
A12306                Sichuan        2.05 [+ or -] 0.03   36
A12307                Sichuan        2.13 [+ or -] 0.06   36
A12308                Sichuan        2.07 [+ or -] 0.05   36
A12309                Sichuan        2.12 [+ or -] 0.02   36
A12310                Sichuan        2.07 [+ or -] 0.03   36
A12311                Sichuan        2.13 [+ or -] 0.05   36
A12312                Sichuan        2.08 [+ or -] 0.05   36
A12313                Sichuan        2.04 [+ or -] 0.05   36
A12314                Chongqing      2.06 [+ or -] 0.06   36
A12315                Shanghai       2.15 [+ or -] 0.06   36
A12316                Shanghai       2.09 [+ or -] 0.03   36
A12317                Shanghai       3.12 [+ or -] 0.05   54 ([dagger])
A12318                Shanghai       2.99 [+ or -] 0.05   54 ([dagger])
A12319                Shanghai       3.08 [+ or -] 0.06   54 ([dagger])
A12320                Sichuan        3.15 [+ or -] 0.04   54 ([dagger])
A12321                Sichuan        2.03 [+ or -] 0.05   36
A12322                Sichuan        2.17 [+ or -] 0.04   36
A12323                Sichuan        2.00 [+ or -] 0.04   36
A12324                Chongqing      2.08 [+ or -] 0.07   36
A12325                Sichuan        2.15 [+ or -] 0.02   36
A12326                Yunnan         1.99 [+ or -] 0.06   36
A12327                Sichuan        2.02 [+ or -] 0.06   36
A12328                Sichuan        2.07 [+ or -] 0.08   36
A12329                Sichuan        2.26 [+ or -] 0.03   36
A12330                Sichuan        2.07 [+ or -] 0.05   36
A12331                Sichuan        2.07 [+ or -] 0.07   36
A12332                Sichuan        2.06 [+ or -] 0.06   36
A12333                Sichuan        2.00 [+ or -] 0.05   36
A12334                Sichuan        2.21 [+ or -] 0.02   36
A12335                Sichuan        2.09 [+ or -] 0.05   36
A12336                Sichuan        2.05 [+ or -] 0.03   36
A12362                Fujian         2.03 [+ or -] 0.06   36
A12363                Jiangsu        2.02 [+ or -] 0.06   36
A12364                Zhejiang       2.07 [+ or -] 0.05   36
A12365                Fujian         2.49 [+ or -] 0.03   45 ([dagger])
A12366                Fujian         2.12 [+ or -] 0.04   36
A12367                Shandong       2.09 [+ or -] 0.07   36
A12368                Beijing        2.01 [+ or -] 0.01   36
A12369                Hainan         1.62 [+ or -] 0.02   27
A12370                Hainan         2.15 [+ or -] 0.05   36
  ([double dagger])
A12371                Hainan         2.10 [+ or -] 0.05   36
A12372                Guangdong      2.00 [+ or -] 0.02   36
93-138                Ymman          2.02 [+ or -] 0.06   36
  ([double dagger])
93-139                Yunnan         2.05 [+ or -] 0.06   36
  ([double dagger])
93-140                Yunnan         2.05 [+ or -] 0.06   36
  ([double dagger])
93-141                Yunnan         2.04 [+ or -] 0.02   36
  ([double dagger])
93-142                Yunnan         2.11 [+ or -] 0.05   36
  ([double dagger])
93-143                Yunnan         2.04 [+ or -] 0.04   36
  ([double dagger])
93-144                Yunnan         2.02 [+ or -] 0.06   36
  ([double dagger])
93-145                Yunnan         2.05 [+ or -] 0.03   36
  ([double dagger])
93-146                Yunnan         2.25 [+ or -] 0.05   36
  ([double dagger])
93-147                Yunnan         2.03 [+ or -] 0.05   36
  ([double dagger])
93-148                Yunnan         2.10 [+ or -] 0.05   36
  ([double dagger])
93-149                Yunnan         2.08 [+ or -] 0.03   36
  ([double dagger])
Tifton 10             Hanna et       2.90 [+ or -] 0.08   54
                        a1., 1990
Tifgreen              Hanson, 1972   1.61 [+ or -] 0.03   27
Uganda                Hanson, 1972   1.05 [+ or -] 0.02   18
  ([double dagger])
Tifway                Hanson, 1972   1.56 [+ or -] 0.05   27

([dagger]) Chromosomes of the accession was determined or confirmed by
cytological observations of 5 to 16 cells.

([double dagger]) The accession was not used in AFLP analysis.

Table 2. Numbers of total bands and polymorphic bands, percentage
polymorphic bands,and polymorphic information content (PIC) for
each of 13 AFLP selective primer pairs.

Selective
amplification
primer pairs         Total bands           Polymorphic bands

eAAC/mCAG          63                       25
  ([dagger])
eAAC/mGAC          40                       17
eACT/mGAC          49                       42
eACT/mCAG          61                       45
eACT/mGAC          62                       38
eACT/mCTG          63                       46
eAGT/mCAA          62                       27
eAGT/mCAC          72                       61
eAGT/mCAG          58                       37
eAGT/mCAT          69                       41
eAGT/mGAC          53                       25
eAGT/mCTG          76                       45
eGCT/mCAG          35                       17
Total             763                      466
Average            58.69 [+ or -] 11.38     35.85 [+ or -] 12.40

Selective
amplification
primer pairs     % polymorphic bands             PIC

eAAC/mCAG               39.68            0.16
  ([dagger])
eAAC/mGAC               42.50            0.20
eACT/mGAC               85.71            0.19
eACT/mCAG               73.77            0.15
eACT/mGAC               61.29            0.15
eACT/mCTG               86.79            0.19
eAGT/mCAA               43.55            0.24
eAGT/mCAC               84.72            0.23
eAGT/mCAG               63.79            0.21
eAGT/mCAT               59.42            0.29
eAGT/mGAC               47.17            0.26
eAGT/mCTG               59.21            0.27
eGCT/mCAG               48.57            0.20
Total
Average                 61.07            0.21 [+ or -] 0.05

([dagger]) e is the preamplification primer sequence for EcoRI site
(5-GACTGCGTACCAATTC) without any selective nucleotides and m is the
preamplification primer sequence for MseI site (5-GATGAGTCCTGAGTAA).

Table 3. Genetic similarity coefficients within and among three
ploidy levels of Chinese Cynodon accessions.

                       Genetic similarity coefficient

                                                   Among ploidy
             No. of        Within ploidy
Ploidy     accessions         (Range)         4x      5x        6x

4x       103              0.80 (0.69-0.99)   1.00   0.71 **   0.78 **
5x         3              0.96 (0.95-0.98)          1.00      0.73 **
6x         8 ([dagger])   0.85 (0.77-0.99)                    1.00

** Indicating significant difference between ploidy levels at a
probability level of P < 0.01.

([dagger]) Including Tifton 10, which was collected in Shanghai, China
(Hanna et al., 1990).
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Author:Wu, Y.Q.; Taliaferro, C.M.; Bai, G.H.; Martin, D.L.; Anderson, J.A.; Anderson, M.P.; Edwards, R.M.
Publication:Crop Science
Geographic Code:9CHIN
Date:Mar 1, 2006
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