Eighteen sugarcane genotypes were involved in this study, including 13 cultivars that were widely grown for sugar production in Guizhou, China and five elite clones that were selected through conventional breeding by the Guizhou Institute of Subtropical Crops (GISC) (Table 1). Healthy apical leaves were collected from the plants of the 18 genotypes from the germplasm nursery at GISC, which were stored at −20˚C prior to DNA extraction. DNA isolation was conducted according to Pan et al. [1] . DNA concentrations were measured on a Shimadzu Model UV-1601 spectrophotometer (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA) and DNA quality was assessed by 0.8% agarose gel electrophoresis using diluted uncut l DNA as the standard. Final DNA concentration was adjusted to five ng/μL and stored at −20˚C for SSR analysis.

SSR analysis was conducted with 21 highly polymorphic ISMC SSR primer pairs following the protocol of Pan [13] . PCR amplification reactions were conducted in a total volume of 10 μL, containing 10 ng of DNA, 0.2 mM each of FAM-labeled forward primer and unlabeled reverse primer, 80 mM each of dNTP, 10 mM Tris-HCl, 2.5 mM MgCl₂, 50 mM KCl, and 1 U Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN, USA). The thermo-cycle program was subsequently conducted as follows: 95˚C for 5 mins, followed by 30 cycles of 94˚C for 30 s, annealing temperature for each pair of primers for 30 s, 72˚C for 30 s, followed by 72˚C for 2 min. FAM-labeled PCR fragments along with the GS500 size standards were separated through capillary electrophoresis (CE) on the DNA Analyzer ABI 3730XL (Applied Biosystems, Foster City, CA, USA) to generate <.fsa> GeneScan files.

The GeneMapper software v3.0 (Applied Biosystems, Foster City, CA, USA) was used to analyze the GeneScan files through visualization of capillary electrophoregrams of

*FN = Funong; GT = Guitang; QT = Qiantang; ROC = Taiwan, China; YT = Yuetang; Xuan 50 = RB72-454.

SSR alleles with sizes automatically calibrated using the GS500 size standards. Irregular Peaks such as “stutters”, “pull-ups”, “dinosaur tails”, and “minus-A peaks” were not scored according to Pan [13] . The presence of a regular peak was scored as 1 and its absence was scored as 0. The polymorphism information content (PIC) of each SSR primer pair was calculated according to the formula of Milbourne et al. [40] : PIC = 1 - ΣPg², where Pg is the frequency of an individual genotype. Genetic distance between each genotype was calculated using the Jaccard similarity coefficient and then a dendrogram was constructed using NTSYSpc-2.2 [41] software on the basis of unweighted pair group method with arithmetic means (UPGMA). A homology tree was built using DNAMAN® (Lynnon Co., Quebec, Canada) to analyze the genetic homology relationship among the 18 sugarcane genotypes where the presence of a regular peak was recorded as A and its absence was recorded as C [42] .

All sugarcane SSRs used in this study were polymorphic yielding a total of 167 alleles, of which 148 alleles (88.6%) were polymorphic. Figure 1 is an example of SSR profiles

of nine sugarcane genotypes based on marker SMC336BS. The number of alleles produced by each SSR primer pair ranged from four to twelve with an average of eight alleles per primer pair (Table 2), slightly higher in comparison with Hameed’s study (6.85) [43] but lower than the average number of alleles per primer pair reported by Chandra (9.2) [21] and Liang (15.6) [44] using the same set of SSR makers. SMC24DUQ and SMC597CS both produced maximum number of alleles, of which 10 were polymorphic. Although SMC486CG and SMC569CS produced the minimum num- ber of alleles, all alleles were polymorphic. The percentage of polymorphic bands for each SSR primer pair was in the range of 66.7% and 100%. Eight SSR primer pairs (38.1%), namely, SMC278CS, SMC31CUQ, SMC334BS, SMC336BS, SMC486CG,

SMC569CS, mSSCIR66, produced 100% polymorphic alleles. The PIC values of the 21 SSR primer pairs ranged from 0.68 to 0.92 with an average of 0.86. SMC31CUQ and SMC334BS were the most informative primer pairs producing the highest PIC value of 0.92.

Eight SSRs were found to be cultivar-specific for nine sugarcane genotypes, amp- lifying a total of 11 unique SSR alleles. Of which, five were specific to QT-series clones, namely, SMC851MS-145, mSSCIR43-229, SM597CS-182, SMC7CUQ-168, and SMC334BS-165. Primer pair SMC851MS produced three cultivar-specific alleles, namely, SMC851MS-127 in Xuan 50, SMC851MS-143 in FN 38, and SMC851MS-145 in QT 08-558. Primer pair mSSCIR43 produced two cultivar-specific alleles, namely, mSSCIR43-229 in QT 4 and mSSCIR43-240 in GT 31. Each of the remaining six SSR primer pairs amplified only one cultivar-specific allele, namely, SM597CS-182 in QT 08-536, SMC7CUQ-168 in QT 06-212, SMC334BS-165 in QT 08-558, SMC24DUQ-151 in FN 38, SMC1604SA-106 in GT 11, and SMC119CG-223 in YT 60. These results implies that a fewer number of primer pair combinations among these eight SSR primer pairs will be able to identify sugarcane genotypes within a large scale of sugarcane germplasm evaluation. Moreover, these primer pairs are also primer pairs of choice for pedigree verification in sugarcane polycross or hybrid identification in bi-parental crosses when the nine sugarcane genotypes are used as the parents.

The performances of each SSR primer pair may vary on different sugarcane cultivars because these cultivars may have different chromosome makeups. The 21 SSR primer pairs used in the present study were chosen from 67 highly polymorphic SSR primer pairs based on the evaluation of five elite U.S. germplasm [13] . These primer pairs were able to distinguish among cultivars and breeding lines in the U.S. [42] . These SSR primer pairs also can detect genetic variations among sugarcane clones originated from China, Bangladesh, South Africa, Mexico, India, and related wild species [21] [44] [45] . Therefore, these 21 SSR primer pairs can be considered as core SSR primer pairs for sugarcane molecular characterization worldwide. However, Pan suggested that these SSR primer pairs be tested first on a local elite sugarcane germplasm collection developed under certain geographical environment prior to a full-scale genotyping exercise [13] .

It is the first report on evaluating genomic diversity among sugarcane cultivars growing in Karst region in China, the 21 SSR primer pairs showed highly polymorphic among both material sugarcane and elite clones, which indicates these 21 SSR primer pairs are suitable for genotyping sugarcane clones bred in Karst climate in China.

Homology analysis showed there were 64-98% homologies among the 18 sugarcane genotypes (Figure 2). Two pairs of genotypes, namely, ROC21 and YT 00-236 and YT 94-128 and ROC25, shared the highest homology value of 98%, followed by ROC22 and YT-60 (97%). The homology values shared between the QT-series sugarcane genotypes were between 67% and 76%. A UPGMA dendrogram was constructed to reveal the extent of genetic diversity among the 18 sugarcane genotypes. Genetic similarity coefficients for all the genotypes ranged from 0.64 to 0.98. The 18 sugarcane genotypes were clustered into three major groups, namely, A, B and C (Figure 3). Group B was the largest group with 10 genotypes (55.6%), followed by Group C with five genotypes (27.8%) and Group A with three genotypes (16.7%). Group B was further divided into two sub-groups, BI and BII. The Sub-group BI consisted of two genotypes, GT11 and QT08-558; while the Sub-group BII included eight genotypes. Genotype ROC21 and YT00-236 in Group C were almost genetically identical. In Group B, both ROC25 and YT94-128 and ROC22 and YT60 were genetically close to each other. This was probably due to the fact that ROC-series cultivars of Taiwan were first introduced into Guangdong province, where these cultivars were used either as cross parents to breed the YT-series clones or directly as cultivars if they well adapted to local climates.

The 18 sugarcane genotypes did not group in accordance with their geographical origins. One reason might be that these sugarcane genotypes were descendants of the early sugarcane Noblization breeding program, thus, sharing a genetic base of a few common progenitor clones. Our result was in agreement with those from Nair’s and Liang’s studies [4] [42] .