Molecular Variation of Sporisorium scitamineum in Mainland China Revealed by RAPD and SRAP Markers Y. Que, L. Xu, J. Lin, and R. Chen, Key Lab of Sugarcane Genetic Improvement, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian Province, China; and M. P. Grisham, United States Department of Agriculture–Agricul- tural Research Service, Sugarcane Research Unit, Houma, LA 70360 Abstract Que, Y., Xu, L., Lin, J., Chen, R., and Grisham, M. P. 2012. Molecular variation of Sporisorium scitamineum in mainland China revealed by RAPD and SRAP markers. Plant Dis. 96:1519-1525. Sugarcane smut caused by Sporisorium scitamineum occurs world- showed that, whereas the molecular variation of S. scitamineum was wide, causing serious losses in sugar yield and quality. To study the associated with geographic origin, there was no evidence of co-evolu- molecular variation of S. scitamineum, 23 S. scitamineum isolates col- tion between sugarcane and the pathogen. The results of RAPD, SRAP, lected from the six primary sugarcane production areas in mainland or RAPD-SRAP combined analysis also did not provide any infor- China (Guangxi, Yunnan, Guangdong, Hainan, Fujian, and Jiangxi mation about race differentiation of S. scitamineum. This suggests that provinces) were assessed by random amplified polymorphic DNA the mixture of spores from sori collected from different areas should be (RAPD) and sequence-related amplified polymorphism (SRAP) mark- used in artificial inoculations for resistance breeding and selection. ers. The results of RAPD, SRAP, and RAPD-SRAP combined analysis Sugarcane smut, caused by Sporisorium scitamineum (Basi- The researchers concluded from the results that the fungus mi- onym: Ustilago scitaminea) (25,33), occurs worldwide and is one grated from Asia to other continents, probably through movement of the most prevalent fungal diseases of sugarcane in China. The of infected plant material. disease can cause serious losses in stalk yield and sucrose content Several molecular methods have proven useful for analysis of in susceptible cultivars (16). However, sugarcane smut does not genetic variation in pathogenic fungi (21,35). For example, rapid always pose a serious problem because incidence and severity vary amplification of polymorphic DNA (RAPD; 26,30), amplified depending on cultivars grown and environmental conditions. In fragment length polymorphism (AFLP; 4), and internal transcribed recent years, the incidence of the disease has increased, resulting in spacer (ITS) sequence analysis (32,33) have been used to assess greater damage to mainland China sugarcane, especially in the the intraspecific diversity within S. scitamineum populations. Un- most important sugarcane production area of Guangxi province, like the traditional polymerase chain reaction (PCR) analysis, where the infection rate was as high as 20% in ratoon cane (L. Xu RAPD does not require any previous DNA sequence information and Y. Que, unpublished). Although several methods have been of the target organism, because the primers will bind anywhere in recommended for the control of smut, only the use of resistant the genome, depending on sequences that are complementary to cultivars is practical and economical (8,36). the primers. This makes it more feasible in the phylogenetic study To increase the understanding of the interaction between sugar- of various plant-pathogenic fungi (5,12). Sequence-related ampli- cane and S. scitamineum and to improve breeding for smut- fied polymorphism (SRAP), which combines simplicity, reliability, resistance cultivars, an investigation of the molecular variation of moderate through-put ratio, and easy sequencing of selected bands, S. scitamineum was studied. An international group of researchers is a relatively new but powerful molecular marker system (20). It led by the International Society of Sugar Cane Technologists was uses two types of primers (forward and reverse) with the sequence established in 2004 to investigate the genetic diversity of S. scita- CCGG in the forward primer and AATT in the reverse primer. This mineum. Raboin et al. (28) analyzed a collection of S. scitamineum method targets the open read frames (ORFs) of genes and can de- populations collected from 15 sugarcane-producing countries for tect polymorphisms due to length variance of introns, promoters, polymorphisms at 17 microsatellite loci. They found that genetic and spacers. Compared with AFLP and inter-simple sequence re- diversity was extremely low among the American and African pop- peat, the higher annealing temperature of 50°C or above and the ulations. Higher genetic diversity, however, was observed among longer length of primers allows for a large number of robust poly- the Asian populations, particularly those from the Philippines. The morphic bands in SRAP amplification, leading to desirable dis- American and African S. scitamineum populations all belonged to a crimination ability (6). The objective of this study was to describe single lineage, which was also found in some populations in Asia. the genetic variation of populations of S. scitamineum isolated from the six primary sugarcane production areas in mainland China (Guangxi, Yunnan, Guangdong, Hainan, Fujian, and Jiangxi Corresponding authors: L. Xu, E-mail: [email protected]; and provinces) with two techniques, RAPD and SRAP, in order to gain Y. Que, E-mail: [email protected] a better understanding of the molecular variation within this spe- Product names and trademarks are mentioned to report factually on availa- cies. ble data; however, the United States Department of Agriculture–Agricul- tural Research Service (USDA) neither guarantees nor warrants the stand- Materials and Methods ard of the product, and the use of the name by USDA does not imply the Test materials. Twenty-three sugarcane S. scitamineum isolates approval of the product to the exclusion of others that may also be suitable. were collected from the six primary sugarcane-producing prov- inces in mainland China (Table 1). Accepted for publication 3 May 2012. DNA extraction and PCR identification. Mycelia derived from a single spore were used in DNA extraction by a procedure de- http://dx.doi.org/10.1094/ PDIS-08-11-0663-RE scribed by Que et al. (26,27). Quality and quantity of DNA sam- © 2012 The American Phytopathological Society ples were determined by 1.0% agarose gel electrophoresis and Plant Disease / October 2012 1519 ultraviolet spectrophotometer analysis. Before RAPD and SRAP Data analysis. For both RAPD and SRAP analyses, the pres- analysis, the identification of all DNA was verified using PCR to ence and absence of each band was coded by 1 and 0, respectively, amplify the bE mating-type gene of S. scitamineum (2,29). and was scored using a binary data matrix (0 to 1). Genetic dis- RAPD analysis. PCR amplification of genomic DNA was per- tances (1 – Sij) among isolates were calculated according to the Nei formed using arbitrary primers listed in Table 2. The S series pri- and Li (24) similarity coefficient Sij = 2a/(2a + b + c), where Sij is mer set was originally obtained from the Shanghai Sangon Bio- the similarity between two individuals i and j, a is the number of logical Engineering Technology & Services Co. Lt.; Operon shared bands, b is the number of bands exclusively amplified by i, primers were purchased from Operon Technologies. RAPD and c is the number of bands exclusively amplified by j. Then, amplification of S. scitamineum DNA was performed using the 13 DPS2000 software (Zhejiang University, China) was used in the random primers in Table 2. PCR amplification was performed in a corresponding data analyses, and resulted in the construction of a final volume of 20 µl containing 50 ng of S. scitamineum DNA and phylogenetic tree and dissimilarity coefficient matrix. During the 0.5 µM 10-mer random primer. The reaction mixture also con- data processing, the distance matrix was subjected to cluster analy- tained a dNTP concentration of 0.15 mM and 2.5 µl of 10× PCR sis by the unweighted pair-group method with arithmetic means reaction buffer (containing 15 mM MgCl2) and 1 unit of Taq DNA (UPGMA). In RAPD-SRAP combined analysis, the data from polymerase (Takara Bio Inc.). The volume was adjusted to 20 µl RAPD and SRAP were combined and analyzed with the same with the addition of sterile distilled water. Amplification was per- procedure as above. formed in an Eppendorf thermal cycler, with an initial denaturation at 96°C for 5 min, followed by 40 cycles each of denaturation at Results 94°C for 1 min, annealing at 37°C for 1 min and extension at 72°C RAPD analysis. Thirteen RAPD primers were used for the for 1.5 min. Immediately after thermal cycling, a final extension at evaluation of genetic relationships among 23 S. scitamineum iso- 72°C for 8 min was done and samples were cooled and held at 4°C lates. In total, 189 bands was scored. Among them, 102 bands until use. PCR products were loaded into a 1.0% agarose gel con- (54%) were polymorphic between at least two individuals. The taining 0.1% EB, subjected to electrophoresis in 1× Tris-acetate- highest and lowest rates of polymorphism (number of polymorphic EDTA (40 mM Tris; 20 mM glacial acetic acid; and 1 mM EDTA, amplified bands/total monomorphic and polymorphic bands within pH 8.0) for about 2 h. The gel image was analyzed using Quantity a paired comparison) among the amplified bands were 88 and One software (version 4.6.2; Bio-Rad Laboratories, Inc.). 31%, respectively. The length of amplification bands ranged from SRAP analysis. From 45 possible primer pairs of five forward 200 to 3,000 bp, with an average band number of 15 per primer. and nine reverse primers (Table 3), 18 combinations that produced Genetic dissimilarities among all isolates ranged from 0.071 to a few strong, reproducible, and polymorphic bands were selected. 0.825 (Table 4). The Nei-Li similarity coefficient and UPGMA These primer combinations were used to amplify the 23 S.