Molecular Insights of Genetic Variation in Erianthus arundinaceus Populations Native to China

Background E. arundinaceus (Retz.) Jeswiet is a warm-season, tall-growing perennial species native to much southern portion in China. The grass has been extensively used in sugarcane breeding and is recently targeted as a bioenergy feedstock crop. However, information on the genetic structure of the Chinese wild germplasm is limited. Knowledge of genetic variation within and among populations is essential for breeding new cultivars in the species. The major objective of this study was to quantify the magnitude of genetic variation among and within natural populations in China. Methodology/Principal Findings In this experiment, we analyzed genetic variation of 164 individuals of 18 populations collected from natural habitats in six Chinese provinces using 20 sequence-related amplified polymorphism (SRAP) primer pairs generating 277 polymorphic bands. Among and within the populations, the percentage of polymorphic bands (PPB) was 80.00% and 27.07%, genetic diversity (HE) was 0.245 and 0.099, effective number of alleles (NE) was 1.350 and 1.170, and Shannon's information index (I) was 0.340 and 0.147, respectively. The populations were clustered into six groups exhibiting a high level of genetic differentiation, which was highly associated with geographic origins of respective germplasm populations, but was not significantly associated with geographic distances between the populations. Conclusions/Significance This is the first report indicating that large genetic variation exists in the Chinese E. arundinaceus germplasm based on the SRAP molecular marker analysis of native populations. The genetic structure of populations in the species has been substantially affected by geographic landforms and environments. The diverse collection will be highly valuable in genetic improvement in the species per se and likely in sugarcane.


Introduction
E. arundinaceus (synonym of Saccharum arundinaceum Retz.) is a warm-season, tall-growing, caespitose perennial species native to China and certain other south and southeast Asian nations of temperate climates to tropical environments [1][2]. As a wild relative of sugarcane (Saccharum officinarum L.), the species has contributed to the genetic improvement in sugarcane breeding [3] and possesses high potential for the development of energy cane interspecific hybrids [4]. It is widely distributed in the Chinese provinces of Anhui, Fujian, Guangdong, Guangxi, Guizhou, Hainan, Henan, Hubei, Hunan, Jiangsu, Jiangxi, Shanxi, Sichuan, Taiwan, Xinjiang, Xizang, Yunnan, and Zhejiang [5]. The species is related to taxa in Miscanthus, Narenga, Saccharum, and Sclerostachya, so is considered to be a member of the ''sugarcane complex'' [6]. Due to its excellent tolerance to abiotic stresses and disease resistance, the species has long been used in sugarcane breeding [7]. Although difficult, breeders have successfully generated fertile Saccharum 6 Erianthus hybrids, which are further crossed to sugarcane clones in the production of wide intergeneric hybrids [8][9][10][11]. Recently, the species has been targeted as a bioenergy perennial because of its high biomass yield potential on marginal lands [12]. With the support from the National High-Tech R&D Program of China, a breeding program has been initiated to improve the species as a bioenergy feedstock crop at the Sichuan Academy of Grassland Science, China since 2011.
Genetic variation in E. arundinaceus has been well documented. Using morphological traits, a high level of variation was reported in E. arundinaceus accessions from China, while the variation from Indonesia was relatively low [13][14]. Karyotype analyses indicated most clones of Chinese E. arundinaceus had 2n = 4x = 40 and 6x = 60 somatic chromosomes while 2n = 2x = 20 was rare [15]. Using DNA markers, the percentage of polymorphic bands ranged from 65 to 99% indicating high molecular diversity in Chinese germplasm [16][17][18], while E. arundinaceus from Indonesia appeared to have a low level of molecular variability [18][19][20]. E. arundinaceus from India was more polymorphic than from Indonesia [18], [21]. Although useful, these reports revealed very limited information on genetic variation among and within populations in the species.
In the last two decades, amplified fragment length polymorphism (AFLP), inter simple sequence repeat (ISSR), random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) but sequence-related amplified polymorphism (SRAP) have been used in characterizing genetic diversity in E. arundinaceus [16][17][18][19][20]. SRAP has been proved to be a reliable molecular marker system based on simple PCR amplifications of genomic DNA [22]. The marker system analyzes DNA polymorphisms with amplifying open reading frames using specifically designed primers. SRAP markers provide a valuable tool to study patterns of genetic variability due to their advantages over other molecular markers, such as less complex and labor-saving procedures and more random sampling of the whole genome.
Information on genetic variation among and within populations could help better understand the natural variation in the species on a large geographic scale, which is useful in sampling and deploying the germplasm in breeding programs. We collected 18 indigenous populations of E. arundinaceus in six provinces of China. Therefore, the major objective of this study was to quantify the magnitude of genetic variation among and within the natural populations.

Ethics Statement
This study was approved by the Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University; Sichuan Academy of Grassland Science; Guizhou Grassland Science Institute; and Grassland Institute, China Agricultural University. No specific permissions were required for collecting Erianthus arundinaceus samples at the locations in China, because the research was funded by the Ministry of Science and Technology and the earmarked fund for China Agriculture Research System of the People's Republic of China, and the species is not an endangered or protected species.

Sample Collection and DNA Extraction
Following the population sampling method by Jing and Lu [23], a total of 164 wild E. arundinaceus individual leaf samples in 18 populations were collected in Sichuan, Yunnan, Guizhou, Guangxi, Guangdong and Hainan provinces, China (see Table 1). Sampled individuals in each population ranged from six to 10. Localities of the collected populations spanned nearly 14uN. latitudes from tropical environments in Hainan to subtropical climates in Sichuan (Table 1 and Figure 1). The leaf tissues were dried using self-indicating silica gel and stored in a freezer at 280uC until DNA extraction. Genomic DNA was isolated using the modified CTAB method of Doyle [24]. Purity and concentration of the genomic DNA were determined with a Nanodrop spectrophotometer (NanoDrop Products, Wilmington, DE).

Data Analysis
Clearly amplified PCR bands were visually scored for presence (1) or absence (0), and then were assembled into an Excel matrix for the following analyses. Use of dominant marker data in genetic diversity analysis can lead to estimation bias with overestimating parameters by as much as 5%, especially with small sample sizes [25][26]. To account for this potential bias, Lynch and Milligan proposed pruning any locus with a band frequency of higher than 1-(3/N), where N is the number of individual samples [25]. Since SRAP markers are dominant, only the marker data of specific loci having a band frequency less than 1-(3/164) = 0.982 were retained for subsequent statistical analyses in this study.
The number of polymorphic loci (Np), percentage of polymorphic bands (PPB), Shannon's information index (I), observed number of alleles(N O ), effective number of alleles (N E ), Nei's gene diversity(H E ), genetic diversity within populations (Hs), total genetic diversity (Ht), genetic differentiation coefficient (Gst), gene flow estimates (Nm), and Nei's genetic distance were calculated using POPGENE [27]. A UPGMA tree based on Nei's [28] genetic distance data was generated by TFPGA (version 1.3) [29] to examine genetic relationships of the populations while a UPGMA tree among individuals was generated by FreeTree program [30]. Bootstrap values were obtained by resampling replacements over loci in 1000 replicates. In addition, a Mantel test was conducted to calculate the correlation between pairwise geographic and Nei's genetic distances using NTSYS software [31]. Finally, WINAMOVA program v.1.55 [32] was used to separate the total genetic variance into within and among populations/groups. The input files for POPGENE and AMOVA were prepared with the aid of DCFA1.1 program [33].

Results
Twenty selected SRAP PPs yielded a total of 365 scorable bands, of which 294 were polymorphic (Appendix S1). Using the method by Lynch and Milligan [25], five loci that each was scored more than 161 of ''0'', were excluded, while 12 loci with each scored more than 161 of ''1'', were changed to monomorphic loci, resulting in 360 scorable and 277 polymorphic bands used in subsequent analyses. The number of amplified bands for each PP ranged from 14 to 22, with an average of 18 bands ( Table 2). The percentage of polymorphic bands (PPB) within each population ranged from 16.94% (Pop3) to 33.33% (Pop4) with an average of 27.07% while PPB was 80.00% at the species level.  (Table 3).

Genetic Distance and Phylogenetic Relationship
Genetic distances (D, Nei's measure) among populations are given in Table 4   between genetic distance and geographic distance among populations (r = 0.77, p = 1. 000).

Genetic Structure and Differentiation among Populations
A highly significant (P,0.001) genetic difference was found among groups, among populations, and within populations ( Table 5). The results from the AMOVA showed that 51.44% genetic variation occurred among populations (P,0.001) and the remaining 48.56% existed within populations (P,0.001). When these populations were classified into six groups based on the results of the clustering analysis, the variance among populations within the groups was 13.06%, whereas the variance among groups was 41.24%. In particular the AMOVA for the populations (Pop11, Pop12, Pop13, Pop14 and Pop15) from Group 4 according to the UPGMA tree showed that 22.0% of genetic variation occurred among populations (P,0.001) and 78.0% occurred within populations (P,0.001) ( Table 4)

Genetic Variation
In previous reports, the genetic diversity of E. arundinaceus was studied using individual clones, which were collected from Southeast Asia and Chinese tropical and subtropical regions. These studies showed the variation level of E. arundinaceus was different in different regions. The genetic diversity of E. arundinaceus clones in Indonesian was studied using morphological traits, demonstrating those clones had low genetic variation [14]. The result was confirmed in later experiments using other E. arundinaceus clones from Indonesia with rDNA, RAPD and RFLP markers [18][19][20]. Clones from India had an intermediate level of diversity [18][19][20][21]. The variation level of clones from the Philippines was similar to that of Indonesian clones, while the variation level of clones from Vietnam was similar to that of India clones [18]. In our study, PPB over 18 natural populations of E. arundinaceus in China was 80.0%, lower than the PPB value (AFLP, 99.3%) in the study of Cai et al. [18], but higher than the values (ISSR, 64.9% and RAPD, 70.1%) by Zhang et al. [16][17], and (AFLP, 69.2%) by Tsuruta et al. [34]. Collectively these reports revealed a high level of genetic diversity in Chinese E. arundinaceus.
Comparisons of the genetic variation levels of E. arundinaceus from the Philippines, Indonesia, India, Vietnam and China, show that E. arundinaceus from pacific Island countries (the Philippines and Indonesia) has lower genetic variation. In contrast, E. arundinaceus collections from continental countries (India, Vietnam and China) have larger genetic variation. We speculated that the low genetic variation of E. arundinaceus from island countries was generated by the effect of ocean isolation and relatively homogenous environments in the countries. The pacific island countries are isolated by the ocean, which may have effectively blocked or minimize gene flow from germplasm outside the islands, consequently reducing genetic diversity [35]. In the current study, the ''isolation effect'' was also evidenced in the genetic diversity of Chinese Those mountains might also isolate the populations in Yunnan (Pop6 and Pop7) from those in other regions. However, the populations (except Pop2 and Pop3) from these isolated regions had higher genetic variation than the mean of all populations from China, suggesting that the effect of isolation by mountains was less than from the ocean. This is the first report characterizing genetic variation in E. arundinaceus through examining Chinese native populations and revealing new biological characteristics of the species. In this study, the average of within population diversity in E. arundinaceus (H E = 0.245) is higher than short-lived perennial (H E = 0.20), mixed-mating species (H E = 0.18) and selfers (H E = 0.12), but similar to outcrossers (H E = 0.27) reported by Nybom [36]. The results were not reported in previous reports. The H E value of Miscanthus floridulus (H E = 0.30) [37] was similar to the results of E. arundinaceus in this report, while the H E value of Saccharum spontaneum (H E = 0.23) [38] was lower than the value in E. arundinaceus. The high He value of E. arundinaceus revealed in this experiment suggests that E. arundinaceus be an outcrossing species.

Genetic Structure of Populations
In this study, the Nei's estimate of E. arundinaceus population substructure (G ST ) was 0.55, indicating more than a half of genetic variation occurred among populations. The results of G ST was similar to the results from AMOVA, which showed that 51.44% genetic variation existed among populations (P,0.001) and the remaining occurred within populations (P,0.001). Chang et al. (2012) reported genetic variation among populations was lower than that within populations in S. spontaneum [38]. Similar results were reported in M. floridulus populations [37]. Interestingly, the AMOVA of the populations from Guangxi and Guangdong (except Pop10) in this study, showed that 22.00% genetic variation occurred among populations (P,0.001) and 78.00% occurred within populations (P,0.001). As the populations are distributed in neighboring and similar environmental conditions without significant landmasses between them, gene flow among the populations may take place more frequently. Consequently, the populations do not differentiate into distinct populations. The result was more similar to the S. spontaneum and M. floridulus populations. Hamrick and Godt [39] pointed out that the genetic variation of outcrossing species occurred among populations was lower than within populations, and a similar result was found by Nybom [36]. Our study suggests that the genetic structure of E. arundinaceus populations is affected by the natural landforms and geographical conditions. Gene flow (Nm) would be able to resist the effect of genetic drift within populations and prevent the differentiation of populations as the value of Nm .1, and when the value of Nm ,1 the genetic drift could lead to genetic differentiation among populations [40]. Outcrossing species have higher levels of gene flow [36], but the Nm value of E. arundinaceus (an outcrossing species) populations in this study was only 0.41, indicating that there was a lower level of gene flow and significant genetic differentiation among the 18 populations. The natural landforms in the sampling areas of E. arundinaceus forming the geographic isolation and heterogeneity of the ecological environment affect gene flow, the genetic and geographical divergence among the populations [41]. Some E. arundinaceus populations in this study were isolated by ocean or mountain. It appears that the isolation affected not only gene flow but also the genetic diversity of E. arundinaceus through natural selection within local environments. In our study 18 E. arundinaceus populations were clustered into six groups, which belonged to different isolated regions. The Mantel tests indicated that there was no significant associated relationship between genetic distance and geographic distances between populations. The result was similar to that in S. spontaneum [38]. Although not statistically significant, the correlation coefficient between genetic and geographic distances may have affected the population structure, but at a magnitude less than geographic isolation.
In addition to diploids (2n = 2x = 20), most Chinese E. arundinaceus plants reported previously are tetraploids (2n = 4x = 40) and hexaploids (2n = 6x = 60) [15]. The altered ploidy might contribute to the genetic variation in the Chinese germplasm since gene flow between plants of altered ploidy is likely limited, consequently genetic divergence would occur. However, the geographic distribution patterns of the three ploidy forms in Chinese E. arundinaceus germplasm are elusive. Further investigation efforts on the association between ploidy forms and genetic variation of the native germplasm in Asian countries, especially China may shed light on the evolution and formation of genetic variability within the species.

Supporting Information
Appendix S1 SRAP data for 18 populations of Erianthus arundinaceus amplified using 20 primer pairs, coded as presence (1) and absence (0). Note: data rows in red color were excluded in data analysis due to more than 161 of ''0'' and data rows in blue color were changed to monomorphic loci due to more than 161 of ''1'' according to Lynch and Milligan [25]. (XLS)