A new method based on SNP of nrDNA-ITS to identify Saccharum spontaneum and its progeny in the genus Saccharum

The identification of germplasm resources is an important aspect of sugarcane breeding. The aim of this study was to introduce a new method for identifying Saccharum spontaneum and its progeny. First, we cloned and sequenced nuclear ribosomal DNA internal transcribed spacer (nrDNA-ITS) sequences from 20 Saccharum germplasms. Analysis of these nrDNA-ITS sequences showed a stable mutation at base 89. Primers (FO13, RO13, FI16, and RI16) were then designed for tetra-primer amplification refractory mutation system (ARMS) PCR based on mutations at base 89 of the nrDNA-ITS sequence. An additional 71 Saccharum germplasms were identified using this tetra-primer ARMS PCR method, which confirmed that the method using the described primers successfully identified Saccharum spontaneum and progeny. These results may help improve the efficiency of modern molecular breeding of sugarcane and lay a foundation for identification of sugarcane germplasms and the relationships among them.

Internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA) contain ITS1, 5.8s rDNA, and ITS2 [5]. In recent years, several features of nrDNA-ITS have made a useful tool for evaluating and analyzing evolutionary relationships at the subspecies level, including rich variances and rapid evolutionary rate, as well as simple PCR amplification and sequencing [6][7][8]. In view of these features, Yang et al. analyzed the nrDNA-ITS sequence characteristics of 19 S. spontaneum germplasms and 11 local sugarcane varieties [9]. The results showed that 11 S. spontaneum germplasms could be divided into several branches, and that local sugarcane varieties were closely related to S. spontaneum. Moreover, the ITS1 sequence could be used as a DNA barcode to further study the genetic diversity of Saccharum and related genera. Liu et al. analyzed differences among nrDNA-ITS sequences from 62 different multiple S. spontaneum materials and showed that 4 species had high variation, especially the nonuploid and decaploid population [10].
As a third-generation molecular marker, single nucleotide polymorphisms (SNPs) are highly stable and widely used for studies of crop molecular genetics [11]. Due to the limited availability of sugarcane genomic maps, research on SNPs in sugarcane lags behind that of rice, rapeseed, and other crops [12]. SNPs for many crops have been discovered through analysis of nrDNA-ITS sequences and in turn have served as valuable molecular markers to identify interspecies germplasms [13] that can contribute to strategies for molecular breeding of crops [14]. Tetra-primer amplification refractory mutation system PCR (tetra-primer ARMS PCR) is a derivative technique based on common PCR that can be specifically used to detect SNPs [15]. Tetra-primer ARMS PCR is rapid, simple, and economical. According to the SNP site, the tetra-primer ARMS PCR technique has been used to identify various germplasm genotypes in rice, wheat, capsicum, and other crops [16][17][18].
Based on previous studies on nrDNA-ITS in sugarcane germplasms, the tetra-primer ARMS PCR technique can be used to analyze genetic diversity and phylogenetic relationships in interspecific and intergenus samples [9,10,19]. However, studies exploring the identification and use of SNPs as molecular markers in Saccharum breeding have not been performed. As such, we cloned, sequenced, and analyzed nrDNA-ITS sequences of 20 Saccharum germplasms to identify a stable SNP. Based on the SNP site, primers were designed according to the principles of tetra-primer ARMS PCR. PCR of 71 materials was performed to identify the presence of Saccharum spontaneum genetic material. This study provides a foundation for improving the efficiency of modern molecular breeding of sugarcane and a molecular basis for identifying sugarcane germplasms.

Reagents and materials
Takala Ex Taq 1 polymerase, Takala LA Taq 1 polymerase, PMD19-T vector, and E. coli DH5α competent cells were obtained from Takara Biotechnology Co., Ltd. (Dalian of China). Primers were synthesized by the Beijing Genomics Institute (Beijing, China).

Genomic DNA extraction
Young leaves from different sugarcane species were collected and powdered after freezing in liquid nitrogen. Genomic DNA was extracted from the leaves using a traditional CTAB method that was performed according to Porebski et al. [20].

Cloning and sequencing
The nrDNA-ITS sequences from 20 clones (Table 1) were amplified using the universal primers ITS1 and ITS4 (ITS1: TCCGTAGGTGAACCTGCGG; ITS4: TCCTCCGCTTATTGATATGC) [21]. The PCR reaction mixtures were prepared on ice (Table 3) and carried out in a thermal cycler (ABI, 9902, USA). The reaction sequences were as follows: pre-denaturation at 95˚C for 5 min followed by 35 cycles of 95˚C for 15 s, 54˚C for 15 s, and 72˚C for 10 s. A final extension was conducted at 72˚C for 5 min. The PCR products were tested by 1.5% agarose gel electrophoresis and purified using an Omega EZNA gel extraction kit. The purified products were then cloned into a PMD19-T vector and transformed into E. coli DH5α competent cells. Recombinant clones were grown in LB medium supplemented with ampicillin (100 μg/mL). Five clones per sample were selected for sequencing by Sangon Biotech Co., Ltd. (Shanghai, China).

Sequence analysis
DNA sequence homology was estimated using a nucleotide BLAST tool in the NCBI database. All DNA sequences were analyzed by DNAMAN 6.0 and BioEdit 7.0.9.0 to obtain variable site information. Identification of Saccharum spontaneum using tetra-primer ARMS PCR

Primer design
Optimized primers for PCR were designed according to the design principle of tetra-primer ARMS PCR primers. Specific reference to the design method for tetra-primer ARMS PCR primers is made in Medrano and de Oliveira [22].

Tetra-primer ARMS PCR procedure
Tetra-primer ARMS PCR of 71 samples (

nrDNA-ITS PCR
The nrDNA-ITS sequences of different samples were obtained by PCR with ITS1 and ITS4 primers. The nrDNA-ITS PCR product from each material tested appeared in the electrophoresis map as a single, intense 678 bp band (Fig 1).

Sequence analysis
All of the clone sequences were analyzed using the BLAST tool in the NCBI database. The homology of all cloned sequences with other germplasm nrDNA-ITS sequences of sugarcane was >98%, which indicated that the clone sequences contained nrDNA-ITS sequences and  2). Among all mutations, those at base 73 and 89 were in a regular form because the mutation occurred only in S. spontaneum clones (Fig 2). However, only the mutation at base 89 was conserved for 10 S. spontaneum clones, which could be a good target region of tetra-primer ARMS PCR.

Discussion
In this study, we found for the first time that base 89 was a stable base mutation in the nrDNA-ITS sequence of the Saccharum genus and was present in S. spontaneum genetic material. We thus designed primers for tetra-primer ARMS PCR based on this nrDNA-ITS sequence SNP. After optimization and identification, the primers FO13, RO13, FI16, and RI16 were found to be suitable for identification of S. spontaneum genetic material and its progeny.
To the best of our knowledge, this is the first instance of the use of tetra-primer ARMS PCR to identify S. spontaneum genetic signatures in the Saccharum genus. These findings will be valuable for classifying sugarcane germplasms and will improve sugarcane hybridization breeding efficiency.
In angiosperms, this entire ITS region (ITS1+5.8S+ITS2) can be easily amplified using universal primers that recognize conserved coding regions to produce a 700 bp amplicon [23]. Here, a 678 bp fragment was amplified using the general primers ITS1 and ITS4 that cover the entire ITS sequence. This ITS sequence is not only widely used for germplasm classification and phylogeny analysis, but also for identification of germplasm resources in sample plants. Previous studies have suggested that this ITS sequence has higher conservation than the medium-height repetitive sequence and non-coded sequences, and that the mutation rate is relatively rapid compared with the coded gene sequence [24]. In allopolyploid plants, ITS sequence evolution is complex, such that ancestral ITS sequences can coexist in some offspring, but in other offspring may evolve in another direction [25,26]. For example, in the polyploid plants of wheat, ancestral nrDNA-ITS sequences coexist in the offspring [27]. In this study, tetra-primer ARMS PCR results showed that the hybrid generation generated between S. officinarum and S. spontaneum yielded three bands (428 bp, 278 bp, 203 bp), the offspring generated between S. officinarum and S. robustum yielded two bands (428 bp, 278 bp), which respectively revealed parents' traits. Therefore, we found that the ancestral nrDNA-ITS sequences could coexist in the offspring in the hybrid process of sugarcane. Our sequence analysis in this study indicated that the nrDNA-ITS sequence in sugarcane is relatively conserved. Moreover, the SNPs we identified were highly stable genetic markers. Therefore, the primers Identification of Saccharum spontaneum using tetra-primer ARMS PCR we developed for tetra-primer ARMS PCR could be used for accurate and reliable identification of S. spontaneum genetic material and progeny in the Saccharum genus.
The identification of Saccharum germplasm collections is mainly based on morphological observation, which can result in misclassification. In recent years, several molecular markers, such as SSR, AFLP, ISSR, and RAPD, have been applied for identification of Saccharum germplasms and progeny identification [28][29][30][31]. Many specific bands can be obtained from the progeny of hybrid offspring using these molecular markers, which contribute significantly to the separation and identification of sugarcane hybrids and germplasms. However, these molecular markers cannot intuitively identify sugarcane germplasms, as they produce multiple amplification bands that complicate interpretation and quantification. Of course, single-copy gene marker had been applied in phylogenetics in many plants with the development of sequencing technology [32,33]. Compare with nrDNA-ITS sequence, the use of a single-copy gene mostly demands development of PCR primers specific for the taxonomic group of interest [34]. Moreover, this can result in the inclusion of paralogous copies in phylogenetic studies in the polyploid plans, resulting in wrong taxon relationships. To avoid this problem, the single-copy nuclear genes that occured mostly only with a single copy in the haploid genome might be preferable in phylogenetic analyses and identification of different species [35]. Therefore, selection of single-copy gene marker is an issue at present, especially in the polyploid plant. Because the sugarcane is an allopolyploids plant, its genome research is not yet complete and it is very difficult to obtain the haploid of sugarcane at present. Thus, selection of a singlecopy gene as molecular marker is very difficult in sugarcane. In a more visual identification of sugarcane germplasm materials, Piperidis et al. used genomic in situ hybridization (GISH) to show that Kokea, Muntok Java, and Bourbonriet suriname were not S. officinarum, but instead were hybridized progeny of S. officinarum and S. spontaneum [36]. Moreover, many breeders believed that Muckche, Canablanca, and Baimeizhe were S. officinarum based on similar morphology. However, in 2016 Wang et al. identified 10 S. officinarum types using GISH technology and found that Muckche, Canablanca, and Baimeizhe were in fact hybridized progeny of S. officinarum and S. spontaneum [37]. These results are consistent with our study, which supports the reliability of the molecular markers we identified. Based on tetra-primer ARMS PCR results, we easily distinguished S. spontaneum and other Saccharum germplasm materials in this study. Moreover, tetra-primer ARMS PCR using the primers FO13, RO13, FI16, and RI16 to identify S. spontaneum and progeny is simpler and less time-consuming than GISH.
The breeding of a sugarcane variety typically requires approximately 10 years. Historically, the breeding process could not be accelerated because plants could not be selected early in the seedling stage due to a lack of molecular markers. The tetra-primer ARMS PCR technology developed in this study could have broad applications for sugarcane breeding in the future. This approach could address the problem of early selection and identify whether seedlings incorporate S. spontaneum genetic material. Germplasms from plants thought to include S. spontaneum as a predecessor could be identified using tetra-primer ARMS PCR technology to determine whether such plants are indeed hybridized progeny of S. officinarum and S. spontaneum.