Sugarcane (Saccharum spp. hybrids) is the most important sugar crop in China, its genetic background is complex. Sucrose industry is of great economic significance in tropical and subtropical countries. Modern sugarcane genome is a complex mixture of aneuploidy and polyploidy (Singh et al., 2010; Yang et al., 2014). Modern sugarcane varieties are bred by hybridization and continuous backcross between a few cultivated original varieties and wild varieties and their related varieties, which leads to the narrow genetic basis of sugarcane, making it difficult for sugarcane varieties to obtain great breakthroughs in commercial traits, such as yield, sugar content, and in breeding, such as disease resistance and adaptability in recent years (Wu et al., 2014).

Jeswiet, a Dutch sugarcane breeder, put forward the Noblization method of sugarcane breeding from 1989 to 1990 after summarizing and innovating, and bred the ‘king of sugarcane’ variety POJ2878, which opened up a new way of sugarcane improvement. The nobility of sugarcane is to use tropical species (noble species Noble Cane) as female parents, hybridize with the wild germplasm resources (Wild cane), and obtain hybrid offspring with wild consanguinity, and then continuously backcross with tropical species (or cultivated species) as male parents to obtain noble hybrids (Chen, 2003).

Saccharum spontaneum L. (2n=40~128), also known as the fine-stem wild species, is the most abundant species and genotypes in the wild species of sugarcane genus. It was first used in the noble breeding of sugarcane, with excellent characteristics such as vigorous growth, strong stress resistance, wide regional adaptability, more tillers, good perennial root and high sugar content. It is the most valuable wild germplasm resources to broaden the genetic basis of sugarcane (Peng, 1990, China Agricultural Press, pp.4-6; Chen et al., 1996; Chang et al., 2011).

In 1916, Barber from India, used the S.spont Co to hybridize with Vellai, Indian species constantly, and finally bred a series of Co varieties with disease resistance, high yield and high sugar (Chen, 2003). In Taiwan, China, sugarcane breeders have also made a breakthrough in the use of Saccharum spontaneum L., and bred Taiwan sugar series and new Taiwan sugar series (Liang, 1988, Sugarcane Industry, (6): 5-6). Hainan Sugarcane Breeding Station used large-stem wild species to introduce the wild Saccharum spontaneum L. and bred Yacheng series of innovative materials (Wu et al., 2014). In these breeding achievements, Saccharum spontaneum L. participated in ‘nobility’ as a male parent to introduce its wild consanguinity, and it was rarely reported that it was used as a female parent to participate in the ‘nobility’ of sugarcane.

There is a bottleneck in the identification of sugarcane varieties and hybrid offspring. Before DNA molecular technology was widely used, breeders studied more on the basis of isozyme markers, karyotype and chromosome analysis, as well as agronomic traits and morphology of sugarcane (Glaszmann et al., 1989; Chandra et al., 2001; Srivastava and Gupta, 2002). However, these technical methods have many constraints and have not been widely used.

Simple sequence repeats (SSR), which exist in the genome of eukaryotes, have the characteristics of wide distribution and random, codominant inheritance and abundant polymorphism (Piperidis et al., 2010; Smith and Devey, 1994; Wang et al., 2017), and tend to be distributed in gene-rich and low-repeat regions (Morgante et al., 2002). SSR molecular marker technology, established by Tautz (1989), has the advantages of good repeatability and simple analysis method. With the further development of DNA molecular marker technology, SSR technology has become an important method for variety identification in crop breeding. At present, SSR technology has been widely used in the identification of sugarcane varieties, true and false hybrids, gene mapping, genetic diversity analysis and assisted breeding (Liang et al., 2010).

The amplified products of SSR markers were mainly detected by capillary electrophoresis (CE), polyacrylamide gel electrophoresis (PAGE), and isotope marker primers. CE detection has better repeatability and higher accuracy, but the cost is also higher. In 2000, capillary electrophoresis was first used for the detection of PCR products of sugarcane SSR markers (Cordeiro et al., 2000). Since then, American breeders Pan et al. (2006; 2007) have used this technology to establish a database of germplasm resources, which has played an important role in sugarcane variety identification and hybrid breeding.

Combined with agronomic traits, a pair of SSR primers and capillary electrophoresis technology were used in this study to identify the hybrid offspring of YN 82-116 and GT 05-3595 and analyze their genetic diversity.

1 Results and Analysis

1.1 Genetic polymorphism of genomic DNA and identification of F₁ generation

After sowing and growing for 2 months, the hybrid offspring of the female parent YN 82-116 and male parent GT 05-3595 survived a total of 162, and 48 offspring with significant differences in agronomic traits between the female parent YN 82-116 were selected. A total of 28 SSR fragments of parents were amplified using MSSCIR1 primers, of which 9 were common bands of parents, 12 were specific bands of male parent, and 7 were specific bands of female parent, the polymorphic bands ratio was 67.9%. The size of SSR fragments ranged from 84 bp to 473 bp, and concentrated between 155 bp and 320 bp (Table 1; Figure 1). All 48 offspring had one or more specific bands of parents, so 48 F₁ hybrids were all real hybrids.

Table 1 SSR fingerprinting bands of YN 82-116 and GT 05-3595

Figure 1 Capillary electrophoresis results of YN 82-116 and GT 05-3595

1.2 Genetic similarity analysis of F₁ generation

According to 28 SSR markers, NTSYSPC2.1 software was used to calculate the genetic similarity coefficient matrix of parents and F₁ hybrids. The similarity coefficient ranged from 0.00 to 0.97, with an average of 0.58, indicating that there were differences in materials. Among them, the genetic similarity coefficient of the original parents YN 82-116 and GT 05-3595 was 0.49, indicating that there was a long genetic distance between the two varieties. The genetic similarity coefficient between offspring 19-5 and 19-22 was the minimum, which was 0.00, indicating that the genetic distance between the two offspring was the largest in all materials. While the genetic similarity coefficient between offspring 19-30 and 19-41 was the maximum, which was 0.97, indicating that the genetic distance between the two offspring was the closest.

1.3 Cluster analysis of F₁ generation

TUPGMA method was used to carry out the cluster analysis of the two parents and 48 F₁ generations. The results showed that the offspring clustered into one group had more similar genetic background (Figure 2). After UPGMA analysis, and the Cophenetic correlation test showed that the correlation coefficient was r=0.777, which indicated that the clustering result was better. When the genetic similarity coefficient is about 0.58, 50 materials can be divided into 6 categories. Among them, Class A contains male parent GT 05-3595 and 37 offspring, Class B only contains female parent YN 82-116, Class C contains 5 materials, Class D contains 19-21 and 19-47, Class E contains 19-4, 19-5, and 19-14, and Class F only contains 19-22. When the genetic similarity coefficient is about 0.62, Class A can be divided into 2 subclasses, and one of them contains female parent and 13 offspring.

Figure 2 UPMAG analysis of 50 materials Note: : Male parent, GT 05-3595; : Female parent, YN 82-116

2 Discussion

In the process of noble sugarcane breeding, breeders selected a single parent, resulting in a series of problems such as narrow genetic background. Although many countries have strengthened the collection of wild germplasm resources, and sugarcane germplasm resources are also very rich, they are not widely used in practical breeding (Chai et al., 2019). Saccharum spontaneum L. participated in ‘nobility’ as a male parent to introduce its wild consanguinity began with Jeswiet (Chen, 2003), which has made many achievements, but it was rarely reported that it was used as a female parent to participate in the breeding. This study used the YN 82-116 as a female parent to hybridize with the male parent GT 05-3595, and obtained wild consanguinity hybrid offspring with good resistance, high yield and high sugar content, and then participated in the process of sugarcane cross breeding.

In the early stage of the experiment, the offspring was screened by agronomic traits, and then SSR identification proved that the 48 hybrids were all true hybrids. According to the analysis of genetic similarity coefficient, the similarity coefficient among parents was only 0.49. There was a great difference. The overall average is 0.58, but the genetic similarity coefficient between hybrids is ranged from 0.00 to 0.97, which brings more choices for later breeding.

The agronomic traits of the 48 selected offspring were significantly different from those of the female parent, which was also verified by the clustering results, indicating that most offspring were closer to the male parent. The difference of genetic distance between the offspring was obvious, which indicated that the offspring of the same combination had great genetic difference. This may be because sugarcane was a highly heterozygous allopolyploid and highly segregated during hybridization (Wu et al., 2014, Molecular Plant Breeding, 12(1): 144-151).

In this study, YN 82-116 was used to introduce its wild excellent trait genes and strengthen the germplasm heterogeneity of sugarcane, so as to improve the consanguinity of sugarcane and select innovative germplasm, parents and new sugarcane varieties with breakthrough, good stress resistance, high efficiency, high yield and sugar content.

3 Materials and Methods

3.1 Test materials

Materials of this study were taken from Hainan Sugarcane Breeding Station of GSIRI (109.171N, 18.357E). The female parent YN 82-116 was planted in the wild resource nursery. The male parent GT 05-3595 was planted in the parent nursery. Two months after sowing and growing, 48 offspring with significant differences in agronomic traits were selected. Taq enzyme and DNA marker (20 000 bp plus DNA Ladder) Beijing TransGen Biotech products were commissioned to synthesize primers by Shanghai Sangon Biotech Co., Ltd. The automatic sample rapid grinding instrument Tissuelyser-96 was produced by Shanghaijingxin Experimental Technology Co., Ltd. PCR amplification instrument is Biometra PE9700, Germany. The nucleic acid protein analyzer is Q5000 ultramicro nucleic acid protein analyzer, which was produced by Quawell. The fragment analyzer is Fragment Analyzer^TM automatic capillary electrophoresis instrument.

3.2 DNA extraction method

DNA was extracted from young leaves by CTAB method. The 0.2g chopped sample was placed in 750 μL 1% CTAB buffer (containing 4 μL mercapto-ethanol), ground to the powder by the grinding instrument, removed in a water bath at 65℃ for 1 h, cooled to room temperature, and added the same volume of chloroform/isopropanol mixture (24:1), centrifuged for 10 min at 12 000 r/min, precipitated with anhydrous ethanol precooled at 4℃, purified with 75% alcohol, and finally dissolved and stored by ddH₂O.

The quality and concentration of the extracted DNA were determined by QUAWELL Q5000 microultraviolet spectrophotometer. The concentration of sample DNA was diluted to 5~10 ng/μL for further use.

3.3 SSR marker amplification and capillary electrophoresis detection

10 μL PCR reaction system, containing DNA template 5~10 ng, 1×BufferⅠ, dNTPs 0.2 mmol/L, TransTaq^® HiFi DNA Polymerase 5 units, forward and reverse primer 0.2 μmol/L. Primer sequence information (Table 2) (Pan, 2006). PCR procedure: 95°C for 3 min, 94°C for 30 s, 54°C for 30 s, 72°C for 40 s, 35 cycles. 72°C for 10 min, stored at 4°C.

Table 2 Primer information

The mixture of 2 μL PCR product and 22 μL PCR product diluent was placed on AATI automatic capillary electrophoresis instrument Fragment Analyzer for electrophoretic detection. During electrophoresis, the fluorescence signal was automatically recorded by the automatic capillary electrophoresis instrument and the fragment size was analyzed.

3.4 Judgement method

DNA samples were amplified by primers, and the size of amplified fragments was obtained by fragment analyzer. The amplified bands of offspring and parents were compared and analyzed. If the amplified bands of the offspring were all from the parents and contained specific bands of the parents, they were true hybrids. If offspring have no specific bands but only contained specific bands of the male parent, they were true hybrids. If the offspring contained specific bands that parents do not have, they were false hybrids. If the amplified bands of offspring only contained female parent, which may be self-bred, and other primers need to be used for identification and analysis (Lu et al., 2012).

3.5 Data analysis and genetic analysis

The fluorescence signal of the recorded SSR marker fragment was compared with the molecular weight standard by the Fragment Analyzer through electrophoresis time, and the size of the fragments at each signal was obtained in proportion. The collected data was analyzed by PROSize 3.0 software, and the final data was obtained by manual analysis combined with the statistical results of the software. SSR marker fragments were recorded as ‘1’, otherwise recorded as ‘0’, and 0-1 data were obtained (Levinson and Gutman,1987; Pan, 2006 ; Schlotterer and Tautz, 1992; Weber and May, 1989). The software NTSYSPC 2.1 was used for calculation and drawing statistics. UPGMA method was used for cluster analysis and genetic tree drawing.

Authors’ contributions

GYQ was the designer and executor of this study. FC and XHY completed the data analysis, drafted the manuscript. CHL and QYS participated in the design of the study, and the analysis of the test results. WQN conceived of the project, directed the design of the study, data analysis, draft and revision. All authors read and approved the final manuscript.

Acknowledgments

The study was supported by the Special Funding Project for the Construction of Domestic First-class Research Institutions of Guangdong Academy of Sciences (2020GDASYL-20200302005), Construction of Modern Agricultural Industrial Technology System (CARS-170107), National Natural Science Foundation of China (31701488), Special Project of Technological System Innovation Team of Sugarcane Sisal Industry in Guangdong Province (GARS-07-02), and Construction Project of Sugarcane Germplasm Resources Bank in Guangdong Province (2019B030316034).

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