Introduction
Sugarcane brown rust caused by Puccinia melanocephala H. & P. Sydow is an important epidemic fungal disease (Rott et al., Reference Rott, Girard and Comstook2013). Since it was first reported in Java in 1890, brown rust has spread, and there have been outbreaks in all the major sugarcane-cultivating countries, causing extensive yield losses and significant economic losses to the sugar industry (Purdy et al., Reference Purdy, Liu and Dean1983; Comstock et al., Reference Comstock, Shine and Raid1992; Raid and Comstock, Reference Raid, Comstock, Rott, Bailey, Comstock, Croft and Saumtally2000; Li et al., Reference Li, Cai, Huang, Fang and Ma2005; Hoy and Hollier, Reference Hoy and Hollier2009; Huang and Li, Reference Huang and Li2018). At present, brown rust disease is common in Guangdong, Guangxi, Fujian, Hainan and Yunnan Provinces of China, causing both germplasm degeneration and yield reduction, and the disease needs to be restricted (Ruan et al., Reference Ruan, Yang and Sun1983; Huang and Li, Reference Huang and Li1998; Li et al., Reference Li, Cai, Huang, Fang and Ma2005; Wei et al., Reference Wei, Deng, Huang, Pan, Wang and Liu2010). In the most affected areas, yield losses caused by brown rust are generally on the order of 15–30%, but can reach 50%, resulting in a decrease of 10–36% in sucrose content, which greatly affects the sustainable and stable development of the sugar industry in China (Raid and Comstock, Reference Raid, Comstock, Rott, Bailey, Comstock, Croft and Saumtally2000; Huang and Li, Reference Huang and Li2018).
The breeding of resistant cultivars and their adoption by growers is the most cost-effective control measure (Raid and Comstock, Reference Raid, Comstock, Rott, Bailey, Comstock, Croft and Saumtally2000). At present, two markers, R12H16 and 9O20-F4, based on the major resistance gene Bru1 from the cultivar ‘R570’, have been developed for brown rust resistance identification (Daugrois et al., Reference Daugrois, Grivet, Roques, Hoarau, Lombard, Glaszmann and D'Hont1996; Asnaghi et al., Reference Asnaghi, Roques, Ruffel, Kaye, Hoarau, Télismart, Girard, Raboin, Risterucci, Grivet and D'Hont2004; Cunff et al., Reference Cunff, Garsmeur, Raboin, Pauquet, Telismart, Selvi, Grivet, Philippe, Begum, Deu, Costet, Wing, Glaszmann and D'Hont2008). Although Bru1 is a major gene for brown rust resistance, reliance on a single gene and planting a single dominant variety across large areas could pose a serious threat to the sugar industry in the event of a disease epidemic (Rott et al., Reference Rott, Girard and Comstook2013). In recent years, some researchers have shown that the Bru1 gene is present in some brown rust-susceptible sugarcane varieties (Parco et al., Reference Parco, Avellaneda, Hale, Hoy, Kimbeng, Pontif, Gravois and Baisakh2014). Thus, discovery of further brown rust resistance genes is necessary.
Following many years of investigation, phenotypic identification and Bru1 gene detection (Li et al., Reference Li, Wang, Huang, Zhang, Shan, Yin and Luo2015a, Reference Li, Wang, Huang, Shan, Zhang, Yin and Luob, Reference Li, Wang, Huang, Zhang, Shan, Yin and Luo2016a, Reference Li, Wang, Huang, Zhang, Shan, Luo and Yinb), elite sugarcane varieties without the Bru1 gene but that were highly resistant to brown rust were selected as male parents, because they may contain novel brown rust resistance genes. Our research group found that ‘Yuetang 03-393’ × ‘ROC 24’ and ‘Liucheng 03-1137’ × ‘Dezhe 93-88’ genetically segregating populations were suitable for linkage genetic map construction and mapping of new brown rust resistance genes (Wang et al., Reference Wang, Li, Huang, Shan, Zhang, Li, Cang, Luo and Yin2019). The F1 hybrid populations derived from these two cross combinations both showed genetic segregation ratios of 3R:1S and 1R:3S, respectively, and because the Bru1 gene was not present, these results indicate that they may contain new brown rust resistance genes (Wang et al., Reference Wang, Li, Huang, Shan, Zhang, Li, Cang, Luo and Yin2019). On this basis, we used these two established genetically segregating populations to construct resistant and susceptible genetic pools and to screen polymorphic simple-sequence repeat (SSR) markers in the resistant and susceptible parents and the resistant/susceptible genetic pools.
Materials and methods
Tested plant materials
Two sugarcane parental lines, resistant male parents ‘ROC 24’ and ‘Dezhe 93-88’, that are highly resistant to brown rust disease and that lack the Bru1 gene, and two lines that are highly susceptible to brown rust, female parents ‘Yuetang 03-393’ and ‘Liucheng 03-1137’, were obtained from the National Germplasm Repository of Sugarcane (NNSGR, Kaiyuan, Yunnan Province, China). The authenticity of extreme high-resistance and extreme high-sensitivity of F1 single plants obtained from the hybrid combinations ‘Yuetang 03-393’ × ‘ROC 24’ and ‘Liucheng 03-1137’ × ‘Dezhe 93-88’, respectively, was confirmed previously (Wang et al., Reference Wang, Li, Huang, Shan, Zhang, Li, Cang, Luo and Yin2019).
DNA extraction and construction of the disease-resistant and -susceptible pools
Total DNA was extracted from the highly rust-resistant parental lines ‘ROC 24’ and ‘Dezhe 93-88’, and the highly susceptible parental lines ‘Yuetang 03-393’ and ‘Liucheng 03-1137’. We used bulked segregant analysis to construct the resistant and susceptible pools from the two genetically segregating populations. The pools were constructed using the following method: 10 individual plants showing an extremely high level of resistance were selected for DNA extraction, and equal amounts of each were mixed to constitute the resistant gene pool (R-pool); similarly, 10 individual plants showing extremely high susceptibility were selected, DNA was extracted and equal amounts from each plant were mixed to constitute the susceptible gene pool (S-pool).
Total DNA was extracted from leaf tissue (0.2 g) using the Easy Pure™ plant Genomic DNA Kit (TransGen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol. The quality of the extracted DNA was assessed using an AG22331 protein and nucleic acid analyzer (Eppendorf, Germany). The DNA concentration of each sample was diluted to 20 ng/μl, and the samples were stored at −20°C.
Screening polymorphic SSR primers in parental lines and genetic pools
For the initial screening to identify SSR markers linked to brown rust resistance and susceptibility, 499 pairs of SSR primers from the International Association of Microsatellites and the International Sugarcane Microsatellite Consortium (Cordeiro et al., Reference Cordeiro, Taylor and Henry2000; Pinto et al., Reference Pinto, Oliveira, Ulian, Garcia and de Souza2004; Pan, Reference Pan2006; Wang et al., Reference Wang, Roe, Macmi, Yu, Murray, Tang, Chen, Najar, Wiley, Bowers, Sluys, Rokhsar, Hudson, Moose, Paterson and Ming2010; Santos et al., Reference Santos, Pinto, Carlini-Garcia, Gazaffi, Mancini, Gonçalves, Medeiros, Perecin, Garcia, Souza and Zucchi2015; Yang et al., Reference Yang, Islam, Sushma, Maya, Hanson, Comstock and Wang2018) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and used in amplification reactions with genomic DNA from the resistant parents, the susceptible parents, the R-pools and the S-pools. First, we screened for polymorphism primers showing clear bands, obvious polymorphism and good repeatability between the parental lines. Then, we screened the polymorphism primers in the R-pools and S-pools.
SSR-PCR amplification and agarose gel electrophoresis
SSR-polymerase chain reaction (PCR) amplifications were performed in 10-μl reaction mixtures containing 4.0 μl ddH2O, 4.5 μl 2× Easy Taq PCR SuperMix for PAGE (TransGen Biotech Co., Ltd., Beijing, China), 0.5 μl total DNA template and 0.5 μl of each primer (10 μmol/l). The amplification conditions were: 4 min at 94°C, followed by 35 cycles of 15 s at 94°C, 15 s at 54°C and 1.0 min at 72°C, with a final extension for 5 min at 72°C.
PCR products were analysed by electrophoresis on an 8.0% modified polyacrylamide gel and stained using the rapid silver staining method (Wang et al., Reference Wang, Li, Huang, Shan, Zhang, Li, Cang, Luo and Yin2019). The band patterns were observed and the gels were photographed.
Results
Estimation of DNA quality
The genomic DNA samples extracted from the parental lines and the hybrid progeny in the genetically segregating populations were examined by 1% agarose gel electrophoresis, which showed that the extracted DNA was intact. DNA concentration and quality were measured using an Eppendorf AG 22331 protein/nucleic acid analyzer, and the A 260/A 280 ratios were about 1.8, which indicated that the DNA samples were suitable for subsequent PCR amplification. DNA concentration and quality of samples are shown in online Supplementary Table 1.
Screening of polymorphic SSR primers in resistant and susceptible parents
DNA extracted from the resistant and susceptible parental lines of each cross combination was individually amplified with each of the 449 pairs of primers. The results identified 25 pairs (Table 1) and 16 pairs (Table 2) of SSR primers that gave stable amplification of discreet DNA fragments with high levels of polymorphism between the parents of ‘Yuetang 03-393’ × ‘ROC 24’ and ‘Liucheng 03-1137’ × ‘Dechang 93-88’, respectively (Fig. 1). Among these, there were five pairs of primers that detected SSR polymorphisms between the resistant and susceptible parents of the two cross combinations: SMC2042FL, SMC286CS, MOLSSR2883, MOLSSR1707 and SCESSR0928. All of these primer pairs detected obvious polymorphisms between the parental lines, and gave stable amplification of DNA fragments and clear bands on the gels. These properties indicated that these polymorphic primer pairs could be used for subsequent polymorphism screening of the brown rust-resistant and -susceptible genetic pools.
SSR primer pairs that identified polymorphisms between the resistant and susceptible parental lines of the two sugarcane F1 cross combinations used in this study. Polyacrylamide gel (8%) showing polymorphic DNA fragments between the resistant and sensitive parents of the cross combination ‘Yuetang 03-393’ × ‘ROC 24’ (lanes 72–310) and the resistant and sensitive parents of the cross combination ‘Liucheng 03-1137’ × ‘Dezhe 93-88’ (lanes 92–249). M, molecular size marker.
Sequences of SSR primer pairs that detected polymorphic markers in ‘Yuetang 03-393’ × ‘ROC 24’
Sequences of SSR primer pairs that detected polymorphic markers in ‘Liucheng 03-1137’ × ‘Dezhe 93-88’
Screening of polymorphic SSR primers in resistant and susceptible genetic pools
The polymorphic SSR primer pairs selected from amplification of genomic DNA from the resistant and susceptible parents were used for further screening of the resistant and susceptible genetic pools. The results showed that four pairs of SSR primers (SMC236CG, SCESSR0928, SCESSR0636 and SCESSR2551) gave consistent amplification of polymorphic DNA fragment between the parental lines and the resistant/susceptible genetic pools derived from the cross combination ‘Yuetang 03-393’ × ‘ROC 24’ (Fig. 2). This indicated that the chromosomal loci corresponding to the polymorphic DNA fragments amplified by these four pairs of SSR primers may be linked to a gene for brown rust resistance in the segregating ‘Yuetang 03-393’ × ‘ROC 24’ population. Therefore, the four pairs of polymorphic SSR primers identified by screening can be used for the localization of novel brown rust resistance genes and the construction of linkage genetic maps in sugarcane. Unfortunately, we did not screen polymorphic SSR primers between the parental lines and the resistant/susceptible genetic pools from the cross combination ‘Liucheng 03-1137’ × ‘Dechang 93-88’.
Amplification of polymorphic DNA fragments between the parental lines ‘ROC 24’ and ‘Yuetang 03-393’ and the resistant and susceptible genetic pools using the four SSR primer pairs SMC236CG (A), SCESSR0928 (B), SCESSR0636 (C) and SCESSR2551 (D). PR, ‘ROC 24’; PS, ‘Yuetang 03-393’; BR, resistant genetic pool; BS, susceptible genetic pool; M, molecular size marker. The arrows indicate DNA fragments polymorphic between the parental lines ‘ROC 24’, ‘Yuetang 03-393’ and the resistant and susceptible genetic pools.
Discussion
Some sugarcane varieties, such as ‘ROC 22’, that once showed resistance to brown rust but are now susceptible are known to contain the Bru1 gene. This indicates a risk that the Bru1 gene may become less effective against P. melanocephala. Therefore, once a P. melanocephala epidemic becomes widespread, it will cause devastating losses to the whole sugar industry. The most effective way to prevent an increase in the prevalence of rust disease is to find novel sugarcane brown rust resistance genes, breed brown rust-resistant varieties and create new resistant, high-yielding germplasm. Previous studies showed that the Bru1 gene was not detectable in some rust-resistant varieties (Molina et al., Reference Molina, Rosales-Longo and Queme2013; Racedo et al., Reference Racedo, Perera, Bertani, Funes, Gonzalez, Cuenya, D'Hont, Welin and Castagnaro2013; Li et al., Reference Li, Wang, Huang, Zhang, Shan, Yin and Luo2015a; Zhang et al., Reference Zhang, Li, Huang, Lu, Wang, Shan, Li, Cang, Yin and Luo2019), indicating the brown rust resistance in these varieties may be not conferred by Bru1 but by other resistance genes.
Mining germplasm for novel genes and using molecular methods to improve varieties has become an effective strategy for plant breeding (Li et al., Reference Li, Lin, Zi, Li, Xu, Wu, Zhu, Liu, Fang and Liu2019). Li et al. (Reference Li, Xu, Su, Wu, Cheng, Sun and Gao2018) reported that, in addition to Bru1, there are still unidentified major genes that control disease resistance in the sugarcane gene pool. Moreover, male parents tend to transmit a higher frequency of rust-susceptible genes than female parents, which was confirmed by the results of this study; the genetic segregation ratio in an F1 hybrid population derived from the cross combination ‘Yuetang 03-393’ × ‘ROC 24’ was 3R:1S, and Bru1 gene was not detected in their F1 hybrids, these indicated that two dominant novel genes controlled brown rust resistance in ‘Yuetang 03-393’ × ‘ROC24’ (Wang et al., Reference Wang, Li, Huang, Shan, Zhang, Li, Cang, Luo and Yin2019). ‘Yuetang 03-393’ was the susceptible female parent and ‘ROC 24’ was the resistant male parent. Sugarcane is a highly heterogeneous polyploid plant with a very large genome, a high number of chromosomes and a complex genetic background. These factors can complicate the discovery and characterization of brown rust resistance genes. In this study, four SSR molecular markers which may be linked to a new brown rust resistance gene were detected by screening the resistant and susceptible genetic pools constructed from a genetically segregating population derived from ‘Yuetang 03-393’ × ‘ROC 24’. The results of our study could provide useful genetic information for the mapping of new brown rust resistance genes in sugarcane germplasm and the development of linked molecular marker loci in the future.
Conclusions
In this study, 449 pairs of SSR primers were designed based on information in the literature. We identified 25 pairs of SSR primers that directed amplification of polymorphic DNA fragments between the parental lines ‘Yuetang 03-393’ and ‘ROC 24’, and 16 pairs of SSR primers that showed polymorphisms between the parental lines ‘Liucheng 03-1137’ and ‘Dezhe 93-88’. Four pairs of SSR primers amplified polymorphic DNA fragments between the parental lines and also in the resistant/susceptible genetic pools derived from the cross combination ‘Yuetang 03-393’ × ‘ROC 24’. Our preliminary results determined that the polymorphic fragments may be linked to the novel brown rust resistance gene, which will be important for the subsequent localization of the gene, the construction of a linkage genetic map and the development of closely-associated molecular markers. Our study also lays a foundation for accelerating the mining of novel sugarcane brown rust resistance genes. The discovery of new brown rust resistance genes is of theoretical and practical significance to prevent the widespread outbreak of disease epidemics and to ensure the safe and sustainable production of sugarcane in China.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262120000428.
Acknowledgements
This study was supported by the Natural Science Foundation of China (31660419), Sugar Crop Research System (CARS-170303), the Yunling Industry and Technology Leading Talent Training Program ‘Prevention and Control of Sugarcane Pests’ (2018LJRC56) and the Yunnan Province Agriculture Research System.
Conflict of interest
The authors declare that they have no conflict of interest.
