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Abstract As an excellent local crop variety of Huanggang, "Huangzhou radish" has been widely promoted and its market influence is increasing day by day. Recently, the purity of "Huangzhou radish" has declined deeply, so it is urgent to improve and strengthen the purity and viability of "Huangzhou radish" including bad phenomena such as degeneration of varieties after self-pollination. At present, there was no SSR molecular marker can be used for genetic diversity identification of "Huangzhou radish". SSR molecular marker can provide a favorable tool to study "Huangzhou radish". In this study, "Xiakang", "Hongbao"and "Huangzhou radish" were used as experimental materials, 100 SSR primers were screened, and 25 pairs of SSR primers were selected. Finally, two pairs of primers were selected for identification of the purity of "Huangzhou radish" by non-denatured polyacrylamide gel electrophoresis (PAGE). The results showed that R-102 and R-112 primer pairs could amplify the special bands for "Huangzhou radish" from above three kinds of radishes. These two pairs of primers could be developed as SSR markers to identify the specificity and purity of "Huangzhou radish". Furthermore, this study provides convenient conditions for promoting the economic and social value of "Huangzhou radish".
Key words "Huangzhou radish"; SSR molecular marker; Marker development
"Huangzhou radish" is a cultivar with local characteristics in Huangzhou District, Huanggang City, Hubei Province, China. In September 2008, "Huangzhou radish" was approved as a national geographical indication protection product. For the development of plant varieties based on DNA variation, molecular marker technology is undoubtedly a relatively efficient and stable means. The most widely used molecular markers are AFLP, SSR, RAPD, RFLP, etc. Among them, SSR marker is a DNA fingerprinting technology based on PCR, which is also a co-dominant marker with good reproducibility, less template required for PCR amplification reaction, convenient detection, high polymorphism, high sensitivity and stable results［1-3］. SSR (simple sequence repeat) is a kind of simple sequence repeat, that is, microsatellite DNA, which is a simple repeat sequence consisting of 1 to 6 base pairs. In recent years, microsatellite DNA has become one of the most commonly used molecular markers in evolutionary biology and population research, and is widely used in genetic linkage maps, bio-hybrid breeding, phylogeny and population genetic diversity［4］. Based on the radish gene sequences obtained from the transcriptome, this study developed "Huangzhou radish"-specific SSR molecular markers, aiming at providing an effective tool and evidence for the identification of "Huangzhou radish" and its purity. Material and Methods
Experimental materials
"Huangzhou radish", "Xiakang radish" and "Hongbao radish" were from the Dabie Mountain biological resource bank. Primers were designed using PRIMER 3 (http://primer3.ut.ee/) and synthesized by Genscript Biotech (https://www.genscript.com.cn/).
DNA extraction
The experimental materials "Xiakang radish", "Hongbao radish" and "Huangzhou radish" were placed in a 25 ℃ incubator for 3 d. After the seed cotyledons formed, the cotyledons (100 mg of leaves) were taken. The TIANGEN plant genome kit was used to extract the total gene of the radish cotyledon according to the kit method. Finally, the quality of the DNA was measured using 1% agarose gel.
PCR amplification and amplification product detection
The total system for PCR amplification was as follows: 12.5 μl of 2×Taq PCR MasterMix, 1 μl of each of the positive and negative primers, 0.5 μl of the template, and finally double-distilled water to 25 μl. The PCR reaction process was as started with pre-denaturation at 94 ℃ for 4 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 55-60 ℃ for 30 s and extension at 72 ℃ for 30 s. The amplification product sizes were determined by agarose gel electrophoresis and polyacrylamide gel after PCR.
PAGE gel analysis of radish purity
According to the results of non-denaturing polyacrylamide gel electrophoresis of figures of "Hongbao radish", "Xiakang radish" and "Huangzhou radish", the purity of "Huangzhou radish" was analyzed to find specific target bands, and the microsatellite DNA sequences corresponding to the specific target bands can be put into use as new SSR molecular markers.
Results and Analysis
Screening of SSR primers by agarose gel electrophoresis
With 25 pairs of primers obtained by preliminarily screened (Table 1), amplified bands of corresponding sizes can be detected in all the three varieties, i.e., "Huangzhou radish", "Xiakang radish" and "Hongbao radish". There was a clear and distinct band between 150 and 250 bp. To further confirm the differences in above band between different varieties, high-resolution detection was performed by polyacrylamide gel electrophoresis based on agarose gel electrophoresis detection.
Differential performance of different SSR primers between varieties
All above 25 pairs of primers were able to identify the detected samples as radish, and the R102 and R112 primer pairs were able to specifically identify whether the detected sample was "Huangzhou radish". The amplification results with R102 primer pair (Fig. 1): There was a significant difference between "Huangzhou radish" and other radishes by PCR amplification and PAGE detection, and only "Huangzhou radish" showed the target band at 227 bp, while other radishes did not exhibit any band at 227 bp. Therefore, the R102 marker can be used as a specific marker to identify "Huangzhou radish". The amplification results with R112 primer pair (Fig. 2): The PCR bands and PAGE bands were significantly different between other radishes and "Huangzhou radish". The above radishes were all able to amplify the target band (251 bp), but only "Huangzhou radish" showed a specific band at 400 bp, while other radishes did not give any band at 400 bp. Therefore, the R112 marker can be used as a specific marker to identify "Huangzhou radish".
In the PAGE gel detection of the red radish ("Hongbao") and white radish ("Xiakang" 40 d), only one set was selected with specificity for each variety, which was R-115 and R-109, respectively. One SSR marker can be used to identify the red radish, and the other can be used to identify the white radish. These markers can be used to identify the purity of white radish, red radish and "Huangzhou radish", respectively, and can also identify the radish species of "Xiakang", "Hongbao" and "Huangzhou radish" from mixed radish lines. The primers used in the whole identification process were also used to detect 11 different varieties of white and green radishes including "Duanye 13" and "Wuqing radish". The results showed that R102 and R112 in above markers could produce specific bands in "Huangzhou radish", indicating R102 and R112 can indeed be developed as SSR molecular markers for identifying the characteristics and purity of "Huangzhou radish".
Conclusions and Discussion
In this study, molecular markers in "Huangzhou radish" were developed based on SSR molecular marker. In order to give full play to the social value and economic value of "Huangzhou radish" as a GI product, the screening of "Huangzhou radish" from "Huangzhou radish", "Xiakang radish" and "Hongbao radish" as experiment materials was explored through SSR molecular marker analysis, aiming at providing various theoretical basis for the protection, utilization and development of germplasm resources of "Huangzhou radish". The development of new SSR molecular markers in crops has been reported in many cash crops such as tea tree［5］, mung bean［6］and peanut［7］.
Previously, some scholars have also conducted extensive research and utilization of white fleshy radish resources［8-12］, but due to the geographical specificity of "Huangzhou radish", the research reports on it are relatively fewer, which restricts the development and utilization of the germplasm resources of "Huangzhou radish". In 2012, Fang et al.［8］also used SSR molecular marker technology to detect the genetic diversity of 37 succulent radish varieties from different regions of China. In 2014, Qiu et al.［13］used SSR molecular marker to identify 75 kinds of radishes, and selected 8 pairs of SSR primers to effectively distinguish the 75 radish germplasms. Wang et al.［14］used 160 pairs of SSR primers to construct the fingerprints of 16 autumn red radish hybrids and their parents. Finally, such 6 pairs of primers as PR104, RSS987, RSS2491, RSS3328, RSS3540 and RSS3830 were selected, and the fingerprints of the 16 autumn red radish hybrids were constructed using the selected primers, thereby realizing the rapid accurate molecular identification of autumn red radish varieties. It can be seen that the SSR molecular marker technology is not only mature in the study on other species［15-16］, but also on radish. Based on above research basis, this study screened the specific molecular markers of "Huangzhou radish", which provide an effective tool for fingerprint analysis in radish, identification of "Huangzhou radish" and purity identification in "Huangzhou radish" population.
Acknowledgements
Wang Pingan and Yao Jinlan are the executors of the experimental design and experimental research of this study; Zengcheng Qiuyu, Li Jingcai, Fang Yuanping, Li Shisheng and Wang Linling completed the data analysis and the writing of the first draft of paper; Li Shisheng carried out the experimental design, paper writing and revision. All authors read and agree to the final text.
References
［1］SMITH JSC, CHIN ECL, SHU H, et al. An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPs and pedigree［J］. Theoretical and Applied Genetics, 1997, 95(1/2): 163-173.
［2］YAO HW, ZHANG LD, SUN JY, et al. Summary of DNA molecular marker technology［J］. Hebei Yuye, 2010, 7(20): 42-46.
［3］LI SS, SUN CY, JIANG SC, et al. SSR molecular markers and its applications in plant genetics and breeding［J］. Jilin Vegetables, 2014, (5): 33-38.
［4］GOLDSTEIN DB, POLLOCK DD. Launching microsatellites: A review of mutation processes and methods of phylogenetic interference［J］. Hered, 1997, (88): 335-342.
［5］TAN LQ. Development of SSR markers and construction of genetic linkage map in tea plant［D］. Yaan: Sichuan Agricultural University, 2013.
［6］WU CS. Development of SSR markers and construction of a genetic linkage map in mungbean (Vigna radiate L.)［D］. Lanzhou: Gansu Agricultural University, 2014.
［7］WANG JY, YANG QL, YU SL. Development and application of SSR Markers in peanut［J］. Chinese journal of oil crop sciences, 2009, 31(3): 401-406. ［8］FANG P, CHEN FB, YAO QL, et al. Genetic diversity of radish (Raphanus sativus L.) with different fleshy colors based on SSR data ［J］. Journal of Plant Genetic Resources, 2012, 13(2): 226-232.
［9］YAMANE K, NA L, OHNISHI O. Chloroplast DNA variations of cultivated radish and its wild relative［J］. Plant Sci, 2005, 168: 627-634.
［10］MADHOU P, WELLS A, PANG ECK, et al. Genetic variation in populations of Western Australian wild radish［J］. Austr J Agric Res, 2005, 56(10): 1079-1087.
［11］LI JC. Analysis of genetic diversity of radish germplasm resourse using SSR and establilashment of the primary core collection［D］. Chongqing: Southwest University, 2006.
［12］YAMANE K, LU N, OHNISHI O. Multiple origins and high genetic diversity of cultivated radish inferred from polymorphism in chloroplast simple sequence repeats［J］. JapaneSoci Breed, 2009, 59(1): 55-65.
［13］QIU Y, LI XX, LI QX, et al. Establishment of the molecular identification for radish germplasm using SSR markers［J］. Journal of Plant Genetic Resources, 2014, 15(3): 648-654.
［14］WANG QB, ZHANG L, ZHENG PJ, et al. Establishment and application of SSR fingerprint in autumn red radish hybrids and their parents (Raphanus sativus L.)［J］. Molecular Plant Breeding, 2015, 13(8):1794-1801.
［15］YANG X, LIU F, ZHANG Y, et al. Study on the genetic diversity of eggplant germplasm with SSR markers［J］. Genomics and Applied Biology, 2016(12):251-258.
［16］DENG CL, GAO J, CAO Y, et al. Development and application of sex-specific EST-SSR marker in spinach (Spinacia oleracea L.)［J］. Genomics and Applied Biology, 2012, 31(5): 467-472.
Key words "Huangzhou radish"; SSR molecular marker; Marker development
"Huangzhou radish" is a cultivar with local characteristics in Huangzhou District, Huanggang City, Hubei Province, China. In September 2008, "Huangzhou radish" was approved as a national geographical indication protection product. For the development of plant varieties based on DNA variation, molecular marker technology is undoubtedly a relatively efficient and stable means. The most widely used molecular markers are AFLP, SSR, RAPD, RFLP, etc. Among them, SSR marker is a DNA fingerprinting technology based on PCR, which is also a co-dominant marker with good reproducibility, less template required for PCR amplification reaction, convenient detection, high polymorphism, high sensitivity and stable results［1-3］. SSR (simple sequence repeat) is a kind of simple sequence repeat, that is, microsatellite DNA, which is a simple repeat sequence consisting of 1 to 6 base pairs. In recent years, microsatellite DNA has become one of the most commonly used molecular markers in evolutionary biology and population research, and is widely used in genetic linkage maps, bio-hybrid breeding, phylogeny and population genetic diversity［4］. Based on the radish gene sequences obtained from the transcriptome, this study developed "Huangzhou radish"-specific SSR molecular markers, aiming at providing an effective tool and evidence for the identification of "Huangzhou radish" and its purity. Material and Methods
Experimental materials
"Huangzhou radish", "Xiakang radish" and "Hongbao radish" were from the Dabie Mountain biological resource bank. Primers were designed using PRIMER 3 (http://primer3.ut.ee/) and synthesized by Genscript Biotech (https://www.genscript.com.cn/).
DNA extraction
The experimental materials "Xiakang radish", "Hongbao radish" and "Huangzhou radish" were placed in a 25 ℃ incubator for 3 d. After the seed cotyledons formed, the cotyledons (100 mg of leaves) were taken. The TIANGEN plant genome kit was used to extract the total gene of the radish cotyledon according to the kit method. Finally, the quality of the DNA was measured using 1% agarose gel.
PCR amplification and amplification product detection
The total system for PCR amplification was as follows: 12.5 μl of 2×Taq PCR MasterMix, 1 μl of each of the positive and negative primers, 0.5 μl of the template, and finally double-distilled water to 25 μl. The PCR reaction process was as started with pre-denaturation at 94 ℃ for 4 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 55-60 ℃ for 30 s and extension at 72 ℃ for 30 s. The amplification product sizes were determined by agarose gel electrophoresis and polyacrylamide gel after PCR.
PAGE gel analysis of radish purity
According to the results of non-denaturing polyacrylamide gel electrophoresis of figures of "Hongbao radish", "Xiakang radish" and "Huangzhou radish", the purity of "Huangzhou radish" was analyzed to find specific target bands, and the microsatellite DNA sequences corresponding to the specific target bands can be put into use as new SSR molecular markers.
Results and Analysis
Screening of SSR primers by agarose gel electrophoresis
With 25 pairs of primers obtained by preliminarily screened (Table 1), amplified bands of corresponding sizes can be detected in all the three varieties, i.e., "Huangzhou radish", "Xiakang radish" and "Hongbao radish". There was a clear and distinct band between 150 and 250 bp. To further confirm the differences in above band between different varieties, high-resolution detection was performed by polyacrylamide gel electrophoresis based on agarose gel electrophoresis detection.
Differential performance of different SSR primers between varieties
All above 25 pairs of primers were able to identify the detected samples as radish, and the R102 and R112 primer pairs were able to specifically identify whether the detected sample was "Huangzhou radish". The amplification results with R102 primer pair (Fig. 1): There was a significant difference between "Huangzhou radish" and other radishes by PCR amplification and PAGE detection, and only "Huangzhou radish" showed the target band at 227 bp, while other radishes did not exhibit any band at 227 bp. Therefore, the R102 marker can be used as a specific marker to identify "Huangzhou radish". The amplification results with R112 primer pair (Fig. 2): The PCR bands and PAGE bands were significantly different between other radishes and "Huangzhou radish". The above radishes were all able to amplify the target band (251 bp), but only "Huangzhou radish" showed a specific band at 400 bp, while other radishes did not give any band at 400 bp. Therefore, the R112 marker can be used as a specific marker to identify "Huangzhou radish".
In the PAGE gel detection of the red radish ("Hongbao") and white radish ("Xiakang" 40 d), only one set was selected with specificity for each variety, which was R-115 and R-109, respectively. One SSR marker can be used to identify the red radish, and the other can be used to identify the white radish. These markers can be used to identify the purity of white radish, red radish and "Huangzhou radish", respectively, and can also identify the radish species of "Xiakang", "Hongbao" and "Huangzhou radish" from mixed radish lines. The primers used in the whole identification process were also used to detect 11 different varieties of white and green radishes including "Duanye 13" and "Wuqing radish". The results showed that R102 and R112 in above markers could produce specific bands in "Huangzhou radish", indicating R102 and R112 can indeed be developed as SSR molecular markers for identifying the characteristics and purity of "Huangzhou radish".
Conclusions and Discussion
In this study, molecular markers in "Huangzhou radish" were developed based on SSR molecular marker. In order to give full play to the social value and economic value of "Huangzhou radish" as a GI product, the screening of "Huangzhou radish" from "Huangzhou radish", "Xiakang radish" and "Hongbao radish" as experiment materials was explored through SSR molecular marker analysis, aiming at providing various theoretical basis for the protection, utilization and development of germplasm resources of "Huangzhou radish". The development of new SSR molecular markers in crops has been reported in many cash crops such as tea tree［5］, mung bean［6］and peanut［7］.
Previously, some scholars have also conducted extensive research and utilization of white fleshy radish resources［8-12］, but due to the geographical specificity of "Huangzhou radish", the research reports on it are relatively fewer, which restricts the development and utilization of the germplasm resources of "Huangzhou radish". In 2012, Fang et al.［8］also used SSR molecular marker technology to detect the genetic diversity of 37 succulent radish varieties from different regions of China. In 2014, Qiu et al.［13］used SSR molecular marker to identify 75 kinds of radishes, and selected 8 pairs of SSR primers to effectively distinguish the 75 radish germplasms. Wang et al.［14］used 160 pairs of SSR primers to construct the fingerprints of 16 autumn red radish hybrids and their parents. Finally, such 6 pairs of primers as PR104, RSS987, RSS2491, RSS3328, RSS3540 and RSS3830 were selected, and the fingerprints of the 16 autumn red radish hybrids were constructed using the selected primers, thereby realizing the rapid accurate molecular identification of autumn red radish varieties. It can be seen that the SSR molecular marker technology is not only mature in the study on other species［15-16］, but also on radish. Based on above research basis, this study screened the specific molecular markers of "Huangzhou radish", which provide an effective tool for fingerprint analysis in radish, identification of "Huangzhou radish" and purity identification in "Huangzhou radish" population.
Acknowledgements
Wang Pingan and Yao Jinlan are the executors of the experimental design and experimental research of this study; Zengcheng Qiuyu, Li Jingcai, Fang Yuanping, Li Shisheng and Wang Linling completed the data analysis and the writing of the first draft of paper; Li Shisheng carried out the experimental design, paper writing and revision. All authors read and agree to the final text.
References
［1］SMITH JSC, CHIN ECL, SHU H, et al. An evaluation of the utility of SSR loci as molecular markers in maize (Zea mays L.): comparisons with data from RFLPs and pedigree［J］. Theoretical and Applied Genetics, 1997, 95(1/2): 163-173.
［2］YAO HW, ZHANG LD, SUN JY, et al. Summary of DNA molecular marker technology［J］. Hebei Yuye, 2010, 7(20): 42-46.
［3］LI SS, SUN CY, JIANG SC, et al. SSR molecular markers and its applications in plant genetics and breeding［J］. Jilin Vegetables, 2014, (5): 33-38.
［4］GOLDSTEIN DB, POLLOCK DD. Launching microsatellites: A review of mutation processes and methods of phylogenetic interference［J］. Hered, 1997, (88): 335-342.
［5］TAN LQ. Development of SSR markers and construction of genetic linkage map in tea plant［D］. Yaan: Sichuan Agricultural University, 2013.
［6］WU CS. Development of SSR markers and construction of a genetic linkage map in mungbean (Vigna radiate L.)［D］. Lanzhou: Gansu Agricultural University, 2014.
［7］WANG JY, YANG QL, YU SL. Development and application of SSR Markers in peanut［J］. Chinese journal of oil crop sciences, 2009, 31(3): 401-406. ［8］FANG P, CHEN FB, YAO QL, et al. Genetic diversity of radish (Raphanus sativus L.) with different fleshy colors based on SSR data ［J］. Journal of Plant Genetic Resources, 2012, 13(2): 226-232.
［9］YAMANE K, NA L, OHNISHI O. Chloroplast DNA variations of cultivated radish and its wild relative［J］. Plant Sci, 2005, 168: 627-634.
［10］MADHOU P, WELLS A, PANG ECK, et al. Genetic variation in populations of Western Australian wild radish［J］. Austr J Agric Res, 2005, 56(10): 1079-1087.
［11］LI JC. Analysis of genetic diversity of radish germplasm resourse using SSR and establilashment of the primary core collection［D］. Chongqing: Southwest University, 2006.
［12］YAMANE K, LU N, OHNISHI O. Multiple origins and high genetic diversity of cultivated radish inferred from polymorphism in chloroplast simple sequence repeats［J］. JapaneSoci Breed, 2009, 59(1): 55-65.
［13］QIU Y, LI XX, LI QX, et al. Establishment of the molecular identification for radish germplasm using SSR markers［J］. Journal of Plant Genetic Resources, 2014, 15(3): 648-654.
［14］WANG QB, ZHANG L, ZHENG PJ, et al. Establishment and application of SSR fingerprint in autumn red radish hybrids and their parents (Raphanus sativus L.)［J］. Molecular Plant Breeding, 2015, 13(8):1794-1801.
［15］YANG X, LIU F, ZHANG Y, et al. Study on the genetic diversity of eggplant germplasm with SSR markers［J］. Genomics and Applied Biology, 2016(12):251-258.
［16］DENG CL, GAO J, CAO Y, et al. Development and application of sex-specific EST-SSR marker in spinach (Spinacia oleracea L.)［J］. Genomics and Applied Biology, 2012, 31(5): 467-472.