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Abstract In this study， Paprika 247 with milk white fruit at marketable maturity stage as male parent and capsicum 246 with green fruit at marketable maturity stage as female parent were selected as experimental materials to screen SSR molecular markers closely related to white fruit character， so as to lay a foundation for the use of SSR technique for molecular marker-assisted breeding and provide reference for breeding of new white capsicum germplasms and varieties. 360 pairs of SSR primers were screened in total， and among them， 3 pairs of primers （ES-89， ES-292， ES-296） exhibited remarkable polymorphic differences. This study will provide a basis for next QTL mapping.
Key words Capsicum； White character； SSR analysis
Capsicum is a kind of important vegetable crop planted worldwide. The planting area of capsicum has exceeded 1 333 333 hm2 in China， and its annual yield accounts for nearly a half of the total yield in the world. Capsicum industry is one of the largest vegetable industries in China. The sales volume of capsicum depends to a large extent on fruit color， which as an important flavor and quality character of capsicum is worthy of research and discussion. Capsicum has various colors， and played a role of model plant in heredity of fruit color in previous studies[1]. Therefore， the research on fruit color and corresponding molecular mechanism of capsicum is of great significance to the genetic breeding of capsicum and the molecular regulation of fruit color of capsicum. Currently， there are more studies on red and green characters， but few studies have been conducted on white character of capsicum. Therefore， it is urgent to study the white character of capsicum， so as to understand the genetic law of the white character， which could provide powerful reference for capsicum breeding for quality. The inheritance of the white character of capsicum was selected as a research object in this study.
Color of ripe fruit of sweet pepper could be identified and selected at seedling stage by molecular marker technology， so as to breed ideal fruit and accelerate the breeding of pure lines of colored capsicum， which is very attractive to breeders who are studying the genetic mechanism of capsicum fruit color[2]. After many years of study， several genes controlling the color of main fruit of capsicum could be determined， for instances， A gene controls purple character， CCS controls yellow character， and PSY gene controls orange character， but the corresponding molecular marker technique applied in experiments was RELP markers. However， this type of markers have more disadvantages such as tedious operation and higher cost， so the application in practical breeding is very difficult. Therefore， the development of other more convenient markers such as SSR and InDel has high value， and is of great significance. In this study， with homozygous inbred lines 246 and 247 as male and female parents， respectively， SSR molecular markers closely related to white character were screened. This study lays a foundation for molecular mark assisted breeding by SSR technique， and enriches the gene pool of capsicum， thereby providing reference for the breeding of new white-color capsicum germplasms and varieties.
Materials and Methods
Tested materials
The tested materials were provided by capsicum research group of Horticultural Research Institute， Anhui Academy of Agricultural Sciences， as shown in Table 1.246 is a high-generation inbred line， and 247 is an inbred line obtained from three generations of backcross of white capsicum variety and 246 followed by 5 generations of selfing and directed screening.
Plug seedlings
The experiment was carried out in College of Horticulture， Anhui Agricultural University and the Experimental Base and Molecular Biology Laboratory of Horticultural Research Institute， Anhui Academy of Agricultural Sciences. The experimental materials were seeded in hole trays， and when each plant had 3 true leaves， capsicum genomic DNA was extracted by improved CTAB method.
Extraction and identification of genomic DNA
DNA extraction by CTAB method
The DNA was extracted by improved CTAB method. Tender leaves of capsicum plants were picked and grinded in a centrifuge tube added with 2% CTAB. The mixture was heated in a water bath at 65 ℃ for 20 min， and after mixing well， centrifugation was performed for 10 min. The supernatant was added with equal volume of chloroform and isoamyl alcohol， followed by mixing and 10 min of centrifugation. Into the obtained supernatant， isopropanol with a volume 2/3 of the supernatant was added， and after freezing， transparent gel precipitate was obtained and flushed with 70% ethanol. Finally， TE was added to dissolve the precipitate， obtaining the extracted DNA which was preserved in a refrigerator at -20 ℃.
DNA quality detection
DNA concentration and OD value were determined with a Spectrophotometer nucleic acid detector， and the according to the detected DNA concentration， all the sample was diluted to 10 ng/μl for later use.
DNA purity was detected by 1.2% agarose gel electrophoresis and nucleic acid dye， and photographing was performed with a gel imaging system. If OD260/OD280>1.8， the protein has a content lower than 0.3%， and could be studied at molecular level， and if OD260/OD280<1.8， the DNA sample has more impurities， and should be extracted and further purified. SSR markers
Source of SSR primers
The SSR primers were designed through related NCBI websites. After dissolution according to instructions， the primers were diluted by 10 times， subpackaged， and placed in an environment at 4 ℃ for later use. The primers were preserved at -20 ℃.
SSR reaction system
The reaction system in this experiment was slightly different from the PCR reaction system improved by Hu and standard SSR reaction system. Taq DNA polymerase， dNTPs， MgCl2， Buffer and dye for PCR were purchased from Tiangen Biotech （Beijing） Co.， Ltd. The SSR reaction system was 10 μl， including buffer 1.0 μl， 2.5 μmol/L dNTP 1.0 μl， 25 mmol MgCl2 1.0 μl， 10 μmol/L forward and reverse primers 1.0 μl in total （0.5 μl each）， 5 μmol/L Taq enzyme 0.1 μl， 10 ng/μl DNA 2.0 μl， and ddH2O 3.9 μl. The PCR was started with pre-denaturation at 4 min for 94 ℃， followed by 35 cycles of denaturation at 94 ℃ for 30 s， annealing at 53 ℃ for 30 s， and extension at 72 ℃ for 1 min， and completed by extension at 72 ℃ for 7 min. Then， 3 μl of PCR product was detected by 8% non-denaturing PAGE[3-5].
Polyacrylamide gel electrophoresis
After the assembling of electrophoresis apparatus， polyacrylamide gel was rapidly poured into the gap of glass plate. Pre-electrophoresis was performed under voltage of 150 V and current of 350 mA for 15 min. Then， sampling application was performed with GM345 as Marker， and 3 μl of sample was applied each time. Finally， electrophoresis was performed under voltage at 150 V and current at 350 mA for 100 min. The polyacrylamide gel contained 30% PA solution 12.15 ml， deionized water 28.13 ml， TBE 4.5 ml， TEMED 36 μl and AP 0.6 ml.
After electrophoresis， silver staining was performed， and after fixation of gel， observation was performed under a UV lamp. Band pattern was recorded and photographed.
Results and Analysis
Detection of capsicum genomic DNA
According to the detection with the Spectrophotometer nucleic acid detector， the OD values of single plants of parents were in the range of 1.8-2.0.
Quality detection was then performed to the extracted capsicum DNA by 1.2% agarose gel electrophoresis. The bands were clear， tidy， complete， without tailing （Fig. 1）. The sample satisfied the requirement by SSR molecular marker amplification.
Screening of capsicum polymorphic markers
With the DNA of high-generation homozygosis inbred line parents 246 and 247 as templates， PCR was performed， obtaining 360 pairs of capsicum SSR primers， among which 3 pairs of polymorphically differential primers， i.e.， ES-89 （Fig. 2）， ES-292 （Fig. 3） and ES-296 （Fig. 4）. The polymorphic rate was 0.84%， and the polymorphic markers were all co-dominant markers. Markers SSR89， SSR292 and SSR296 exhibited clear bands with remarkable polymorphic differences. This result provides a basis for subsequent QTL mapping. Discussion
In previous studies about the inheritance of fruit color of agricultural products， most scholars attributed it to quality character， but it could be seen from the genetic laws of multiple crops that the inheritance of fruit color often has the characteristics of continuous variation， and fruit color is not a simple quality character[7-8]. The inheritance of capsicum color is mainly controlled by main genes， and less affected by polygene heritability and environment effect， and therefore， fruit color of capsicum at marketable maturity stage could be selected during segregative generation at early stage， thereby providing a basis for breeding for capsicum color[9].
Molecular marker technology develops the understanding of quantitative characters to the analysis of quantitative trait loci （QTL）[10]. With the application of molecular marker technology， known DNA fragments could be amplified to obtain analysis data for the creation of conditions for deep research on the genetic mechanism of capsicum color， with an attempt to improve research possibility， accuracy and foreseeability.
In this study， high-generation inbred lines with different colors were subjected to SSR molecular marker analysis， 360 pairs of SSR capsicum primers were screened， and 3 pairs of primers among them showed specific bands. This study provides certain theoretical basis for heterosis breeding and molecular mark-assisted breeding for capsicum color， and lays a basis for QTL mapping.
References
[1] ZHANG FF， WANG LH， HU H， et al. Review of fruits color and related pigments in hot pepper[J]. Journal of China Capsicum， 2010（2）： 51-97. （in Chinese）
[2] LI DW， HAO WJ， GONG ZH. Advances in genetics and molecular biology of fruit colour in hot pepper[J]. Journal of China Capsicum， 2014（2）： 1-5. （in Chinese）
[3] LUO B， SUN HY， XU GM， et al. Research progress of SSR molecular markers[J]. Journal of Anhui Agricultural Sciences， 2013， 41（12）： 5210-5212， 5246. （in Chinese）
[4] LIU ZJ， LI JT， YANG Y， et al. SSR molecular markers identification of pepper hybrid and comparative analysis with phenotypes[J]. Acta Agriculturae Boreali-Sinica， 2014， 29（1）： 69-72. （in Chinese）
[5] FU HF， LYU XH， CHEN JY， et al. Progress of application of SSR molecular marker technique in capsicum breeding research in China[J]. Bulletin of Agricultural Science and Technology，2016，（11）：168-171. （in Chinese）
[6] LI HJ. Genetic analysis and QTL mapping in tomato cracking[D]. Haerbin： Northeast Agricultural University， 2016. （in Chinese）
[7] LI B， QIN ZW， ZHOU XY. Genetic analysis and SSR molecular marking of flesh color trait in cucumber[J]. Journal of Northeast Agricultural University， 2010， 41（12）： 21-25. （in Chinese）
[8] PANG WL. Genetic study on fruit shape， color and calyx color traits of eggplant[D]. Beijing： Chinese Academy of Agricultural Sciences， 2008. （in Chinese）
[9] CHEN P， LIU TG， JIANG HK， et al. Genetic analysis on white color of pepper fruit[J]. China Vegetables， 2017，（7）： 37-42. （in Chinese）
[10] LIU L. SSR molecular marker technique and crop QTL mapping[J]. Science & Technology Ecnony Market， 2010（6）： 12-14. （in Chinese）
Key words Capsicum； White character； SSR analysis
Capsicum is a kind of important vegetable crop planted worldwide. The planting area of capsicum has exceeded 1 333 333 hm2 in China， and its annual yield accounts for nearly a half of the total yield in the world. Capsicum industry is one of the largest vegetable industries in China. The sales volume of capsicum depends to a large extent on fruit color， which as an important flavor and quality character of capsicum is worthy of research and discussion. Capsicum has various colors， and played a role of model plant in heredity of fruit color in previous studies[1]. Therefore， the research on fruit color and corresponding molecular mechanism of capsicum is of great significance to the genetic breeding of capsicum and the molecular regulation of fruit color of capsicum. Currently， there are more studies on red and green characters， but few studies have been conducted on white character of capsicum. Therefore， it is urgent to study the white character of capsicum， so as to understand the genetic law of the white character， which could provide powerful reference for capsicum breeding for quality. The inheritance of the white character of capsicum was selected as a research object in this study.
Color of ripe fruit of sweet pepper could be identified and selected at seedling stage by molecular marker technology， so as to breed ideal fruit and accelerate the breeding of pure lines of colored capsicum， which is very attractive to breeders who are studying the genetic mechanism of capsicum fruit color[2]. After many years of study， several genes controlling the color of main fruit of capsicum could be determined， for instances， A gene controls purple character， CCS controls yellow character， and PSY gene controls orange character， but the corresponding molecular marker technique applied in experiments was RELP markers. However， this type of markers have more disadvantages such as tedious operation and higher cost， so the application in practical breeding is very difficult. Therefore， the development of other more convenient markers such as SSR and InDel has high value， and is of great significance. In this study， with homozygous inbred lines 246 and 247 as male and female parents， respectively， SSR molecular markers closely related to white character were screened. This study lays a foundation for molecular mark assisted breeding by SSR technique， and enriches the gene pool of capsicum， thereby providing reference for the breeding of new white-color capsicum germplasms and varieties.
Materials and Methods
Tested materials
The tested materials were provided by capsicum research group of Horticultural Research Institute， Anhui Academy of Agricultural Sciences， as shown in Table 1.246 is a high-generation inbred line， and 247 is an inbred line obtained from three generations of backcross of white capsicum variety and 246 followed by 5 generations of selfing and directed screening.
Plug seedlings
The experiment was carried out in College of Horticulture， Anhui Agricultural University and the Experimental Base and Molecular Biology Laboratory of Horticultural Research Institute， Anhui Academy of Agricultural Sciences. The experimental materials were seeded in hole trays， and when each plant had 3 true leaves， capsicum genomic DNA was extracted by improved CTAB method.
Extraction and identification of genomic DNA
DNA extraction by CTAB method
The DNA was extracted by improved CTAB method. Tender leaves of capsicum plants were picked and grinded in a centrifuge tube added with 2% CTAB. The mixture was heated in a water bath at 65 ℃ for 20 min， and after mixing well， centrifugation was performed for 10 min. The supernatant was added with equal volume of chloroform and isoamyl alcohol， followed by mixing and 10 min of centrifugation. Into the obtained supernatant， isopropanol with a volume 2/3 of the supernatant was added， and after freezing， transparent gel precipitate was obtained and flushed with 70% ethanol. Finally， TE was added to dissolve the precipitate， obtaining the extracted DNA which was preserved in a refrigerator at -20 ℃.
DNA quality detection
DNA concentration and OD value were determined with a Spectrophotometer nucleic acid detector， and the according to the detected DNA concentration， all the sample was diluted to 10 ng/μl for later use.
DNA purity was detected by 1.2% agarose gel electrophoresis and nucleic acid dye， and photographing was performed with a gel imaging system. If OD260/OD280>1.8， the protein has a content lower than 0.3%， and could be studied at molecular level， and if OD260/OD280<1.8， the DNA sample has more impurities， and should be extracted and further purified. SSR markers
Source of SSR primers
The SSR primers were designed through related NCBI websites. After dissolution according to instructions， the primers were diluted by 10 times， subpackaged， and placed in an environment at 4 ℃ for later use. The primers were preserved at -20 ℃.
SSR reaction system
The reaction system in this experiment was slightly different from the PCR reaction system improved by Hu and standard SSR reaction system. Taq DNA polymerase， dNTPs， MgCl2， Buffer and dye for PCR were purchased from Tiangen Biotech （Beijing） Co.， Ltd. The SSR reaction system was 10 μl， including buffer 1.0 μl， 2.5 μmol/L dNTP 1.0 μl， 25 mmol MgCl2 1.0 μl， 10 μmol/L forward and reverse primers 1.0 μl in total （0.5 μl each）， 5 μmol/L Taq enzyme 0.1 μl， 10 ng/μl DNA 2.0 μl， and ddH2O 3.9 μl. The PCR was started with pre-denaturation at 4 min for 94 ℃， followed by 35 cycles of denaturation at 94 ℃ for 30 s， annealing at 53 ℃ for 30 s， and extension at 72 ℃ for 1 min， and completed by extension at 72 ℃ for 7 min. Then， 3 μl of PCR product was detected by 8% non-denaturing PAGE[3-5].
Polyacrylamide gel electrophoresis
After the assembling of electrophoresis apparatus， polyacrylamide gel was rapidly poured into the gap of glass plate. Pre-electrophoresis was performed under voltage of 150 V and current of 350 mA for 15 min. Then， sampling application was performed with GM345 as Marker， and 3 μl of sample was applied each time. Finally， electrophoresis was performed under voltage at 150 V and current at 350 mA for 100 min. The polyacrylamide gel contained 30% PA solution 12.15 ml， deionized water 28.13 ml， TBE 4.5 ml， TEMED 36 μl and AP 0.6 ml.
After electrophoresis， silver staining was performed， and after fixation of gel， observation was performed under a UV lamp. Band pattern was recorded and photographed.
Results and Analysis
Detection of capsicum genomic DNA
According to the detection with the Spectrophotometer nucleic acid detector， the OD values of single plants of parents were in the range of 1.8-2.0.
Quality detection was then performed to the extracted capsicum DNA by 1.2% agarose gel electrophoresis. The bands were clear， tidy， complete， without tailing （Fig. 1）. The sample satisfied the requirement by SSR molecular marker amplification.
Screening of capsicum polymorphic markers
With the DNA of high-generation homozygosis inbred line parents 246 and 247 as templates， PCR was performed， obtaining 360 pairs of capsicum SSR primers， among which 3 pairs of polymorphically differential primers， i.e.， ES-89 （Fig. 2）， ES-292 （Fig. 3） and ES-296 （Fig. 4）. The polymorphic rate was 0.84%， and the polymorphic markers were all co-dominant markers. Markers SSR89， SSR292 and SSR296 exhibited clear bands with remarkable polymorphic differences. This result provides a basis for subsequent QTL mapping. Discussion
In previous studies about the inheritance of fruit color of agricultural products， most scholars attributed it to quality character， but it could be seen from the genetic laws of multiple crops that the inheritance of fruit color often has the characteristics of continuous variation， and fruit color is not a simple quality character[7-8]. The inheritance of capsicum color is mainly controlled by main genes， and less affected by polygene heritability and environment effect， and therefore， fruit color of capsicum at marketable maturity stage could be selected during segregative generation at early stage， thereby providing a basis for breeding for capsicum color[9].
Molecular marker technology develops the understanding of quantitative characters to the analysis of quantitative trait loci （QTL）[10]. With the application of molecular marker technology， known DNA fragments could be amplified to obtain analysis data for the creation of conditions for deep research on the genetic mechanism of capsicum color， with an attempt to improve research possibility， accuracy and foreseeability.
In this study， high-generation inbred lines with different colors were subjected to SSR molecular marker analysis， 360 pairs of SSR capsicum primers were screened， and 3 pairs of primers among them showed specific bands. This study provides certain theoretical basis for heterosis breeding and molecular mark-assisted breeding for capsicum color， and lays a basis for QTL mapping.
References
[1] ZHANG FF， WANG LH， HU H， et al. Review of fruits color and related pigments in hot pepper[J]. Journal of China Capsicum， 2010（2）： 51-97. （in Chinese）
[2] LI DW， HAO WJ， GONG ZH. Advances in genetics and molecular biology of fruit colour in hot pepper[J]. Journal of China Capsicum， 2014（2）： 1-5. （in Chinese）
[3] LUO B， SUN HY， XU GM， et al. Research progress of SSR molecular markers[J]. Journal of Anhui Agricultural Sciences， 2013， 41（12）： 5210-5212， 5246. （in Chinese）
[4] LIU ZJ， LI JT， YANG Y， et al. SSR molecular markers identification of pepper hybrid and comparative analysis with phenotypes[J]. Acta Agriculturae Boreali-Sinica， 2014， 29（1）： 69-72. （in Chinese）
[5] FU HF， LYU XH， CHEN JY， et al. Progress of application of SSR molecular marker technique in capsicum breeding research in China[J]. Bulletin of Agricultural Science and Technology，2016，（11）：168-171. （in Chinese）
[6] LI HJ. Genetic analysis and QTL mapping in tomato cracking[D]. Haerbin： Northeast Agricultural University， 2016. （in Chinese）
[7] LI B， QIN ZW， ZHOU XY. Genetic analysis and SSR molecular marking of flesh color trait in cucumber[J]. Journal of Northeast Agricultural University， 2010， 41（12）： 21-25. （in Chinese）
[8] PANG WL. Genetic study on fruit shape， color and calyx color traits of eggplant[D]. Beijing： Chinese Academy of Agricultural Sciences， 2008. （in Chinese）
[9] CHEN P， LIU TG， JIANG HK， et al. Genetic analysis on white color of pepper fruit[J]. China Vegetables， 2017，（7）： 37-42. （in Chinese）
[10] LIU L. SSR molecular marker technique and crop QTL mapping[J]. Science & Technology Ecnony Market， 2010（6）： 12-14. （in Chinese）