Screening of SSR Core Primers and Construction of Molecular ID for Flue-cured Tobacco Germplasm Resources in Hunan

Hui LI, Yinghong TANG, Shipeng XIANG, Guang HUO, Chutian HUANG

Abstract [Objectives] This study was conducted to establish a reliable and unique molecular ID for flue-cured tobacco germplasm resources in Hunan Province， further improving the efficiency of germplasm collection and identification， and laying a solid material foundation for flue-cured tobacco breeding.

[Methods]Twelve pairs of SSR primers with stable amplification and rich polymorphism were screened out from 816 pairs of SSR primers by a step-by-step screening method. As core primers of the SSR core primer library， the polymorphism of SSR primers was analyzed， the genetic relationship of 162 flue-cured tobacco germplasm resources was identified， and the molecular ID cards were constructed.

[Results] The result of SSR primer polymorphism analysis showed that a total of 57 alleles were detected by 12 pairs of SSR primers in 165 tobacco germplasm resources， with an average of 4.75 alleles per pair of primers; the average diversity of SSR primers was 0.649; and the average value of Shannons index was 1.235. The results of cluster analysis showed that 162 flue-cured tobacco germplasm resources were divided into five groups. The members of each group were divided based on genome information， which had nothing to do with their geographical origin. Meanwhile， 12 pairs of SSR primers gave each flue-cured tobacco germplasm resource a unique molecular ID code.

[Conclusions]From the above results， we can see that the 12 pairs of SSR primers obtained by screening have stable amplification polymorphism， and can serve as the primers of the core primer library， and can be used to construct the unique molecular ID of flue-cured tobacco germplasm resources.

Key words Tobacco; SSR primer; Molecular ID

Received： April 8， 2022  Accepted： June 10， 2022

Supported by Changsha Tobacco Company Project （20-24A01）; Hubei Tobacco Company Project （027Y2022-011）.

Hui LI （1980-）， male， P. R. China， associate professor， PhD， devoted to research about research and utilization of tobacco germplasm resources.

*Corresponding author. E-mail： xiangshipeng@qq.com.

Tobacco （Nicotiana tabacum L.） is an annual or limited perennial herb of Nicotiana of Solanaceae. Tobacco is one of the important economic crops in China and occupies an important position in the national economy. High-quality tobacco leaves are the raw material basis of the Chinese cigarette industry， and the variety is one of the key factors to highlight the characteristics of tobacco leaves[1]. Germplasm resources with rich genetic diversity are the material basis for the selection and breeding of fine varieties[2]. In the process of collecting germplasm resources， the phenomenon of "homonym" or "synonym" always occurs， which has caused great trouble to the collection of germplasm resources. SSR （simple sequence repeats） markers have the advantages of rich polymorphism， good repeatability， and wide distribution of genomic loci. SSR markers have been applied to construction of genetic maps of various crops （wheat[3]， maize[4]， purple potato[5]）， genetic diversity analysis （tobacco[6]， cotton[7]， peanut[8]）， construction of molecular ID of germplasm resources （soybean[9-10]， sesame[11]） and so on. Therefore， establishing a reliable and unique molecular ID for germplasm resources is an important task for the collection and identification of flue-cured tobacco germplasm resources. At present， many researchers have used SSR primers to construct molecular ID of different germplasm resources. Xu et al.[12] screened 8 pairs of SSR primers with good stability and rich polymorphism from 286 pairs of SSR primers for the construction of 80 molecular ID cards for tobacco germplasm resources. Liu et al.[13] screened 25 pairs of SSR primers with rich polymorphism and good stability from 2 000 pairs of SSR primers， which were used for the construction of molecular ID for tobacco germplasm resources. Xu et al.[14] screened 48 pairs of SSR primers with clear bands and good polymorphism from 175 pairs of SSR primers evenly distributed in the tobacco genome， which were used for the construction of molecular ID cards for tobacco germplasm resources. It can be seen that SSR primers with rich polymorphism and good stability can be used for the construction of molecular ID cards for germplasm resources. However， researchers have screened and identified different SSR primers when constructing the molecular ID cards for tobacco germplasm resources. Due to the different primers used， the same species may have different molecular ID cards， which further increases the workload of germplasm resource identification. Therefore， building a core primer library of tobacco germplasm resources and establishing unique molecular ID for tobacco germplasm resources has become an important task for the collection and identification of tobacco germplasm resources. Through screening of a large number of SSR primers， SSR primers with rich polymorphism， stable amplification and clear bands were obtained， and used as the primers of the SSR core primer library， in order to lay a foundation for the construction of the core primer library of tobacco germplasm resources and unique molecular ID.

Materials and Methods

Experimental materials

The seeds of flue-cured tobacco germplasm resources in this experiment were provided by Changsha Company of Hunan Tobacco Company， 162 parts in total （Table 1）， including 108 parts from China， 46 parts from the United States， 1 from Japan， 5 from Canada， 1 from Thailand， and 1 from Zambia. In March 2021， all varieties of flue-cured tobacco were transplanted to the Ningxiang Experimental Base in Hunan Province， and the young leaves of each variety were cut off in June， and stored in a -80 ℃ refrigerator for later use after quick freezing in liquid nitrogen.

Experimental methods

Extraction of genomic DNA

The collected young leaves of each flue-cured tobacco variety were quick-frozen in liquid nitrogen and ground in a sample grinder. After grinding， the genomic DNA was extracted with a plant genomic DNA extraction kit （Tiangen Biotech （Beijing） Co.， Ltd.）. DNA concentration was measured with a NanoDrop2000 micro-nucleic acid analyzer， then diluted to 50 ng/μl and stored in a -20 ℃ refrigerator.

Screening of SSR core primers

According to the sequences of 816 pairs of SSR primers published by Bindler et al.[15-20]， they were synthesized by Tsingke Biotechnology Co.， Ltd. First， 816 pairs of SSR primers were preliminarily screened with 10 different flue-cured tobacco varieties， and then 26 pairs of SSR primers obtained were screened again with 64 different flue-cured tobacco varieties. Finally， 19 pairs of SSR primers obtained were screened with 165 flue-cured tobacco varieties， and 12 pairs of polymorphism-rich and amplification-stable SSR primers were obtained （Table 2）. PCR amplification system was 25 μl in volume， containing PCR Mix 12.5 μl， upstream primer 1μl， downstream primer 1 μl， template 100 ng， and ddH2O to 25 μl. The PCR amplification program was started with pre-denaturation at 94 ℃ for 5 min， followed by 33 cycles of denaturation at 94 ℃ for 30 s， annealing at 55 ℃ for 30 s and extension at 72 ℃ for 1 min， and completed with extension at 72 ℃ for 10 min. The product was storage at 4 ℃. From the PCR product， 2 μl was taken for electrophoresis detection on 7.5% polyacrylamide gel， and the gel was stained with silver nitrate， and photographed after developing.

Polymorphism analysis of SSR core primers

The electrophoresis band information was read in a binary manner， with clear electrophoresis bands on the gel image marked as "1"， the condition of no clear electrophoresis band in the same position marked as "0"， and the missing electrophoresis band marked as "9"， finally forming a 0，1 matrix. The 0，1 matrix was converted by DataFormater[21] software into the data format required by Popgene[22] software， and then the number of observed alleles， the number of effective alleles， expected heterozygosity， gene diversity and Shannons index were calculated.

Cluster analysis of flue-cured tobacco germplasm resources based on SSR core primers

The 0，1 matrix was converted by DataFormater[21] into the data format required by Powermarker[23] software. Cluster analysis was performed using Powermarker[22] and MEGA[24] software.

Construction of molecular ID of flue-cured tobacco germplasm resources

According to the top-to-bottom order of the electrophoretic bands， they were assigned as 1， 2， 3， 4， 5， and 6， respectively. 0 indicated that there was no electrophoretic band in the lane， and 7 indicated that the lane was a heterozygous band. Finally， the matrix information of electrophoresis bands was formed， and then the molecular ID of flue-cured tobacco germplasm resources was constructed by IDanalysis1.0[25] software.

Results and Analysis

Screening and polymorphism analysis of SSR core primers

According to the SSR primer sequences provided in the reference， 816 pairs of SSR primers were synthesized by Tsingke Biotechnology Co.， Ltd. After multiple times of screening， 12 pairs of SSR primers with rich polymorphism， clear bands and stable amplification were obtained （Fig. 1， Table 1）. The 12 pairs of SSR primers detected a total of 57 allele loci in 162 tobacco germplasm resources. The number of effective alleles detected by SSR primers varied from 1.53 to 4.61， with an average of 4.75 alleles detected per pair of primers. The gene diversity detected by SSR primers ranged from 0.345 to 0.783， with an average value of 0.649; and the Shannons index varied from 0.56 to 1.63， with an average value of 1.235. In summary， the 12 pairs of SSR primers obtained by screening were polymorphic primers， which can be used as the core primers for tobacco germplasm resources and for the construction of molecular ID of germplasm resources.

Cluster analysis of flue-cured tobacco germplasm resources

According to the electrophoresis band information， a 0，1 matrix was obtained， and the results of cluster analysis by Powermarker and MEGA software are shown in Fig. 2. It can be seen from Fig. 2 that the 162 flue-cured tobacco germplasm resources were divided into 5 groups. Group I included 35 germplasm resources; Group II comprised 37 germplasm resources; Group III included 7 germplasm resources; Group IV comprised 31 germplasm resources; Group V consisted of 52 germplasm resources. Among them， group V included the most germplasm resources， accounting for 32.09% of the total resources， while group III comprised the least amount of germplasm resources， accounting for 0.04% of the total resources. Combined the producing areas of flue-cured tobacco with the clustering results， it was found that flue-cured tobacco resources from the same geographical origin were not completely classified into the same cluster group， but found in all five cluster groups. It indicated that the clustering results were divided based on the genomic information of flue-cured tobacco itself， which was not directly related to the geographic origin of flue-cured tobacco.

Construction of molecular ID for flue-cured tobacco germplasm resources

According to the electrophoretic band information amplified by the 12 pairs of SSR primers from 162 germplasm resources， molecular ID for flue-cured tobacco germplasm resources were constructed by ID analysis software， and the results are shown in Table 4. The order of the 12 pairs of SSR primers in the construction of molecular ID was PT20213， TM10679， PT30394， PT20189 PT30417， TM10899， PT54339， TM11166 PT51976， PT51234， PT20287 and HX1. It can be seen from Table 4 that each resource had a string composed of 12 pairs of SSR primers， endowing each germplasm resource with a unique molecular ID. These 12 pairs of SSR primers could be used as the primers of the core primer library.

Conclusions and Discussion

The construction of the unique molecular ID for tobacco germplasm resources is an important work in the collection and identification of tobacco germplasm resources. The construction of library including a certain number of SSR core primers with stable amplification and rich polymorphism is the basis for the construction of the unique molecular ID for germplasm resources.   In this study， 12 pairs of SSR core primers with abundant polymorphism and stable amplification were obtained from 816 pairs of SSR primers by layer-by-layer screening and identification. Among them， the polymorphic primers PT20189， PT20287 and TM10899 are the same as the results of Ding et al.[6]， Xu et al.[12] and Xiang et al.[20]; and the polymorphic primer PT54339 is the same as the results of Liu et al.[3] and Xiang et al.[17]， and the polymorphic primers PT51976 and PT51234 are the same as the results of Wang et al.[19]. It can be seen that the polymorphic primers obtained in this study are the same as those obtained by different researchers from different germplasm resources. Therefore， these 12 pairs of polymorphic primers could be used as SSR primers for the SSR core primer library of flue-cured tobacco germplasm resources.

The construction of molecular ID for germplasm resources generally adopts the primer combination method， that is， the amplification bands of different primers are assigned values to form a unique string to identify different germplasm resources. In this study， 12 pairs of polymorphic SSR primers were obtained to construct molecular ID for 162 tobacco germplasm resources， and each germplasm resource obtained its own unique molecular ID.

In this study， 12 pairs of SSR primers with stable amplification， clear bands， and rich polymorphism were obtained by screening a large number of SSR primers， and cluster analysis was performed on 162 germplasm resources， and each germplasm resource was given a unique analysis ID. However， with the continuous collection of tobacco germplasm resources， the number of SSR primers needs to be further increased. Therefore， we still need to continue the screening and identification of polymorphic SSR primers， and constantly enrich the number of SSR primers in the SSR core primer library.

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