Zea mays L. is an annual herbaceous plant of the genus Zea in Gramineae, which is one of the important crops in China with multiple uses of food crop, economy, fruit, forage and energy. By 2012, the total output of corn in China surpassed that of cereals and became the largest grain crop in China (Wu and Lu, 2020, Anhui Agricultural Science Bulletin, 26(2-3): 13-15). Maize is the main grain crop in Baoshan city, with an annual planting area of 8.67×10⁴ hm². Because Yunnan has a low-latitude mountain monsoon climate and diverse regional climate types, the demand for maize varieties in different regions is also diverse (Duan, 2016, China Seed Industry, 7: 31-33). However, due to the frequent utilization of a few excellent populations in China and the decline of self-crossing, maize breeding is faced with many problems, such as the narrow genetic base of germplasm resources, the slow growth of yield per unit area, and the decline of disease resistance and stress resistance. Therefore, only after analyzing the genetic diversity of the maize varieties promoted in Baoshan city, can we have a clear understanding of the existing germplasm base and cultivate more suitable new varieties with the existing germplasm resources.

Early studies on genetic diversity of maize varieties were mostly based on morphological, physical and biochemical methods (Zhu, 2013). In recent years, with the development of biotechnology, molecular markers have been widely used to identify genetic relationships and study genetic diversity in maize. Wang et al. (2014) used SSR markers to analyze the genetic diversity and differentiation characteristics of 328 maize varieties, and showed that the genetic diversity index of varieties developed in recent years had little change between different years, and there was little difference between other regional test groups except the Jing-Jin-Tang group. Zhang et al. (2016) amplified 213 fresh-eaten inbred maize from 40 selected SSR primers, and the cluster analysis results were consistent with the pedigree origin of inbred lines. Lu et al. (2019) used 29 pairs of SSR primers to analyze the genetic diversity of 87 waxy maize inbred lines, and the results showed that the genetic variation of most waxy maize inbred lines in the tested population was relatively simple. Zhao et al. (2018) used 60 pairs of SSR core primers to analyze the genetic diversity and identify the genetic relationship of 68 inbred waxy corn lines. The results showed that 68 inbred waxy corn lines had rich genetic diversity and far genetic relationship, which could be used to screen high-quality waxy corn germplasm resources and provide a basis for variety identification. Zhang et al. (2019) used 30 pairs of SSR polymorphic primers to conduct allele diversity and cluster analysis on 42 maize inbred lines commonly used in Yunnan, and the results showed that 42 maize inbred lines commonly used in Yunnan had close genetic distance and close genetic relationship, so their genetic diversity was narrow and germplasm resources were not rich enough. Compared with other methods, SSR markers are not affected by environment and growth period, and are characterized by simple operation, good polymorphism, high throughput, high sensitivity, automation and good stability (Guichoux et al., 2011). 

In this study, SSR core primers recommended by Chinese agricultural industry standard (NY/T1432-2014) were used to analyze genetic diversity and build fingerprints of 15 maize varieties widely planted in Baoshan, Yunnan province. The genetic distance between different varieties was elaborated to provide theoretical and technical support for the breeding and promotion of maize germplasm resources in Baoshan, Yunnan Province.

1 Results and Analysis

1.1 Polymorphism analysis of SSR primers

Seventeen pairs of primers with good specificity and clear bands were screened from the SSR core primers recommended by Chinese agricultural industry standard (NY/T1432-2014). A total of 72 bands were detected by 17 pairs of primers in 15 Baoshan common maize varieties, and each pair of primers detected 3~6 specific polymorphic fragments. The primers umc1335y5, phi072k4, umcl545y2 and umc1432y6 were the least, and 3 bands were detected. bnlg439w1 and bnlg2305k4 had the most polymorphic loci, with 6 polymorphic loci detected. The 17 pairs of SSR primers could amplify 4.3 polymorphic loci on average. The results showed (Table 1) that Nei's genetic diversity index varied from 0.176 to 0.418 with an average of 0.283, and phi072K4 had the highest genetic diversity index of 0.418. Shannon information index varied from 0.315 to 0.605 with an average of 0.444, among which the Shannon information index of phi072k4, umc1705wl and umc1492y13 were all above 0.5. The PIC range of 17 SSR primers ranged from 0.512 to 0.812, among which phi080k15 had the highest PIC (0.812) and umc1335y5 had the lowest PIC (0.512). PIC values are affected by the frequency and number of amplified alleles, and PIC average values are positively correlated with the abundance of genetic resources of materials, which can directly reflect the differences between varieties (Botstein et al., 1980; Gu et al., 2014), when PIC<0.25, it indicates that the genetic basis of the material is low polymorphism; 0.25≤PIC<0.5 was moderate polymorphism; When PIC≥0.5, the genetic basis of material was highly polymorphic. In this study, the average PIC value of 17 pairs of primers detected against 15 maize varieties was 0.686, indicating that 15 maize varieties have rich genetic basis and are highly polymorphic.

Table 1 Polymorphism evaluation of 17 SSR core primers

1.2 Genetic diversity analysis of 15 maize varieties

Genetic similarity analysis was performed on the atlas data of 15 maize varieties using NTSYS 2.10e software. The results showed that the range of genetic similarity coefficient was 0.417~0.819 and the average genetic similarity coefficient was 0.632 (Table 2). The genetic similarity coefficient between Tongyu 3 and Jinyu 1 was 0.417, and the genetic basis difference between Tongyu 3 and Jinyu 1 was the smallest, while the genetic similarity coefficient between Tongyu 3 and Huayu 1 was 0.819, indicating that the genetic similarity between Tongyu 3 and Jinyu 1 was high, and the genetic basis difference between Tongyu 3 and Huayyu 1 was the smallest.

Table 2 The genetic similarity coefficient of 15 Maize materials

1.3 Cluster analysis of SSR molecular markers in maize

Based on genetic similarity coefficient, unweighted pair-group method with arithmetic mean (UPGMA) was used to perform genetic clustering analysis for 15 maize species (Hu et al., 2017). The results showed that the 15 cultivars could be divided into five groups with a threshold value of 0.65, and the first group contained five cultivars: Yunrui 88, Rongyu 168, Kangnong 808, Jiadan 6 and Gaokang 808. There is only one variety in class Ⅱ, Jinyu 1; There were two varieties in class Ⅲ, which were Haixuan 1 and Chudan 5. Category Ⅳ includes 3 varieties, including Meijiayu 1, Meiyu 1 (waxy) and Rodan 299; The Ⅴ category includes 4 varieties, Huiyu 0806, Qiangxin 66, Tongyu 3 and Huayu 1 (Figure 1).

Figure 1 The clustering analysis of 15 maize materials

2 Discussion

The traditional genetic breeding method divides inbred lines into groups according to field phenotypes, and selects the dominant species through field hybridization among different groups. This method has some problems such as long breeding cycle, heavy workload and unstable characteristics. With the application and rapid development of molecular marker technology in genetic breeding, it can not only be used to construct the DNA fingerprint of maize germplasm, but also determine the genetic relationship according to the genetic similarity, so as to divide and screen out the dominant population, and guide the hybrid breeding of the dominant population, shorten the breeding cycle. It has played a rapid role in promoting the development of maize genetics and breeding (Zhu and Wang, 2015). SSR molecular marker technology is developed in recent years, biological technology, and has high polymorphism, less dosage, codominance, repeatability, and low cost, good in crop breeding is conducive to the molecular level, reveal the relationship between genetic and phenotype, facilitate the rapid development of maize germplasm resources efficient rating, the exploration of new art gene (Wu and Lu, 2020, Anhui Agricultural Science Bulletin, 26(02-03): 13-15)

In this study, 17 pairs of SSR primers screened in the Chinese agricultural industry standard NY/T1432-2014 were used to analyze the genetic diversity of 15 maize varieties promoted in Baoshan city, Yunnan Province. A total of 72 polymorphic loci were detected, with an average of 4.3 polymorphic loci per pair of primers. It is higher than the average 3.97 polymorphic loci of 120 maize inbred lines from the United States and Serbia and 2 maize inbred lines from China by Liu et al. (2018), and the average polymorphism index (PIC) is 0.686, which is also higher than the average polymorphism information index (PIC) of 120 Maize inbred lines from Europe and the United States is 0.500. Showed that the genetic polymorphism of 15 maize varieties in Baoshan, Yunnan was better than that of European and American maize varieties, probably because Liu et al. (2018) used less molecular markers for 122 European and American and Chinese maize varieties, some SSR primers could not be effectively amplified. In this study, the SSR primers with good specificity were screened, and then the genetic diversity was analyzed to ensure the accuracy and reliability of the research results. The average polymorphism index (PIC) 0.686 and genetic similarity coefficient 0.632 of 15 maize in this study were close to the average polymorphism index (0.71±0.16) and genetic similarity coefficient (0.60) of 135 maize inbred lines in Southwest China studied by Li et al. (2007) using 62 pairs of SSR primers. It indicated that the genetic diversity of Baoshan maize in Yunnan was consistent with the characteristics of southwest maize inbred lines (Li et al., 2007): A relatively narrow germplasm base in southwest China, and between corn and corn in Sichuan area in Yunnan region has relatively large genetic diversity, also showed relatively large difference of geographical source materials are relatively rich, which is also consistent with the high genetic similarity between the American NSS group and the American SS and Serbian populations (Liu et al., 2018). Because gene exchange between the United States and Serbia inbred lines is frequent, close relatives, the inbred lines and the NSS and Serbia group of SS than America and China the backbone is a group of genetic distance far away, because the location result in maize genetic exchange is less, a further sign of richness of genetic diversity associated with the difference of geographical origin. In this study, the average polymorphic loci, average polymorphic index (PIC) and average genetic similarity coefficient of 17 pairs of SSR primers were all lower than that of Hu et al. (2017). Results of genetic diversity analysis of 84 maize hybrids in Yunnan by SSR molecular marker technology and UPMGA method: The average polymorphic loci was 10.23, the average polymorphic index (PIC) was 0.779, and the average genetic similarity coefficient was 0.790, indicating that the genetic diversity of maize planted in Baoshan was lower than that of Yunnan hybrid, and the genetic base of Yunnan hybrid was narrower, indicating that the genetic base of maize varieties planted in Baoshan was narrower. The abundance of germplasm resources is insufficient.

In this study, the genetic similarity coefficient varied from 0.417 to 0.819, and the average genetic similarity coefficient was 0.632. Using 0.65 as the threshold, 15 maize varieties could be divided into 5 groups, which could provide theoretical and technical support for the breeding and promotion of maize germplasm resources in Baoshan city. It will provide scientific basis for introducing different varieties in maize breeding and improving the richness and quality of germplasm resources in Yunnan province in the future

3 Materials and Methods

3.1 Experimental material

The test materials were 15 corn varieties popularized and planted in Baoshan city of Yunnan Province collected by Yuxi Seed Management Station (Table 3).

Table 3 The tested maize materials

3.2 Main reagents and instruments

Plant Genome DNA Extraction Kit (HP Plant DNA Kit) were purchased from Omega. PCR supermixture (D331B), 6×Loading Buffer, and agarose were from TransGen Biotech, and 40% polyacrylamide solution was from Finc.

Gradient PCR Instrument (Model: CT006248) was purchased from BIO-RAD; The electrophoresis apparatus (Model: DYY-10C) from Beijing Liuyi Biotechnology Co. Ltd; Gel Imager (Model: ChemicDoc XRS+) of BIO-RAD; High speed bench centrifuge from ThermoFisher (Model: D-37520); Biosafety cabinet from ThermoFisher (Model: LFPM58-68); Decolorizing shaker from QILINVEIER Instruments (Model: TS-200); High pressure steam sterilizing pot from ZEALWAY (Model: AUTOCLAVE·GI54DW); Thermostatic water bath cauldron from Shanghai Jinghong Factory (Model: PK-80).

3.3 PCR amplification

The seeds of 15 maize samples were extracted with reference to the method of plant genome DNA extraction kit. Dissolve in 50 μL TE solution and store at -20℃.

DNA concentration was adjusted to 50 ng/μL for PCR amplification of specific products. The reaction system was 25 μL: template DNA 2 μL, positive and reverse primers 10 pmol/μL 0.5 μL each, Easy Taq Mix 12.5 μL, double distilled water 9.5 μL. PCR amplification procedure: pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 40 s, annealing at 60℃ for 35 s, extension at 72℃ for 45 s, 35 cycles, and storage at 4℃.

3.4 Product detection and screening

Each amplified sample was mixed with 4 μL 6×Loading Buffer, and the amplified products were detected by agarose gel electrophoresis at a concentration of 2%.

In this study, 40 pairs of core primers recommended by Chinese agricultural industry standard NY/T1432-2014 were used for PCR amplification with each primer and DNA template of 15 samples, and specific bands were screened according to agarose gel electrophoresis detection results. After denaturation at 95℃ for 7 min and cooling at 4℃ for 15 min, the PCR products of each sample were detected by electrophoresis with 6% polyacrylamide gel at a voltage of 1 500 V for 45 min. After rinsing with deionized water for 10 s, the gel was fixed in 10% glacial acetic acid for 10 min. After rinsing with deionized water, the gel was dyed in 0.1% AgNO₃ solution for 10 min. After rinsing with deionized water, the gel was placed in developing solution of 1% sodium hydroxide and 0.5% formaldehyde.

3.5 Data statistics

According to the electrophoresis results, the polymorphism bands separated by SSR electrophoresis (Deng et al., 2015) were counted and compared with each other based on the migration positions of the bands amplified by primers. The bands with the same migration were marked as "1" and the bands without bands were marked as "0". Then Excel software was used to record and data. The polymorphism, polymorphism information content (PIC) (Smith et al., 1997), Nei's genetic diversity index and Shannon's information index (I) (Yeh et al., 1999) of the selected SSR primers was analyzed using Popgene 1.32 software. Similarity analysis among varieties was carried out by Similarity program in NTSYS 2.10e (Rohlf et al., 1987), and genetic similarity coefficient was obtained. Clustering program was used for Clustering analysis of varieties according to UPGMA, and Clustering tree graph was drawn.

Authors’ contributions

LS is the experimental designer and executor of this study. SDL completed data analysis and wrote the first draft of the paper; LDJ participated in experimental design and analysis of experimental results; SYD is the architect and principal of the project, directing experimental design, data analysis, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgement

This study was supported by the Joint Youth Project of Yunnan Provincial Undergraduate Universities (2017FH001-099) and the National Natural Science Foundation of China (31960371).

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