Cucurbitais is an annual tendril herbaceous plant of genus Cucurbita, Cucurbitaceae, which is abundant in biological diversity, ecological diversity, species diversity and genetic diversity (Wang, 2002). China has a long history of pumpkin plantation. It is one of main pumpkin cultivating countries around the world and the cultivation is widely distributed in north as well as south of China by Li et al. (2018). As an important index for species quality determination, the purity of hybrid species is the key to insure better popularization of improved cultivars which has positive correlation with economic benefit. Therefore, purity identification of hybrid species is a necessary process in hybrid seed production (Liu et al., 2013). Traditional purity identification is mainly through field plant morphology, while it costs long time and plenty of human resources. It is also easy to be influenced by environmental and artificial factors which lead to low accuracy (Wang et al., 2011; Liu et al., 2013; Zhang et al., 2013). With the continuous development of SSR molecular marker technology, it has developed into a fast and mature DNA molecular genetic marker that can be not only applied to analyze the germplasm resource diversity and genetic relationship of pumpkin (Liu, 2012; Wang et al., 2016), but also used for purity identification of pumpkin seeds (Han et al., 2014a; Han et al., 2014b). Compared with the traditional field morphology identification, SSR molecular marker is usually with short period and good repeatability. The result by this method is accurate, reliable and reusable that is not limited by environmental factors. The standard of DNA quality is in a relatively low level and its result is easy to analyze (Liu et al., 2013; Zhang et al., 2013). Until now, SSR molecular marker technology has been applied to the purity identification of various hybrid species, such as Zea mays (Liang et al., 2017), Helianthus annuus (Liu et al., 2017) and Cucumis melo (Zhao et al., 2017).
Nenzao 2 was the Nenzao pumpkin cultivar selected and bred by the Hunan Vegetable Research Institute in 2012. The cultivar had advantages of early maturity and cold tolerance. The tender fruit of this species was tall and round, and the peel was green with good luster. It also had high rate of female flower as well as fruit-setting. Nenzao Jiamei was the Nenzao pumpkin cultivar selected and bred by Hunan Vegetable Research Institute in 2015. The cultivar had the advantages of good cold tolerance, strong growth potential and fast fruit expansion. The tender fruit of this cultivar was white green, tall and round. Nenzao Heiniu was Nenzao pumpkin cultivar selected and bred by Hunan Vegetable Research Institute in 2014. The cultivar was precocious with strong cold tolerance and good commodity. The fruit was round and the meat was thick, compact and durable. The peel was dark green with dark spots and good luster. The cultivated Nenzao 2, Nenzao Jiamei and Nenzao Heiniu were used as test materials to select primers for the identification of three species, so as to accurately and effectively identify the purity of hybrid generation of three cultivars combined with field plant purity identification, which would provide references of purity identification for further application of three pumpkin cultivars.
1 Results and Analysis
1.1 Primer selection of SSR purity identification
Primers used for purity identification of different species or different cultivars of the same species are generally different. In the early stage of hybrid purity identification by SSR molecular marker technology, selecting appropriate primers is very important. 27 pairs of SSR primers were applied for polymorphic selection of parents of Nenzao 2, Nenzao Jiamei and Nenzao Heiniu in this research, respectively. The electrophoretic results showed that 3 pairs of primers (NG05, NG74, NG88) amplified specific complementary bands in Nenzao 2 parents. 5 pairs of primers (NG05, NG26, NG74, NG80, NG04) amplified specific complementary bands in Nenzao Jiamei parents. 1 pair of primers (NG05) amplified specific complementary bands in Nenzao Heiniu parents. According to the primer selection result, based on the significance of difference of each primer band in parents, NG05, NG26 and NG05 were used to identify the purity of F1 seed of Nenzao 2, Nenzao Jiamei and Nenzao Heiniu, respectively (Figure 1).
Figure 1 The screening of primers
Note: M: 100 bp DNA marker; P1, P2: Male parent and female parent of Nenzao 2 by primer NG05, respectively; P3, P4: Male parent and female parent of Nenzao Jiamei by primer NG26, respectively; P5, P6: Male parent and female parent of Nenzao Heiniu by primer NG05, respectively
1.2 Purity identification of hybrids in Nenzao 2, Nenzao Jiamei and Nenzao Heiniu
SSR identification was applied on 192 F1 samples from Nenzao 2, Nenzao Jiamei and Nenzao Heiniu by the selected primers NG05, NG26 and NG05, respectively. Through the band characteristics of hybrids and two parents, sample number and false hybrid number of F1 sample with parent complementary bands in three hybrids were calculated. The electrophoresis results showed that the F1 seed purity of Nenzao 2 was 95.7%, and that of Nenzao Jiamei was 99.4% as well as that of Nenzao Heiniu was 96.6%. Partial electrophoresis results referred to Figure 2.
Figure 2 The purity detection of hybrids
Note: M: 100 bp marker; 1: Male parents; 2: Female parents; 3-26: Hybrids; A: Parents and hybrids of Nenzao 2 using the primer NG05; 22: False hybrids; B: Parents and hybrids of Nenzao Jiamei using the primer NG26; 18: False hybrids; C: Parents and hybrids of Nenzao Heiniu using the primer NG05; 24, 26: False hybrids
1.3 Correlation analysis of SSR purity identification and field purity identification
Through the result comparison analysis of SSR identification and field morphological detection of Nenzao 2, Nenzao Jiamei and Nenzao Heiniu, the result of Nenzao 2 and Nenzao Heiniu by SSR identification were both lower than that by field morphological detection. The result of Nenzao Jiamei by SSR identification was the same as that by field morphological detection. The average deviation of these three cultivars was 0.4% (Table 1). Correlation analysis was carried out on the two methods of testing results, of which the difference was significantly correlated at 0.05 level and the correlation coefficient was 0.997, indicating that the results were completely linear correlation. Regression equation was established: y=-23.962+1.241x, illustrating a significant linear regression relationship between the field planting pattern identification and SSR identification. In general, SSR molecular markers could be rapidly and accurately used for the purity identification of three Nenzao pumpkin cultivars.
Table 1 Hybrid identification of purity
Due to the large quantity of hybrids, alkaline lysis method was used to extract DNA in this research. Compared with CTAB method, the storage time of DNA extracted by alkaline lysis method was shorter but the method greatly simplified the test procedure and saved more time. It had no significant effect on identification result, which made the purity identification test faster and more effective.
The study used SSR molecular marker combined with field morphological detection to determine the purity of 3 Nenzao pumpkin cultivars. The results of SSR purity identification were 95.7%, 99.4% and 96.6%, respectively, and that of field morphological detection were 96.5%, 99.4% and 97.0%, respectively. The correlation coefficient of results by these two methods reached a significantly correlated level of 0.997, which was similar with the identification result of pumpkin hybrid seed by Han et al. (2014). The result indicated the feasibility of purity identification of Nenzao pumpkin hybrids by SSR molecular marker, which not only saved time and money, but also overcame problems of field detection caused by human and environmental factors.
When using SSR molecular markers for the purity identification of hybrid, all hybrid progenies can be identified by 1 pair of primers by Li et al. (2018). While in this study, Nenzhao 2 and Nenzao Jiamei could be identified by 3 pairs of primers and 5 pairs of primers, respectively. The result illustrated the high efficiency of SSR purity identification of hybrid, of which the result was similar with that of purity identification of tomato hybrid by Xie et al. (2008) and eggplant hybrid by Wang et al. (2015). However, a large number of primer selections were required in the early stage of purity identification of hybrid seeds by using SSR molecular marker technology, and the consumed cost increased with the increase of screening primers. Therefore, for a particular crop or species, corresponding specific primers should be selected to reduce the cost of hybrid purity identification.
3 Materials and Methods
3.1 Plant materials and reagents
Materials in the test were Nenzao 2, NenzaoJiamei, NenzaoHeiniu and their parents. All materials were provided by eggplant and pumpkin project of Hunan Vegetable Research Institute. All primers used in the test were from Gong et al. (2008a; 2008b) and was compounded by Dingguo Biotechnology CO. Ltd., Beijing (all primers concentrations: 10 µmol/L). The test was carried out in the laboratory of experimental building of Hunan Vegetable Research Institute from March to May in 2017.
Taq DNA Polymerase (ET101), 100 bp DNA Ladder (MD109) and dNTP Mixture (CD111) were bought from Tiangen BioTech (Beijing) CO., Ltd. The rest chemical reagents were all domestic analytical pure.
3.2 DNA extraction
Three F1 generation and their parents of pumpkin seed to be detected were cultivated in the greenhouse of the base of Hunan Vegetable Research Institute in early March. After seed germination, 0.5 cm seed plumular axis which was germinated for 3 to 4 days was used for DNA extraction. DNA extractions of parents and F1 generation of tested materials all referred to the alkaline lysis method by Han et al. (2014). 192 single DNA were extracted from hybrid seed, which were used for SSR analysis.
3.3 SSR detection
The reaction system of PCR amplification was 0.1 µL dNTP Mixture, 0.1 µL Taq DNA Polymerase and 1 µL 10×Buffer. The template was 0.5 µL DNA with 1 µL primer as well as 7.3 µL ddH2O. The reaction procedure was 94℃ for 5 min, 94℃ for 30 s, 59℃ for 30 s, and 72℃ for 30 min, repeating for 30 times. Then it was processed under 72℃ for 5 min and preserved in 4℃.
Amplified products were separated through the electrophoresis in 0.5% non-degenerated PAGE gel. 2 µL PCR products were taken for sample application at first and then electrophoresis was carried out for 1.5 h under 180 V. After electrophoresis, the sample was detected for silver staining and the procedures were simplified as follows: Fixed in 0.4% glacial acetic acid solution for 8 min, stained with 0.2% silver nitrate solution for 10 min and colored in the mixed solution of 1.5% sodium hydroxide and 0.5% formaldehyde for 8 min. The product was cleaned in pure water after each procedure and the whole process was carried out on horizontal rotators. After silver staining, the product was imaged under the X-ray film observation lamp, photographed and stored for later data analysis.
3.4 Field purity identification
Pumpkin samples were cultivated in field directly on August 29, 2016. The furrow was 1.4 meters wide. Every sample was seeded 2 lines in each field and each point was planted with 2 seeds. 100 points were needed in total and these seeds were cultivated on bamboo shelves of the shape of Chinese character “human”. 10 days after seedling emergence, seedling raising fertilizer was applied for one time and the urea concentration was 0.3%. The total number of plants and hybrid plants in each sample was counted in the field survey, and the purity of cultivars was calculated on October 8th.
3.5 Statistical analysis
Pumpkin seed purity (%) = total detection number-false hybrid number/total detection number
SPSS 17.0 was used to analyze the correlation coefficient of data of SSR purity identification and field purity identification. A regression equation was established as y=a+bx, and t identification was carried out.
LQ is the executor of the experimental design and research; LQ and WDH completed the data analysis and the first draft of the paper; YJG and WDH participated in the result analysis; LQ is the conceiver and director of the project, guiding the experimental design, data analysis, thesis writing and revision. All the authors read and approved the final manuscript.
This study is funded by Hunan Collection, Preservation and Application of Vegetable Germplasm Resources (Coalition subproject 2017LM0101).
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