Jacaranda mimosifolia is a tall deciduous tree of Jacaranda in Bignoniaceae, which is famous for its blue-purple flowers and has high ornamental value. This tree species is native to South America, Australia and Africa, widely distributed in Peru, Mexico, Bolivia, Brazil, Argentina, South Africa and other countries. There are 50 species in the world, and two species of them, Jacaranda mimosifolia D.Don and Jacaranda cuspidifolia Mart. are introduced and cultivated in China, which are mainly introduced in Guangdong, Guangxi, Sichuan, Fujian, Hainan, Yunnan (Liu, 2015). Jacaranda mimosifolia is sensitive to the geographic climate and environmental conditions, and its growth, especially the phenological phases of leaves and flowers, are quite different in different habitats (Sun, 2016). Mastering the growth adaptability of Jacaranda mimosifolia, especially the rule of flowering phenological phases, will be the key technical bottleneck in the industrial development and application of Jacaranda mimosifolia, and carrying out genetic diversity research will provide a theoretical basis for the breeding of the improved varieties.

The early introduction of Jacaranda mimosifolia in China was rare and precious, and there were only single plants in some cities. Because the detailed source of its germplasm resources could not be verified, the current Jacaranda mimosifolia varieties on the market were intermingled, and the relationship between provenances was chaotic. Therefore, studying the genetic background of Jacaranda mimosifolia germplasm resources and revealing its genetic diversity are of great value to the protection of Jacaranda mimosifolia germplasm resources and the genetic improvement of varieties. The industrialization of Jacaranda mimosifolia in China began in the 1980s, but there were few research reports on this tree species at home and abroad, mainly focusing on breeding technology (Zaouchi et al., 2013; Miyajima et al., 2013; Zhang et al., 2016), landscape value and wood properties (Xie et al., 2004, Southwest Horticulture, 32(6): 31-32; Zhang et al., 2016). Li (2015) analyzed the distribution of germplasm resources and garden application of Jacaranda mimosifolia in Sichuan, and put forward suggestions on the suitable area of Jacaranda mimosifolia in Sichuan; Zhou et al. (2013) carried out the sequence analysis of Jacaranda mimosifolia JaCBF1 transcription factor fragment, cloned a new CBF-like transcription factor fragment from Jacaranda mimosifolia, and verified the cold tolerance function. There are few reports on the germplasm resources and genetic diversity of Jacaranda mimosifolia.

ISSR (inter-simple sequence repeat) molecular marker is a molecular marker technique developed on the basis of SSR (simple sequence repeat) molecular marker (Ewa et al., 1994). This technique is widely used in the study of plant genetic diversity and relationship identification (Igwe et al., 2017; Kalia et al., 2017; Takata et al., 2019). Therefore, in order to study the genetic diversity and relationship of Jacaranda mimosifolia from the molecular level, this study carried out cluster analysis of Jacaranda mimosifolia from different areas through ISSR molecular markers, and identified the germplasm from the DNA level. The results showed that the germplasm resources of Jacaranda mimosifolia introduced in China can be clustered into two groups, and have rich genetic diversity, which will provide a theoretical basis for the protection of germplasm resources, breeding of improved varieties and innovative utilization of Jacaranda mimosifolia.

1 Results and Analysis

1.1 ISSR primer screening and polymorphism of amplified products

From the 33 ISSR primers, 10 primers that can amplify clear bands and have polymorphisms were screened. Figure 1 showed the amplification results of 10 ISSR primers in sample 5.

10 ISSR primers was used to perform PCR amplification on 21 samples of Jacaranda mimosifolia, and the DNA amplification effect was good. The PCR amplification detection results of primer UBC855 in 21 samples of Jacaranda mimosifolia (Figure 2) showed that the amplified band number and fragment size were polymorphic.

ISSR molecular marker technique was used to manually read bands from 21 samples of Jacaranda mimosifolia. A total of 53 clearly identifiable bands were obtained from 10 primers, of which 44 bands were polymorphic, with an average polymorphism ratio of 83.02%. Each primer could amplify 5.3 bands in average, and the average polymorphic band was 4.4 (Table 1). The highest polymorphism was 100.00% and the lowest was 50.00%. Among them, the amplification polymorphisms of primers UBC836 and UBC876 were the lowest, which were 50.00%, while the amplification polymorphisms of primers UBC834, UBC855, UBC856 and UBC864 were 100.00%. The amplified DNA fragments were 100~1200 bp. Therefore, the 21 tested germplasm resources of Jacaranda mimosifolia had high genetic polymorphism.

1.2 Genetic diversity analysis

The genetic diversity index of ISSR (Table 2) was analyzed by PopGen32 software. The total number of tested alleles (Na) of 21 samples of Jacaranda mimosifolia was 1.83 in average, the total number of effective alleles (Ne) was 1.00~2.00, with an average of 1.45, the total Nei’s gene diversity index (H) was 0.00~0.50, with an average of 0.27, and Shannon’s information index (I) was 0.00~0.69, with an average of 0.40. Among them, the average number of effective alleles Ne, which can better reflect the real situation of genetic diversity, was 1.45 in 21 germplasms from 19 provenances. Therefore, it can be seen that the genetic diversity of 21 tested germplasm resources of Jacaranda mimosifolia was high.

1.3 Genetic distance and genetic relationship analysis

The amplification results of 10 ISSR primers of 21 Jacaranda mimosifolia samples were assigned 1 and 0 respectively according to the primers with or without bands to obtain the genetic distance and genetic similarity (Table 3). The results showed that the average genetic similarity coefficient of 21 Jacaranda mimosifolia samples was 0.72, and the range of genetic similarity coefficient between different samples was 0.57~0.91. Among them, the genetic similarity between Jm4 and Jm15 samples was the largest, which was 0.91, indicating that there was a small difference between Jm4 and Jm15 samples, and the genetic distance was the smallest (0.10); The genetic similarity coefficient between Jm3 and Jm6 samples was the smallest, which was 0.57. Moreover, the genetic similarity coefficient between Jm3 and Jm11 and Jm12 samples was also the smallest, indicating that Jm3 was quite different from Jm6, Jm11 and Jm12, and the genetic distance was farther.

Cluster analysis was performed on 21 samples of Jacaranda mimosifolia by unweighted pair group with mathematic average (UPGMA) (Figure 3). 21 germplasm resources of Jacaranda mimosifolia were clustered into two groups, of which Jm10 (Putian 2, Fujian), Jm11 (Putian 1, Fujian) and Jm16 (Kaiyuan, Yunnan) were clustered into group B, and the average genetic similarity coefficient was 0.72; While group A included 18 other samples of Jacaranda mimosifolia, and its average genetic similarity coefficient was 0.73. In group A, Jm6 (Panzhihua, Sichuan), Jm12 (Kunming, Yunnan) and Jm20 (Guangzhou, Guangdong) were clustered into a small group, the average genetic similarity coefficient of these three samples was 0.74, and the other 15 samples were clustered into another small group. Among them, the genetic similarity between Jm4 from Yibin, Sichuan and Jm15 from Xiamen, Fujian was the highest, which was 0.91. The genetic similarity between these two and Jm13 from Xunwu, Jiangxi was higher and the genetic distance was closer. The genetic similarity between Jm14 from Quanzhou, Fujian and Jm17 and Jm19 from Kaiyuan and Mengzi, Yunnan respectively was higher, and the genetic distance between this sample and Jm4, Jm13 and Jm15 samples was closer, and the average genetic similarity coefficient was 0.82.

There were 21 germplasms from 19 provenances, and their female parent germplasms came from 7 provinces (autonomous regions and municipalities directly under the central government) in China. The analysis of habitat conditions showed that 21 tested germplasms of Jacaranda mimosifolia spanned 8 latitudes from north to south, among which Jm1, Jm4 and Jm5 were at relatively high latitudes, N30.62º, N28.45º and N27.89º respectively, and Jm15, Jm20 and Jm21 were at relatively low latitudes, N24.48º, N23.13º and N22.82º respectively, but none of them showed relatively close genetic relationship; Jm7, Jm15 and Jm20 were at relatively low altitudes, at 15 m, 0 m and 14 m respectively, and Jm5 and Jm12 were at relatively high altitudes, at 1 513 m and 1 908 m respectively, but none of them showed relatively close genetic relationship. It was inferred that the genetic distance of Jacaranda mimosifolia introduced and cultivated in China was not strongly correlated with administrative region or geographical distance, and there may be randomness in the layout of introduction and cultivation.

According to the investigation of germplasm resources, the Jm11 from Putian, Fujian was identified as Jacaranda cuspidifolia Mart. by relevant local departments, but through field observation, it was not found that its leaves had the characteristics of Jacaranda cuspidifolia Mart., which can be further confirmed from the results of this study that Jm11 was not clustered into a single group.

2 Discussion

Genetic diversity can reflect the origin and evolution, reproductive vitality and adaptability to environmental changes of a species, and can largely determine the number, geographical distribution and future survival and development of the species (Lin et al., 2019). According to the results of genetic distance and genetic similarity, the average genetic similarity coefficient of 21 samples of Jacaranda mimosifolia was 0.72, the variation range of genetic similarity coefficient between different samples was 0.57~0.91, and the average Nei’s gene diversity index was 0.27, which was higher than the average genetic diversity level of plants (0.22~0.23) published by Nybom (2004), indicating that Jacaranda mimosifolia had rich genetic diversity. There were obvious genetic differences in genomic DNA of Jacaranda mimosifolia from different provenances, and great genetic changes had taken place, which may be the genetic evolution affected by ecological environment, climatic conditions and natural hybridization, which was consistent with Nevo (2001). The tested Jacaranda mimosifolia was basically from the female germplasm of Jacaranda mimosifolia that was introduced and cultivated in China in the early stage. The earliest one has a history of more than 100 years. The surviving ones have gone through a long time and natural selection in different environments. They were not only very rare and precious, but also further verify the genetic evolution and strong adaptability of Jacaranda mimosifolia. There were only two varieties of Jacaranda mimosifolia introduced in China, Jacaranda mimosifolia D.Don and Jacaranda cuspidifolia Mart.. The survey found that the Jm11 from Putian, Fujian was identified as Jacaranda cuspidifolia Mart., but through field observation, it was not found that its leaves had the characteristics of Jacaranda cuspidifolia Mart., which can be further confirmed from the results of this study that Jm11 was not clustered into a single group.

At present, the analysis of genetic diversity at the molecular level has not been reported. The identification of Jacaranda mimosifolia germplasm resources was limited to traditional identification methods, such as judging according to new shoots, finalized leaves, phenological phases of vegetative buds, flowers, fruits and other characters. However, the phenological characteristics of plants growing in different regions often change with climate factors such as temperature, light and water (Wang et al., 2010). Sun (2016) analyzed the growth of Jacaranda mimosifolia, especially the expression analysis of flowering phenological characteristics, and found that there were significant differences in the four different habitats. Therefore, it was difficult to make accurate judgment based on the phenotypic characteristics of Jacaranda mimosifolia by traditional identification methods. ISSR can effectively reveal the genetic diversity, genetic relationship and genetic differentiation of plant germplasm resources (Zhang and An, 2015). This study identified the germplasm resources at the molecular level, defined the genetic relationship of the tested Jacaranda mimosifolia, divided 21 germplasms from 19 provenances into different groups, obtained the genetic relationship of Jacaranda mimosifolia from different provenances, and distinguished different groups. The results showed that Jacaranda mimosifolia had rich genetic diversity, and its genetic relationship was not strongly related to the geographical source of the cultivation area, which will provide a theoretical reference for discussing the adaptability and viability, the protection of germplasm resources, breeding of improved varieties and industrialized application of Jacaranda mimosifolia. In view of Jacaranda mimosifolia being famous as viewing flowers and its sensitivity to the geographical and climatic environment (Sun et al., 2015), in future research, we can deeply explore the genetic variation rules, flowering and fruiting rules of Jacaranda mimosifolia, and use genetic engineering means to regulate flower color and shape and improve flower quality (Wang et al., 2003), so as to improve the competitiveness of Jacaranda mimosifolia in the application market of similar industries.

3 Materials and Methods

3.1 Test materials

The tested Jacaranda mimosifolia samples were all young plants, which were collected from the mother trees of Jacaranda mimosifolia introduced and cultivated in 21 different regions of China, including Sichuan, Chongqing, Fujian, Jiangxi, Yunnan, Guangdong, Guangxi and so on (Table 4). The annual seedlings were sown and cultivated in the South National Forest Seedling Demonstration Base (21°30′N, 111°38′E) in Zhanjiang, Guangdong. After the young plants were collected in June, 2018 and brought back to the laboratory, fresh leaves were taken, washed with deionized water, dried, ground into powder with liquid nitrogen, transferred into a centrifuge tube, numbered and stored in a -20℃ refrigerator for later use.

3.2 Genomic DNA extraction and detection

The total genomic DNA of the tested Jacaranda mimosifolia was extracted by the plant genomic DNA extraction kit provided by Tiangen Biotech (Beijing) Co., Ltd., and the operation steps were carried out according to the instructions and adjusted appropriately. 1.0% agarose and EB staining gel were used to detect the electrophoresis quality. The nucleic acid protein analyzer was used to detect the concentration and purity of DNA, and then the concentration was diluted to about 30 ng/μL and stored in the refrigerator at -20℃ for later use.

3.3 ISSR primer screening

From the 100 ISSR primer sequences published by Columbia University (UBC Primer Set#9), 33 characteristic primers with clear and rich amplification bands were selected for the study of genetic diversity of Jacaranda mimosifolia, which were synthesized by GenScript (Table 5).

3.4 Detection of ISSR amplification products

The ISSR-PCR reaction system and procedure were designed according to relevant literature (Fan et al., 2011; Li et al., 2013; Wu et al., 2015; Liu et al., 2015). The optimized PCR reaction system was 20 µL: 1 µL 30 ng DNA plate, 10 µL 2×Taq PCR MasterMix, 1 µL primer, and make up to 20 µL with ddH₂O. PCR amplification conditions were as follows: pre-denaturation at 94℃ for 4 min, denaturation at 94℃ for 45 s, annealing at 52℃ for 45 s, extension at 72℃ for 90 s, and for 36 cycles; and store at 4℃ after extension at 72℃ for 7 min.

The electrophoresis detection of PCR amplification products in 1.0% agarose horizontal gel containing ethidium bromide (EB) was carried out. The electrophoresis conditions were as follows: sample loading 20 µL, 1×TAEe electrophoresis buffer, 120V voltage electrophoresis to the appropriate position. After electrophoresis, the gel imaging system was observed and photographed.

3.5 Data statistical analysis

The electrophoresis results of fully amplified primers were quantified and compared with DNA ladder marker to determine the size of amplified DNA fragments and sort them. The primers with bands were assigned 1, while the primers without bands were assigned 0, and the binary metadata matrix of 1 and 0 was constructed, and then the genetic diversity parameters were calculated by Popgene32 (Abbasi et al., 2017; Peng et al., 2013). The genetic similarity coefficients between the tested samples were calculated by software Ntsys 2.10e, the SHAN tree was constructed by unweighted pair group with mathematic average (UPGMA), and the genetic relationships between different groups and different varieties were analyzed.

Authors’ contributions

ZGW and LXF were designers of the experiments and executors of this study; LXF and LG participated in data collation and wrote the first draft of the manuscript; LXM, ZPJ, HM, GLQ and FL participated in some experiments; ZGW was the person in charge of the project, guiding experimental design, data statistics, thesis writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by Special Fund Project of Central Public-Interest Scientific Institution (CAFYBB2019MB004) and Special Fund Project of Forestry Science and Technology Innovation in Guangdong Province (2018KJCX024).

References

Abbasi S., Afsharzadeh S., and Saeidi H., 2017, Genetic diversity of Potamogeton pectinatus L. in iran as revealed by ISSR markers, Acta Botanica Croatica, 76(2): 177-182
https://doi.org/10.1515/botcro-2017-0008

Nevo E., 2001, Evolution of genome–phenome diversity under environmental stress, Proc. Natl. Acad. Sci. USA, 98(11): 6233-6240
https://doi.org/10.1073/pnas.101109298

Ewa Z., Antoni R., and Damian L., 1994, Genomic fingerprinting by simple sequence repeat (SSR)-anchored poly-merase chain reaction amplification, Genomics, 20(2): 176-183
https://doi.org/10.1006/geno.1994.1151

Fan H.Y., Cao F.X., Peng J.Q., Long J.X., Deng M., and Si S.B., 2011. Establishment and optimization of ISSR—PCR reaction system of Camellia gigantocarpa, Zhongnan Linye Keji Daxue Xuebao (Journal of Central South University of Forestry & Technology), 31(4): 97-103

Igwe D.O., Afiukwa C.A., Ubi B.E., Ogbu K.I., Ojuederie O.B., and Ude G.N., 2017, Assessment of genetic diversity in Vigna unguiculata L. (Walp) accessions using inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) polymorphic markers, BMC genetics, 18(1): 98
https://doi.org/10.1186/s12863-017-0567-6
PMid:29149837 PMCid:PMC5693802

Kalia P., Saha P., and Ray S., 2017, Development of RAPD and ISSR derived SCAR markers linked to Xca1Bo gene conferring resistance to black rot disease in cauliflower (Brassica oleracea var. botrytis L.), Euphytica, 213(10): 232
https://doi.org/10.1007/s10681-017-2025-y

Li Q., 2015, Study on distribution and landscape application of Jacaranda mimosiflia D. Don in Sichuan region, thesis for M.S., Sichuan Agricultural University, supervisor: Shi J.A., pp.

Li X.H., Zhang H., Wang D.Y., Zhang L., Shao J.W., Zhang X.P., 2013, The genetic structure of endemic plant Pteroceltistatarinowii by ISSR markers, Shengtai Xuebao (Acta Ecologica Sinica), 33(16): 4892-4901
https://doi.org/10.5846/stxb201212191825

Liu J., Liao K., Zhao S.R, Cao Q., Sun Q., and Liu F., 2015, The core collection construction of Xinjiang wild apricot based on ISSR molecular markers, Zhongguo Nongye Kexue (Scientia Agricultura Sinica),48(10): 2017-2028

Lin L., Zhang M., Du Z., Zhu T.T., Sun S.B., and Jin L., 2019, Analysis on genetic diversity of 16 populations of Lycium ruthenicum Murr. in middle and lower reaches of Heihe river basin based on ISSR molecular markers, Zhongguo Zhongyiyao Xinxi Zazhi (Chinese Journal of Information on Traditional Chinese Medicin), 26(11): 62-67

Miyajima I., Takemura C., Kobayashi N., SotoG M.S., and Facciuto G., 2013, Flower bud initiation and development of Jacaranda mimosifolia (Bignoniaceae) in Japan, Acta Horticulturae, (1000): 71-76
https://doi.org/10.17660/ActaHortic.2013.1000.7

Nybom H., 2004, Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants, Molecular Ecology, 13(5): 143-1155
https://doi.org/10.1111/j.1365-294X.2004.02141.x
PMid:15078452

Peng J.Q., Cao F.X., and Fan H.Y., 2013, Study on genetic diversity of Camellia gigantocarpa detected by ISSR markers, Zhongnan Linye Keji Daxue (Journal of Central South University of Forestry & Technology), 33(7):62-66

Sun Y., Shi J.A., Shao X.P., Li F., Li Q., Gao X., Zhong Q., Zhao W.Z., 2015, Effects of light intensity on photosynthetic pigment content and flowering of Jacaranda mimosifolia D. Don in different habitats, Yingyong yu Huangjing Shengwu Xuebao (Chinese Journal of Applied and Environmental Biology), 21(6): 1150-1156

Sun Y., 2016, Growth and Development of Jacaranda mimosiflia in different habitats, thesis for M.S., Chengdu: Sichuan Agricultural University, Supervisor: Shi J.A., pp.

Takata K., Taninaka H, Nonaka M., Iwase F., Kikuchi T., Suyama Y., Nagai S., Yasuda N., 2019, Multiplexed ISSR genotyping by sequencing distinguishes two precious coral species (Anthozoa: Octocorallia: Coralliidae) that share a mitochondrial haplotype, PeerJ, 7: e7769
https://doi.org/10.7717/peerj.7769

Wu Z.D., Ni H.T., Wang M.Q., Wang H.Z., and Ma L.B., 2015, Screening of ISSR core primer suitable for identifying beet cultivar, Zhongguo Nongxue Tongbao (Chinese Agricultural Science Bulletin), 31(17): 48-52

Wang J.X., Chen H.L., Li Q., and Yu W.D., 2010, Research advances in plant phenology and climate, Shengtai Xuebao (Acta Ecologica Sinica), 30(2): 447-454

Wang X.Q., Meng X.C., and Peng J.Z., 2003, Regulation of flower growth and pigmentation, Xibei Zhiwu Xuebao (Acta Botanica Boreali⁃Occidentalia Sinica), 23(7): 54-59

Zhang G.W., Yin Q., Li R.P., Liu X.F., Xu Y.X., Deng X.M., 2016, A Study on tissue culture of Jacaranda mimosifolia, Linye yu Huanjing Kexue (Forestry and Environmental Science), 32(6): 86-89

Zhang G.W., Zhang P.J., Liu X.F., Cai L.R., Xu Y.X., and Sun J., 2016, Wood anatomical characteristics of introduction Jacaranda mimosifolia, Linye yu Huanjing Kexue (Forestry and Environmental Science), 32(2): 27-30

Zhou J., Zhang W., Li D.M., Zhang X.J., and Chen Q.B., 2013, Sequence analysis and chilling resistance about a new CBF-like transcription factor fragment from Jacaranda acutifolia, Linye Kexue Yanjiu (Forest Research) 26(4): 414-419

Zhang Q., and An H.M., 2015, Genetic diversity of Pyrus pyrifoliare sources distributed in Guizhou province revealed by ISSR markers, Guoshu Xuebao (Journal of Fruit Science), 32(1): 6-12

Zaouchi Y., Bahri N.B., Rezgui S., and Bettaieb T., 2013, Effects of arbuscular mycorrhizal inoculation and fertilization on mycorrhizal statute of Jacaranda mimosifolia D.Don cultivated in nurseries, Comptes Rendus Biologies, 336(10): 493-499
https://doi.org/10.1016/j.crvi.2013.09.005
PMid:24246891