Outline
Comptes Rendus

Biodiversity/Biodiversité
Genetic analysis of Canarium album in different areas of China by improved RAPD and ISSR
Comptes Rendus. Biologies, Volume 340 (2017) no. 11-12, pp. 558-564.

Abstract

To lay the foundation of the classification of Canarium album (C. album), and C. album from Terminalia Chebula (T. chebula) in different areas of China, improved RAPD and ISSR analysis were performed to analyze polymorphism and genetic relationship. Ten samples were collected from different locations in China. A total of 221 fragments were detected by improved RAPD, out of which polymorphic fragments accounted for 82.3% with average amplification bands of 10.05 per primer. ISSR markers revealed a total of 147 alleles, where polymorphic fragments accounted for 83.5%, with average amplification bands of 7.35 per primer. The sizes of fragments ranged from 200 to 2500 bp and from 150 to 2000 bp in RAPD and ISSR markers, respectively. The similarity coefficient ranged from 0.46 to 0.86 with RAPD markers and 0.36 to 0.89 with ISSR markers. The results indicated that improved RAPD and ISSR methods are useful for genetic diversity study of C. album. Thus, this study provides us the theoretical basis for the breeding and classification of C. album in South and Southwest China.

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DOI: 10.1016/j.crvi.2017.09.006
Keywords: Canarium album, RAPD, ISSR, Clustering analysis, Identification

Zhiliang Mei 1; Xianqin Zhang 1; Xiaoyan Liu 1; Saber Imani 1; Junjiang Fu 1, 2

1 The Research Center for Preclinical Medicine, Southwest Medical University, Luzhou, Sichuan Province 646000, PR China
2 Hunan Normal University Medical College, Changsha, Hunan Province 410013, PR China
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     author = {Zhiliang Mei and Xianqin Zhang and Xiaoyan Liu and Saber Imani and Junjiang Fu},
     title = {Genetic analysis of {\protect\emph{Canarium} album} in different areas of {China} by improved {RAPD} and {ISSR}},
     journal = {Comptes Rendus. Biologies},
     pages = {558--564},
     publisher = {Elsevier},
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Zhiliang Mei; Xianqin Zhang; Xiaoyan Liu; Saber Imani; Junjiang Fu. Genetic analysis of Canarium album in different areas of China by improved RAPD and ISSR. Comptes Rendus. Biologies, Volume 340 (2017) no. 11-12, pp. 558-564. doi : 10.1016/j.crvi.2017.09.006. https://comptes-rendus.academie-sciences.fr/biologies/articles/10.1016/j.crvi.2017.09.006/

Version originale du texte intégral

1 Introduction

Canarium album (Calbum), or Calbum (Lour.) Raeusch, Qingguo in Chinese, is a native cultivar in the Southeast of China, particularly in Sichuan, Guangdong, Guangxi, Hainan, and Fujian provinces. Calbum has been introduced to other Asian tropical and semi-tropical regions, including Vietnam, Malaysia, Japan, etc. It is a very good fruit cultivar because of its tolerance to poor soils, including saline or alkaline soils and rocky hillsides. Calbum in China is different from the European olive, which is basically for oil. Therefore, we also call it as Chinese olive (Ochinese). The fruit, nut, seed, and root of Ochinese have been used as Chinese medicines for a long time for swollen and sore throat [1], excessive thirst, hematemesis due to cough, lacillary dysentery, epilepsy, puffer poisoning, alcoholism. The dried fruits are also traditional medical materials that have some pharmacological functions, such as antioxidation, anti-virus, anti-bacterium, anti-inflammation and detoxification activities [2,3]. Therefore Calbum is extensively cultivated in China, producing an edible drupe fruit that is sold fresh on markets.

However, it is difficult to distinguish the different cultivars for Calbum only through their morphology. To understand the genetic variation between the different cultivars, the most effective tool for genetic analysis is DNA fingerprinting or genetic maker technique [4,5]. However, despite some advantages, the polymorphic sites detected by single DNA fingerprinting methods are restricted and genetic relationship among experimental materials cannot be fully elucidated. The number of polymorphic sites could be increased and the coverage of genome could be improved, if the data achieved based on several DNA fingerprinting technologies was integrated and analyzed, thus contributing to abundant genetic information. Random amplification polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are dominant markers for DNA fingerprinting detection in various cultivars, while the combination of RAPD and ISSR can provide a better understanding of genetic variation. In this study, we distinguish the different cultivars of Calbum from different regions of China by improved RAPD and ISSR, and study their genetic relationships. Importantly, Terminalia chebula (Tchebula), easily misclassified into Calbum, was introduced to explore their genetic relationship as well. Thus, in this study, improved RAPD-PCR and ISSR analyses were performed to analyze polymorphism and genetic relationships for classifying Calbum, and differentiating Calbum from Tchebula in different areas of China.

2 Materials and methods

2.1 Sample collection and reagents

The geographic distributions of the samples collected in this study were as shown in Fig. 1 and Table 1, including one sample from Luzhou city, Hejiang county in Sichuan province, which was described previously [6]. The fresh leaves and fruits were carefully identified and named according to the fruit size and weight, leaf size and colour (Table 1 and data not shown), and the specimens have been deposited at the source bank of Southwest Medical University. The DNAs of fresh leaf samples for each accession were collected from plants and was extracted with the modified CTAB method [7]. DNA quality was determined after electrophoresis on 1% agarose gels. DNA concentration was measured by spectrophotometry at 260 and 280 nm [8,9]. The final concentration of all DNA samples was adjusted to 10 ng/μL for PCR analysis and stored at –20 °C till use [9].

Fig. 1

The locality of Calbum samples from China. The blue line in the enlarged map indicates the Yangtze River.

Table 1

Sources of the samples used in this study.

Table 1
Sample Cultivars Source No.
MZ Canarium album (Lour.) Raeusch Meizhou, GuangDong 001
MM Canarium album (Lour.) Raeusch Maoming, Guangdong 002
QY Canarium album (Lour.) Raeusch QingYuan, GuangDong 003
FZ Canarium album (Lour.) Raeusch Fuzhou, Fujian 004
ZZ Canarium album (Lour.) Raeusch ZhangZhou, Fujian 005
GL Canarium album (Lour.) Raeusch Guilin, Guangxi 006
QZ Canarium album (Lour.) Raeusch Qinzhou, GuangXi 007
LZ Canarium album (Lour.) Raeusch Luzhou, Sichuan 008
CD Canarium album (Lour.) Raeusch ChengDu, Sichuan 009
PE Fructus Terminaliae chebulae Immaturi PuEr, Yunnan 010

2 × PCR Taq Master Mix was purchased from Tian Gen Biotech Co. Ltd (Beijing, China). The random RAPD primers were purchased from SBS Genetech Corporation (Beijing, China). DNA Marker (DL2000) was purchased from SinoBio (Beijing, China). Other reagents were analytical grade reagents used previously [10].

2.2 PCR amplification

The PCR amplification system with a total volume of 10 μL, including 1 μL of 2.5 μmol/L primers for RAPD (Table 2) or ISSR (Table 3), 1.5 μL of DNA template (about 15 ng), 5 μL of 2 × PCR TaqMastermix, and 3.5 μL of sterile water. PCR reactions of each accession were executed by means of the Applied Biosystems Veriti® 96-Well Thermal Cycler (Life Technology, NY, USA) [11,12]. All the PCR reactions were repeated at least three times for ten samples.

Table 2

Information of improved RAPD primers.

Table 2
Primer Sequence 5′–3′
SBS-A1 CAGGCCCTTC
SBS-A9 GGGTAACGCC
SBS-A15 TTCCGAACCC
SBS-A20 GTTGCGATCC
SBS-I10 ACAACGCGAG
SBS-I19 AATGCGGGAG
SBS-M15 GACCTACCAC
SBS-N5 ACTGAACGCC
SBS-Q4 AGTGCGCTGA
SBS-Q7 CCCCGATGGT
SBS-A8 GTGACGTAGG
SBS-A12 TCGGCGATAG
SBS-A19 CAAACGTCGG
SBS-I2 GGAGGAGAGG
SBS-I18 TGCCCAGCCT
SBS-M5 GGGAACGTGT
SBS-N3 GGTACTCCCC
SBS-N10 ACAACTGGGG
SBS-Q5 CCGCGTCTTG
SBS-Q11 TCTCCGCAAC
Table 3

Information of ISSR primers.

Table 3
Primer Sequence 5′–3′
807 AGA GAG AGA GAG AGA GT
810 GAG AGA GAG AGA GAG AT
814 CTC TCT CTC TCT CTC TA
818 CAC ACA CAC ACA CAC AG
825 ACA CAC ACA CAC ACA CT
834 AGA GAG AGA GAG AGA GYT
840 GAG AGA GAG AGA GAG AYT
842 GAG AGA GAG AGA GAG AYG
876 GAT AGA TAG ACA GAC A
889 DBD ACA CAC ACA CAC AC
808 AGA GAG AGA GAG AGA GC
811 GAG AGA GAG AGA GAG AC
815 CTC TCT CTC TCT CTC TG
823 TCT CTC TCT CTC TCT CC
828 TGT GTG TGT GTG TGT GA
836 AGA GAG AGA GAG AGA GYA
841 GAG AGA GAG AGA GAG AYC
855 ACA CAC ACA CAC ACA CYT
886 VDV CTC TCT CTC TCT CT
898 CCC TCC CTC CCT CCC T

PCR amplification for ramp-PCR was performed as follows: initial denaturation at 95 °C for 90 s, followed by 40 cycles of 94 °C for 40 s, 36 °C for 90 s, 72 °C for 90 s, and final extension of 5 min at 72 °C. The ramp time from annealing to extension was adjusted from 2.5 °C/s (100%) (default time) to 0.125 °C/s (5%) to compare the resolution and production of the two methods by ramp-PCR. The ramp rate from annealing to extension was adjusted to 0.125 °C/s (5% ramp rate). This was carried out in triplicate for all 10 samples [13].

The PCR condition for ISSR was as follows: initial denaturation at 95 °C for 90 s, followed by 35 cycles of 40 s at 94 °C, 30 s at 50 °C, 90 s at 72 °C, and final extension during 5 min at 72 °C. DNA was amplified by a total of 20 ISSR primers (Table 3) with valuable polymorphic bands.

2.3 Agarose electrophoresis

The amplified PCR products by ramp-PCR or ISSR were resolved by electrophoresis on a 1.5% agarose gel in 0.5 × TAE buffer. Gels were visualized by staining with 0.5 μg/mL ethidium bromide (EtBr), and the images were documented using the ChemiDoc XRS system (Bio-Rad, USA). Bands that were unambiguous and reproducible in successive amplifications were selected for scoring [11,12].

2.4 Statistical analysis

All the bands with signals clearly in the gel profiles were recorded as present (1) or absent (0). The similarity matrix (SM) and the similarity index (SI) were calculated using the SM coefficient, and the dendrogram based on UPGMA (unweighted pair group method with arithmetic mean algorithm) was generated using the SAHN module in NTSYS-pc 2.1 packages, which was described previously [13,14].

3 Results

3.1 Improved RAPD technique in analysis of Calbum

To establish the condition of improved RAPD in Calbum, SBS-A8 and SBS-I19 primers were used and the ramp time was adjusted from annealing to extension with 2.5 °C/s to 0.125 °C/s in 10 samples collected (Fig. 1, Table 1). As seen in Fig. 2, all samples demonstrated similar repeatable fingerprints; each sample amplified less bands in average with the ramp time 2.5 °C/s (Fig. 2, left); the amount of PCR products (mainly large pieces of DNA) and DNA bands was obviously increased when the ramp time was adjusted from 2.5 °C/s to 0.125 °C/s (Fig. 2, right). For example, the sample No. 3 only had 2 bands with regular PCR (Fig. 2A, left), while the number of bands increased to 5 with the improved RAPD technique (Fig. 2A, right), indicating that the production, resolution, and reproducibility of RAPD were significantly improved. Therefore, the improved RAPD technique, prolonging the ramp time from annealing to extension 0.125 °C/s, was applied to complete other RAPD primers.

Fig. 2

The results of improved RAPD. A. Primer SBS-A8. B. Primer SBS-I19. Regular PCR represents regular RAPD amplification, while ramp-PCR represents improved RAPD amplification. The number represents different samples shown in Table 1. “M” means DL2000 DNA marker with the indicated molecular weight size (bp).

3.2 Amplification results of improved RAPD analysis

Twenty primers (Table 2) were used to conduct improved RAPD analysis, and RAPD results were improved. PCR reaction for each sample was repeated three times, and generated reproducible polymorphic amplification bands. The representative fingerprints are shown in Fig. 3. The band sizes of the PCR products ranged from 200 bp to 2500 bp (Fig. 3). A total of 221 bands were obtained, among which polymorphic fragments accounted for 82.3%, indicating high genetic diversity among the10 experimental materials with average amplification bands of 10.05 per primer. A maximum of bands (14) appeared when primer SBS-Q7 was used (data not shown). The results indicated that these primers are useful to identify DNA polymorphisms and perform the analysis of the genetic relationships in Calbum samples.

Fig. 3

The different banding patterns of Calbum samples using the improved RAPD method. The number represents the different samples shown in Table 1. “M” means DL2000 DNA marker with the indicated molecular weight size (bp). Only partial results are shown.

3.3 Genetic distance and cluster analysis by improved RAPD

The genetic relationship dendrogram was obtained by conducting cluster analysis to the similarity coefficient of the above-mentioned ten cultivars by improved RAPD (Fig. 4). Their similarity coefficient ranged from 0.46 to 0.86 (Fig. 4B). Among the similarity coefficients of the cultivars, the similarity coefficients of samples QZ and GL were the highest at 0.86, and clustered together, whereas the similarity coefficients of samples PE and FZ were the lowest at 0.46, which showed the biggest genetic distances (Fig. 4A and B). The similarity coefficient of sample PE was lower than those of the other nine samples (Fig. 4A).

Fig. 4

Dendrogram of Calbum samples based on improved RAPD. A. Dendrogram of Calbum samples based on 221 PCR bands amplified by the 20 primers. The bar on the bottom indicates the similarity index based on the SM coefficient. B. Genetic distance for Calbum samples using improved RAPD. The number represents the different samples shown in Table 1.

3.4 Amplification results of the ISSR analysis

Twenty primers (Table 3) were used to conduct the ISSR analysis, and improved RAPD results were obtained. The PCR reaction for each sample was repeated three times, and generated reproducible polymorphic amplification bands. The representative fingerprints are shown in Fig. 5. The band sizes of PCR products ranged from 150 bp to 2000 bp (Fig. 5). A total of 147 bands were obtained, among which polymorphic fragments accounted for 83.5%, indicating high genetic diversity among the 10 experimental materials, with an average amplification band of 7.35 per primer. A maximum of bands (12) appeared when primer 841 was used. The results indicated that these primers are useful in identifying DNA polymorphisms and performing genetic relationship analysis in Calbum samples.

Fig. 5

The different ISSR banding patterns in Calbum samples. The numbers 1 to 10 indicate the different samples showed in Table 1. “M” means DL2000 DNA marker with the indicated molecular weight size (bp). Only partial results are shown.

3.5 Genetic distance and cluster analysis by ISSR

The genetic relationship dendrogram was obtained by conducting cluster analysis to the similarity coefficient of the above-mentioned ten cultivars by ISSR (Fig. 6). Their similarity coefficient ranged from 0.36 to 0.89 (Fig. 4B). Among the similarity coefficients of the cultivars, the similarity coefficient of samples QZ and GL was highest at 0.89, and clustered together. Whereas the similarity coefficient of samples PE and LZ was lowest at 0.36, which showed the biggest genetic distances (Fig. 4A and B). The similarity coefficient of sample PE was lower than those of the other nine samples (Fig. 4A).

Fig. 6

Dendrogram of Calbum samples based on ISSR. A. Dendrogram of Calbum samples based on 147 PCR bands amplified by the 20 primers. The bar on the bottom indicates the similarity index based on the SM coefficient. B. Genetic distance dendrogram for Calbum samples. The number represents the different samples shown in Table 1.

4 Discussion

Since the 1990s, a number of molecular marker techniques have been developed, including random RAPD [9,15–17], ISSR [18,19], and amplified fragment length polymorphism (AFLP) [20], etc., which has been widely used in the genetic identification of various plants and animals [21,22]. ISSR frequently occurs in most eukaryotic genomes and can be very informative, reliable (reproducible), co-dominant, multi-allelic, and highly polymorphic, making them well suited for detecting variations among closely related varieties [23]. However, the application of the ISSR technique to plants is only possible when the microsatellite markers suitable for that plant were available [24]. It has been proved that ISSR is available for studying Calbum [25,26]. RAPD is characterized by fewer requirements on template DNA, no pollution, and inexpensive, but also by disadvantages as poor reproducibility and low production. Fu et al. [10] reported that the resolution and production of RAPD are greatly increased by prolonging the ramp time from annealing to extension. To examine the effectiveness of RAPD method in Calbum, an improved RAPD technique has been developed and applied by prolonging the ramp time from 2.5 °C/s to 0.125 °C/s. Here, 2.5 °C/s was the default ramp time, we called it regular RAPD-PCR or regular PCR; 0.125 °C/s ramp time was the reduced rate of ramp time, we called it improved RAPD-PCR or ramp-PCR. In our analysis, we obtain 221 bands in randomly selected 20 primers by ramp-PCR.

The production of RAPD was improved, and the number of DNA amplified bands was increased by prolonging the ramp time. Specifically, in SBS-A8 amplification patterns, the numbers of band and productions are enhanced obviously in sample No. 1 by ramp-PCR from 3 to 6 (Fig. 2A); in SBS-I19 amplification patterns, the band numbers and productions are enhanced obviously in sample No. 1 by ramp-PCR from 3 to 7, respectively (Fig. 2A, B). Therefore, the Calbum accessions are distinguishable using our improved RAPD technique according to the abundant DNA polymorphisms. The method described here can amplify more specific DNA bands from the primers (Fig. 2). The reason may be that prolonging the ramp time is beneficial to DNA template binding with primers. Furthermore, the low heating speed was beneficial to increasing the bond stability of the primer with template DNA, preventing the primer from detaching from the template [22], thus increasing the number of bands and the PCR product yields. Therefore, RAPD with ramps of slower speed can be used to increase the yields, resolutions, and numbers of polymorphic bands, thereby increasing the stability and accuracy of the experimental results.

Tchebula, commonly known as yellow or chebulic myrobalan, is often confused with Calbum from the point of view of the phenotype, whereas they are different from each other at the gene level based on our current study. The medical value can be highlighted when Tchebula is distinguished from Calbum. Our cluster analysis reveals that all 10 samples were clustered into two groups by both RAPD and ISSR methods. Sample PE (Tchebula) form an own group itself, showing lowest genetic similarity and highest genetic distance compared with the other nine samples (Calbum). Our study confirmed that Calbum is a different cultivar than Tchebula, and revealed a low genetic relationship between sample PE and the other ones.

This study may provide a useful source of genetic diversity in Calbum, because it included genotypes from different parts of China. The genetic level of differentiation may connect with the geographical distribution. Samples CD and LZ, sampled from two places in the Sichuan Basin, are clustered together, showing close genetic relationship. The other 7 samples, whose locations are close to each other, were clustered together, exhibiting the existence of high genetic similarity and low genetic distance. These genotypes can be applied in future breeding selection to maximize the genetic diversity in Calbum.

Despite the discrimination of PCR amplified profiles in the two marker assays [25], some differences could be detected between RAPD and ISSR [27]. In our study, similar results were obtained by both methods, indicating that ISSR is obviously a useful tool to study Calbum. Actually, the polymorphic fragments detected by ISSR (83.5%) were higher than that (82.3%) detected by RAPD, although less bands were achieved, revealing that the ISSR makers selected in this study are suitable to study the genetic diversity of Calbum. Due to the traits that ISSR markers link to SSR loci, ISSR markers are likely to mark gene-rich regions [28,29], which might be useful in breeding selection. To our best knowledge, this is the first report of a genetic diversity study of Calbum combining both improved RAPD and ISSR technique.

5 Conclusion

In this study, we show that improved RAPD and ISSR methods are useful for studying the genetic diversity of Calbum. Our study provides a theoretical basis for the breeding and classification of Calbum in China. The improved RAPD and ISSR could be effective techniques to analyze the polymorphism and genetic relationships in a variety of applications, including Calbum.

Disclosure of interest

The authors declare that they have no competing interest.

Acknowledgments

The work was supported in part by the Science and Technology Innovation Team of Colleges and Universities of Sichuan Province (13TD0032), and the Research Foundation of the Science and Technology Department of Sichuan Province (14JC0797, 2015JY0038).


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