Scholarly article on topic 'ISSR analysis of Fusarium oxysporum Schl. in Hebei province'

ISSR analysis of Fusarium oxysporum Schl. in Hebei province Academic research paper on "Biological sciences"

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{"Fusarium oxysporum Schl." / ISSR / "Reaction system optimization" / "Cluster analysis" / "Genetic diversity"}

Abstract of research paper on Biological sciences, author of scientific article — Yuan Lin, Lu Fuping, Liu Shanshan, Wang Xiao, Zhang Ruixuan, et al.

Abstract An Orthogonal design is applied to the optimization of ISSR-PCR reaction system for Fusarium oxysporum Schl. The effect of the five main factors was tested. A most suitable ISSR-PCR reaction system for Fusarium oxysporum Schl. was established. The optimized reaction system consists of 20 ng template DNA, 1 U Taq DNA polymerase, 0.4μmol/L primer, 0.2 mmol/L dNTPs, 4.0 mmol/L Mg2+ and 2.5μL 10× buffer. Optimal annealing temperature was decided by different primers. 2 primers with stable amplification and rich polymorphism for ISSR were selected. Based on the optimizing, 30 isolated strains from Hebei were tested in order to analyse the genetic diversity between of Fusarium oxysporum Schl. The isolated 30 strains were clustered into 2 genetic line-ages at 0. 86 genetic similarity. The genetic lineages of 30 strains showed no obvious relation with their geographical originals.

Academic research paper on topic "ISSR analysis of Fusarium oxysporum Schl. in Hebei province"

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Environmental Sciences

Procedia Environmental Sciences 12 (2012) 1237 - 1242

2011 International Conference on Environmental Science and Engineering

(ICESE 2011)

ISSR analysis of Fusarium oxysporum Schl. in Hebei province

12 1 2 2 2 Yuan Lin ' ,Lu Fuping ,Liu Shanshan ,Wang Xiao ,Zhang Ruixuan ,Li

Ziqin3,Zhang Hui3

1Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300222,

2College of Life Science, Inner Mongolia University, Huhhot 010021, China 3Inner Mongolia Academy of Agriculture and Animal Husbandry, Huhhot 010021, China

Abstract

An Orthogonal design is applied to the optimization of ISSR-PCR reaction system for Fusarium oxysporum Schl. The effect of the five main factors was tested. A most suitable ISSR-PCR reaction system for Fusarium oxysporum Schl. was established. The optimized reaction system consists of 20 ng template DNA, 1 U Taq DNA polymerase, 0.4 ^mol/L primer, 0.2 mmol/L dNTPs, 4.0 mmol/L Mg2+ and 2.5^L 10* buffer. Optimal annealing temperature was decided by different primers. 2 primers with stable amplification and rich polymorphism for ISSR were selected. Based on the optimizing, 30 isolated strains from Hebei were tested in order to analyse the genetic diversity between of Fusarium oxysporum Schl. The isolated 30 strains were clustered into 2 genetic line-ages at 0. 86 genetic similarity. The genetic lineages of 30 strains showed no obvious relation with their geographical originals.

© 2011 Publishedby Elsevier B.V. Selection and/or peer-review underresponsibility ofNational University of Singapore. Keywords:Fusarium oxysporum Schl.; ISSR; Reaction system optimization; Cluster analysis; Genetic diversity;

1. Introduction

China is the second flax producing countries in the world. However, flax production in China is fall behind than other areas. Since 1990s, the flax production is declining year by year. The flax production and flax products mostly be jeopardized by flax disease[1]. It is important to understand the epidemiology and genetic diversity of the pathogen population to control plant diseases. The pathogenic isolates of Fusarium oxysporum cause fusarium wilt and it can significantly decrease the crop production. Fusarium wilt in flax is caused by Fusarium oxysporum Schl., one of the most destructive strains of the vascular wilt fungus. The pathogens extremely variation and their pathogenic are multiple in different geographic regions[2], mainly distributed in Hebei, Shanxi, Inner Mongolia, Heilongjiang, Xinjiang, Gansu and Ningxia provinces.

ELSEVIER

1878-0296 © 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of National University of Singapore. doi:10.1016/j.proenv.2012.01.414

Assessing the genetic variability and analyzing the mechanism of pathogenic have practical significance1^. In the present study, the isolates were collected from Hebei province and the ISSR technique was used to evaluate the genetic diversity and the genetic variation in Fusarium oxysporum Schl.

2. Material and methods

2.1 Strain Collection

To analyze genetic diversity of Fusarium oxysporum Schl. populations, 30 isolates were collected from six locations in Hebei province. The isolates and their origins were listed in Table 1.

Table 1. The isolates and their origins_

No. Isolate Locality No. Isolate Locality

1 5-5 Youlougou 16 28-5 Dahonggou

2 6-2 Youlougou 17 29-8 Kangbao

3 7-4 Youlougou 18 32-10 Kangbao

4 8-7 Youlougou 19 33-3 Kangbao

5 10-2 Dongfangzi 20 35-16 Kangbao

6 12-8 Dongfangzi 21 36-3 Kangbao

7 13-2 Dongfangzi 22 37-5 Kangbao

8 14-2 Dongfangzi 23 38-10 Kangbao

9 15-3 Dongfangzi 24 42-7 Seedling Nursery

10 18-5 Dongfangzi 25 43-8 Seedling Nursery

11 20-1 Caonianpo 26 48-4 Seedling Nursery

12 21-4 Caonianpo 27 56-6 Seedling Nursery

13 22-2 Caonianpo 28 61-4 Seedling Nursery

14 24-2 Caonianpo 29 62-1 Seedling Nursery

15 26-2 Dahonggou 30 70-7 Seedling Nursery

2.2 DNA extraction

Genomic DNA was extracted using the method described by Fernando, W. G. D.[4, 5], which slightly improved. The fungal mycelium was precooled at -80 °C, then grinded in liquid nitrogen. 600 ^L of TES buffer (100 mM Tris, 10 mM EDTA, and 2% sodium dodecyl sulfate) was added. When the fungal mycelium melt, transferred to a 1.5-ml microcentrifuge tube. Then, 140 ^L of 5 M NaCl and 70 ^L of 10% cetyltrimethylammonium bromide were added to the tube and vortexed. The mixture was incubated at 65 °C for 30min. Next, 600 ^L of a mixture of chloroform and isoamyl alcohol (vol/vol, 24:1) was added and then centrifuged at 10,000 rpm for 15 min. The supernatant was transferred into a new tube. The latter step was repeated. Then, 80 ^L of 5 M NaCl and 1,000 ^L of 100% ethanol were added to precipitate the DNA, and the solution was centrifuged at 13,000 for 5 min. The DNA pellet was washed using 200 ml of cold 80% ethanol. After drying, the pellet was suspended in 100 ^L of warm sd water (37°C). The DNA was quantified using 1% agarose gel and diluted to 20 ng/^L and stored at -20 °C.

2.3 Program of PCR amplifications [6]

The program with an initial denaturing temperature of 94°C for 5 min, 35 cycles of denaturation (30 s at 94°C), annealing (30 s at the proper temperature), extension (2 min at 72°C) and the final extension step

(10 min at 72°C). Amplification products were resolved electrophoretically on 1% agarose gels in TAE 1 ^buffer.

2.4 PCR reaction system optimizing

Sixteen experiments were carried out using an orthogonal design for the study of the five independent variables, Taq DNA polymerase, Mg2+, dNTPs, DNA template and primer, each volume at four levels (Table

2, Table 3). PCR amplifications were performed in a total volume of 25 ^L containing 2.5 ^L 10 X PCR buffer, other elements were conducted as the table 3. Primer UBC811 and the seventh isolate were used to do PCR reaction system optimizing. Orthogonal experiments were replicated thrice for each reaction.

3. Results and discussion

3.1 Collection and purification of isolates

30 isolates were collected and the isolates have varying appearances. The aerial mycelium first appears white, and then may change to a variety of colors - ranging from violet to dark purple - according to the strain in solid media culture, such as potato dextrose agar (PDA). Under the microscope, we saw the macroconidia are three to five celled, gradually pointed and curved toward the ends.

Table 2. The elements and levels of ISSR reaction system

Element Final Concentration

1 2 3 4

Taq polymerase (U) 0.5 1.0 1.5 2.0

Mg2+ (mmol/L) 2.5 3.0 3.5 4.0

dNTPs (mmol/L) 0.10 0.15 0.20 0.25

Primer (^mol/L) 0.20 0.40 0.60 0.80

DNA template (ng/25 ^L) 20 40 60 80

Table 3. Lj6i (45) orthogonal experimental design for ISSR-PCR reaction system

No. Taq polymerase Mg2+ dNTPs Primer DNA template

(U) (mmol/L) (mmol/L) (l^mol/L) (ng/25 |^L)

1 0.5 2.5 0.10 0.20 20

2 0.5 3.0 0.15 0.40 40

3 0.5 3.5 0.20 0.60 60

4 0.5 4.0 0.25 0.80 80

5 1.0 2.5 0.15 0.60 80

6 1.0 3.0 0.10 0.80 60

7 1.0 3.5 0.25 0.20 40

8 1.0 4.0 0.20 0.40 20

9 1.5 2.5 0.20 0.80 40

10 1.5 3.0 0.25 0.60 20

11 1.5 3.5 0.10 0.40 80

12 1.5 4.0 0.15 0.20 60

13 2.0 2.5 0.25 0.40 60

14 2.0 3.0 0.20 0.20 80

15 2.0 3.5 0.15 0.80 20

16 2.0 4.0 0.10 0.60 40

3.2 ISSR-PCR reaction system optimizing

Table 3 contains the levels of independent variables form according to the experimental design and each row represents one experiment. The sixteen results were showed in figure 1, respectively. The direct analysis to the amplified bands were according He Zhengwen[7]. As it is shown in figure 1, the bands were obviously distinct with each other in case of the combination of different elements. The first, fourth, seventh,

thirteenth and fourteenth combinations only got weak bands, even got nothing; the second, third, fifth, ninth, fifteenth and sixteenth combinations got integrated bands, but they had a weak signal with the primary bands; the tenth, eleventh and twelfth combinations, their background were diffusion because of the high Mg2+ concentration. The sixth and eighth combinations were up to the standard. Both of two combinations had clearly separated bands, stable amplification and rich polymorphism, especially the eighth. We can draw conclusion that the optimized reaction system were performed in a total volume of 20 ng template DNA, 1 U Taq DNA polymerase, 0.4 ¡imol/L primer. 0.2 mmol/L dNTPs. 4.0 mmol/L Mg2+ and 2.5^L 10* buffer.

1 2 g? 4 5 6 7

9 10 11 12 13 14 IE 16

1-16: No.1-16 PCR products; M: DNA marker (250bp ladder Marker) Figure 1. Orthogonal experimental results for ISSR-PCR reaction system

3.3 Selection of Primers

According to the 100 universal primer sequences designed by University of British Columbia, two primers with clearly separated bands, stable amplification and rich polymorphism were selected. The sequences of primers were shown as Table 4.

Table 4. Sequences, annealing temperatures(°C) of primers employed in ISSR analysis

No. Primer sequence Annealing temperature( °C )

UBC855 ACA CAC ACA CAC ACA CYT 52 °C

UBC856 ACA CAC ACA CAC ACA CYA 52 °C

Note: Y=Pyrimidine; R=Purme; B=C,G,orT; H=A,C,ORT

Ml ; 3 4 S 6 7 S 9 10 11 12 13 14;, IS 16 17

M: 250bp DNA Ladder Marker; 1: UBC807; 2: UBC810; 3: UBC811; 4: UBC825; 5: UBC864; 6: UBC809; 7: UBC812; 8: UBC834; 9: UBC855; 10: UBC856; 11: UBC858; 12: UBC885; 13: UBC841; 14: UBC813; 15: UBC846; 16: UBC853;

17: UBC835

Figure 2. The products using different ISSR primers

3.4 ISSR fingerprints analysis

The optimized reaction system and two selected primers were used to analyze total 30 isolates (Figure 3). 30 isolates were clustered using the ISSR data by UPGMA dendrograms(Figure 4).

Ml 2 3 4 5 6 7 8 9 10 11 12 13 14 15

M: 250bp DNA Ladder Marker; 1: 5-5; 2: 6-2; 3: 7-4; 4: 8-7; 5: 10: 18-5; 11: 20-1; 12: 21-4; 13: 22-2;

Figure 3. ISSR fingerprints of partial isolates

10-2; 6: 12-8; 7: 13-2; 14: 24-2; 15: 26-2

8: 14-2; 9: 15-3;

Figure 4. Dendrogram of UPGMA cluster analysis constructed by using Dice similarity coefficients calculated for total isolates of Fusarium oxysporum Schl. based on ISSR fragments

With few exceptions in distribution of isolates within groups, the dendrogram of ISSR datasets was observed to get a similar distribution among Fusarium oxysporum Schl. isolates with about 0.830 to 0.931. Group 1 formed the large group with 26 isolates (5-5, 6-2, 7-4, 8-7, 10-2, 14-2, 15-3, 18-5, 20-1, 22-2, 24-2, 26-2, 28-5, 29-8, 32-10, 33-3, 35-16, 36-3, 37-5, 38-10, 42-7, 43-8, 56-6, 61-4, 62-1, 70-7) at an arbitrary level of 0.860 similarity. Group 2 included 2 isolates (12-8, 13-2) collected from the Dongfangzi. Group 3 only included 48-4 from Seedling Nursery and group 4 only included 21-4 from Caonianpo. It showed that the genetic variation within populations of Fusarium oxysporum Schl. were significant, although the value of genetic identity was high. ISSR markers are much more effective because the amplification of ISSRs is based on SSRs, which are extremely variable in eukaryotes.

4. Conclusion

In our study, we observed that there was no clearly relationship between the genetic distance of populations and the geographic distance. The isolates from different locations clustered together and the isolates from same location clustered separated from each other. One example is 7-4 from Youlougou and 26-2 from Dahonggou clustered together, although they were collected from different

locations. However, the two isolates had a high genetic identity value. It can be concluded that the relationship between the genetic distance of populations and the geographic distance is not obvious among total isolates. It may because their locations are close with each other and they have similar ecotype. As a result, a close similarity exists between the total isolates. The similar measures are essential for flax breeding programme against the Fusarium wilt.

5. Acknowledgements

This research was supported by the talent training fund (J0730648) from National Natural Science Foundation of China.

References

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[2] Wang Haiping, Li Xinwen, Li Jingxin, et al. "Studies on Ecotype of Flax Wilt-inducing Pathogenic Organisium (Fusarium oxysporum f. sp. lini)", Acta Agriculture Boreali-sinica, vol 19, no 2, pp. 115-118, 2004.

[3] Li Dan. "Taxonomic Study of Fusarium Species in Middle and Western Regions of Gansu Province", Gansu Agricultural University, 2008.

[4] Fernando, Zhang. J. X. "Genetic diversity of Gibberella zeae isolates from Manitoba", Plant Disease, vol 90, pp. 1337-1342, 2006.

[5] X. W. Guo, W. G. D. Fernando, and H. Y. Seow-Brock. "Population structure, chemotype diversity, and potential chemotype shifting of Fusarium graminearum in wheat fields of Manitoba", Plant Disease, vol 92, no 5, pp. 756-762, 2008.

[6] H.Bayraktar, F.S.Dolar, S.Maden. "Use of RAPD and ISSR Markers in Detection of Genetic Variation and Population Structure among Fusarium oxysporum f.sp.ciceris Isolates on Chickpea in Turkey", Phytopathology, vol 13, pp. 146-154, 2008.

[7] He Zhengwen, Liu Yunsheng, Chen Lihua, et al. "Orthogonal design-direct analysis for PCR optimization", Bulletin of Hunan Medical University, vol 23, no 4, pp. 403-404, 1998.