1. Development of Microsatellite Markers for Population Structure Analysis of Phytophthora nicotianae
Auliana Afandi1, Supriyono Loekito2, Haruhisa Suga3, and Koji Kageyama4
1The United Graduate School of Agricultural Sciences, Gifu University; 2Great Giant Pineapple Company, Indonesia; 3Life Science Research Center, Gifu University; 4River Basin Research Center, Gifu University
Conclusion
Results and Discussion
On this study 12 primer sets were generated from genome sequence data to specifically amplify 12 microsatellite loci of P. nicotianae. Nine of 12 selected primer sets are suitable for population structure analysis since they were amplified in all isolates and highly polymorphic.
Locus
Motif
Repeated number of microsatellites
Total alleles
GUGC 5631
GUGC 5632
GUGC 5633 A1
AGT 4 1 A2
CAG 14 12 2 A3
GAA
16, 15
14, 16
10, 11, 12 6 A4
GTA
10, 7
16, 15, 25, 13, 26
10, 7, 26 7 A5
TAA A6
(TCC)n(TCT)n 18 17
17, 14 3 A7
TGCTGT 5 A8
GGT A9
GCT
5, 6
A10
AAC 9
9, 8, 7
A11
GTC
A12
TTA
4, 5
Total number of alleles 32
Introduction
Microsatellite or often also referred as simple sequence repeat (SSR) is tandemly repeating units of DNA consist of 1 or 2-6 bp in length that are widely distributed in the nuclear genome of eukaryotes (Kelkar et al., 2010). Since microsatellites are highly polymorphic, it can serve as a genetic marker for DNA fingerprinting, parentage analysis, genetic mapping, conservation, and population genetic (Wang et al., 2013; Widmark et al., 2011). Considering these reason, microsatellites marker can be a very useful tool for population characterization of plants pathogen.
Phytophthora nicotianae is an important soil-borne pathogen in tropical, subtropical and temperate regions. This species was first isolated by De Haan in 1896 in Indonesia from tobacco. Up until now, the host range are more than 255 species in all over the globe and the pathogen still causes yield losses of approximately 6~12% and economic losses of at least 37 million USD/year (Drenth and Guest 2004). The diversity of its hosts and ecological niches makes P. nicotianae population interesting to be observed. The objection of this study is to develop microsatellite markers reliable for P. nicotianae population genetic analysis.
The aim of this study was to develop reliable microsatellites primer sets to study the population structure of P. nicotianae. Previous study conducted by Schena et al. (2008) offered several primer sets to study P. nicotianae and P. cinnamomi but the developed primer was failed to show any amplification while applied at the isolates of P. nicotianae from Japan in preliminary experiment. This study use three different isolates originated from Chiba prefecture which isolated from different hosts. Isolate GUGC 5631, 5632 and 5633 were originated from Alstoroemeria sp., Ornithogalum sp. and Strelitzia sp. The alleles variability observed on this study show that host play important role in P. nicotianae diversity.
On this study 12 primer sets were generated from genome sequence data to specifically amplify 12 microsatellite loci of P. nicotianae. The availability of genome sequence data plays important role in developing microsatellite primers. If the genome sequence were unavailable, suppression and tail PCR are required which is very coasty and time consuming (Yin-Ling et al, 2009)
Nine of 12 selected primer sets were suitable for population structure analysis since they were amplified in all isolates and highly polymorphic. According to Bruberg et al. (2011) seven loci were already enough to reveals genetic variability within species of Phytophthora spp. In 2010, Montarry et al. use 25 alleles to study P. infestans population, meanwhile the number of observed alleles in this study was 32. Hence, this primer sets will be enough for further population analysis of P. nicotianae.
This study observed that three loci (A3, A4 and A10) were have more than two alleles in one isolates. The presence of more than two alleles on a diploid P. nicotianae will be possible due to heterokaryon that often happened on oomycetes (Fry and Grunwald, 2010).
In conclusion, the primer sets generated in this study were enough for P. nicotianae population analysis because they are highly polymorphic and amplified in all representatives isolates.
Materials and Methods
Generate Primer sets using Primer Blast
Find the microsatellite region using Tandem Repeat Finder
Download complete genome sequence
1. Design the primers in silico
2. DNA Amplification and Sequencing
2 µl of 1 ng/ µl DNA, 2.5 µl of 10 x PCR Buffer with Mg (Takara), 2.5 µl of 4 mg/ ml BSA, 2.5 µl of 10 mM Primer forward and reverse, 2 µl of 2.5 mM dNTP mix (Takara), 0.1 µl rTaq polymerase (Takara), and 10.9 µl ddH2O
1 cycle of 94OC for 5 min, followed by 35 cycles of 94OC for 30 s, 58OC for 30s, and 72OC for 30s and final extension 72OC for 7 min
Amplified fragment then validated to check the microsatellite motif by sequencing.
References
Bruber, M. B., Elameen, A., Le, V. H., Nearstad, R., Hermansen, A., Lehitnen, A., Hannukkala, A., Neilsen, B., Hansen, J., Andersson, B., and Yuen, J. (2011) Genetic analysis of Phytophthora infestans populations in the Nordic European countries reveals high genetic variability. Fungal Biology. 115 (4-5):
Drenth, A. and Guest, I Diversity and Management of Phytophthora in South East Asia. ACIAR Monograph no Pp
Fry, W. E. and Grunwald, N. J. (2010). Introduction to Oomycetes. The Plant Health Instructor. DOI: /PHI
Kelkar, Y. D., Strubczewski, N., Hile, S. E., Chiaromonte, F., Eckert, K. A., and Makova, K. D. (2010) What is a microsatellite: a computational and experimental definition based upon repeat mutational behavior at A/T and GT/AC repeats. Genome Biology and Evolution. 2:
Schena, L., Cardle, El Cooke, D. (2008) Use of genoe sequence data in the design and testing of SSR markers for phytophthora species. BMC Genomics 9:
Wang, Z., Liao, L., Yuan, X., Guo, H., Guo, A., and Liu, J. (2013) Genetic diversity analysis of Cynodon dactylon (bermudagrass) accessions and cultivars from different countries based on ISSR and SSR markers. Biochemical systematics and Ecology 46:
Widmark, A. K., Anderson, B., Sandstrom, M. and Yuen, J. E., (2011) Tracking Phytophthora infestans with SSR markers within and between seasons –a field study in Sweden. Plant Pathology 60:
Yin-Ling, Zhou, W., Motohashi, K., Suga, H., Fukui, H., and Kageyama, K. (2009) Development of microsatellite markers for Pythium helicoides. FEMS Microbiol Letter 293: 85- 9
Cloning and Sequencing
Linear vector of E. coli +
PCR Product as DNA Inserts
Sequencing
Colony of recombined E. coli for colony PCR
Assembled DNA
Table 1.
No.
Isolate
Host
Origin 1.
GUGC 5631
Alstoroemeria sp.
Chiba 2.
GUGC 5632
Ornithogalum sp. 3.
GUGC 5633
Strelitzia sp.