Investigating the population structure of
Xanthomonas
citri
pv.
citri
in Vietnam. Which molecular markers to
discrimi-nate among low polymorphic bacterial populations ?
Xanthomonas citri
• pv. citri (Xcc)
causes Asiatic citrus canker, a
ma
-jor threat of citrus worldwide
two main pathotypes with a broad
-(Xcc-A) and a restricted (Xcc-A*) host ranges
a quarantine organism in many countries.
-low genetic diversity (rep-PCR, PFGE)
-Analyses of large collections or populations require
•
highly discriminant, typeable, reproducible and hi-gh-throughput techniques.
Investigating the population structure is valuable to understand :
•
epidemic situations at small geographical or temporal scales (identification of
-inoculum sources and pathway)
evolution of pathogenic populations and identification of evolutive factors
-Material & methods
557 strains isolated in 2006 in Vietnam, an
ende-•
mic situation
IS-LM-PCR : targets regions between insertion
se-•
quences present as several copies in the genome of Xcc-strain 306 and a Msp site
MLVA : 14 primer pairs targeting single-VNTR locus
•
designed from the strain 306 sequence and used in multiplex PCR format
Population structure
Both markers produced a
•
similar population structure and individuals assignment as given by STRUCTURE
Two “ancestral” populations
•
constituted the Vietname-se X. citri pv. citri contem-porary strains (based on Structure analyse). Circles present the average pro-portion of ancestral popu-lations by province as in-ferred by structure (I for
IS-LM-PCR and M for MLVA data)
Discriminatory power of IS-LM-PCR and MLVA
marker Simpson index number of haplotypes polymorphism IS-LM-PCR 0.959 190 54% MLVA 0.998 463 100%
Hierarchical AMOVA of
Xcc
from 9 province collections
variance of
components % total variation F statistics
marker IS-LM-PCR MLVA IS-LM-PCR MLVA IS-LM-PCR MLVA among regions (N-S) 0.012 0.40 3 7 0.028*** 0.072***
among provinces 0.050 0.63 11 12 0.108*** 0.120***
within provinces 0.415 4.38 87 81 0.133*** 0.183***
Genetic differentiation
Ha Noi Ha Tinh Hung Yen Nghe An Can Tho Dong Thap Lam Dong Long An Tien Giang
Ha Noi 0.361 0.003NS 0.099 0.331 0.453 0.356 0.274 0.296 Ha Tinh 0.176 0.419 0.472 0.529 0.626 0.567 0.441 0.521 Hung Yen 0.013NS 0.220 0.128 0.369 0.505 0.391 0.297 0.335 Nghe An 0.073 0.227 0.102 0.319 0.426 0.360 0.274 0.295 Can Tho 0.127 0.143 0.181 0.100 0.129 0.100 0.145 0.040 Dong Thap 0.213 0.229 0.256 0.172 0.093 0.317 0.324 0.215 Lam Dong 0.226 0.202 0.278 0.199 0.097 0.160 0.116 0.054 Long An 0.164 0.158 0.223 0.120 0.049 0.112 0.079 0.106 Tien Giang 0.152 0.155 0.210 0.126 0.036 0.100 0.076 0.035
FST values for pairwise comparisons of 9 province collections based on IS-LM-PCR data (above) and MLVA data (be-low). Pairwise comparisons of populations within a same region have the same color NORTH & SOUTH
Highly significant differentiation between northern and southern
pro-•
vinces
Low to moderate differentiation between southern populations
•
Variable differentiation between some northern provinces
•
Congruence (Mantel test) between:
Distance matrices from IS markers
• (P<0.001)
Distance matrices from both techniques
• (P<0.001)
Conclusions
IS-LM-PCR MLVA Discriminatory power + ++ Intra-laboratory reproducibility + ++ Labor intensive technique + -Ease of data scoring + ++ Both markers produced congruent results•
MLVA markers: great interest in molecular epidemiology •
of Xcc at small scale and population structure analysis
IS-LM-PCR: more appropriate for a global surveilance sys-•
tem of X. citri pv. citri
Lan Bui Thi Ngoc1,2, Christian Verniere2,Karine Vital2
and Olivier Pruvost2
1Southern Horticultural Research Institute (SOFRI) –
Long Dinh, Tien Giang, VietNam
2CIRAD- UMR Peuplements Végétaux et Bioagresseurs
en Milieu Tropical, Saint-Pierre, La Réunion, France
Objectives
Evaluation of two typing techniques, insertion
sequence ligation-mediated PCR (IS-LM-PCR)
and multilocus variable number tandem repeat
analysis (MLVA) for epidemiological and
popula-tion structure analysis based on a large collecpopula-tion
from nine provinces in Viet Nam
Multidimensional scaling of the distances between 554 strains from 12 provinces based on (A) IS-LM-PCR and (B) MLVA
population 2 population 1
IS-LM-PCR:
ISXac1, ISXac2, ISXac3 336 markers genome of Xcc 306 VNTR: 14 loci Funding : ➀ Ha Noi ➁ Ha Tinh ➂ Hung Yen ➃ Nghe An ➄ Phu Tho ➊ Can Tho ➋ Dong Nai ➌ Dong Thap ➍ Lam Dong ➎ Long An ➏ Tien Giang ➐ Vinh Long ➀ ➀ ➀➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➁ ➁ ➁ ➁ ➁ ➁ ➁ ➁ ➁ ➁ ➂ ➂ ➂ ➂ ➂ ➂ ➂ ➂ ➂ ➂ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➄ ➄ ➄ ➄ ➄ ➄ ➄ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊➊ ➊ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➎ ➎ ➎ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➐ ➐ ➐ ➐ ➐ ➐ −0.3−0.2 −0.2 −0.1 0.0 0.1 0.2 −0.1 0.0 0.1 0.2 axis 2 axis 1 ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➃ ➄ ➄ ➄ ➄ ➄ ➄ ➄ ➄ ➄ ➄ ➄ ➄➄ ➄ ➄ ➄ ➄ ➄ ➄➄ ➄ ➄ ➄ ➄ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➀ ➁ ➁ ➁ ➁ ➁ ➁ ➁ ➁ ➁ ➁ ➁ ➂ ➂ ➂ ➂ ➂ ➂ ➂ ➂ ➂ ➂ ➂➂➂ ➂ ➂ ➂ ➂➂ ➂ ➂ ➂ ➂ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊ ➊➊➊➊ ➊ ➊ ➊ ➊ ➊ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋ ➋➋ ➌ ➌ ➌ ➌➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➌ ➎ ➎ ➎ ➎ ➎ ➎ ➎ ➐ ➐ ➐ ➐ ➐ ➐ ➐ ➐ ➐ ➐ ➐ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍ ➍➍ ➍ ➍ ➍ ➍ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ ➏ −20 0 20 40 −20 −10 0 10 20 30 axis 1 axis 2
