全 文 :植物病理学报
ACTA PHYTOPATHOLOGICA SINICA 44(5): 536 ̄541(2014)
Received date: 2013 ̄09 ̄08ꎻ Revised date: 2014 ̄06 ̄07
Foundation item: Financial support by the Ministry of Science and Technology (2011CB100403)ꎬ the Ministry of Agriculture
(200903035)ꎬ National Natural Science Foundation (31260417) and Yunnan Academy of Agricultural Sciences (2007yaas07) .
Corresponding author: CHEN Wan ̄quanꎬ Professorꎬ major in wheat disease researchꎻ Tel:+86-10-62815903ꎬ
E ̄mail: wqchen112@ippcaas.cnꎻ
DUAN Xia ̄yuꎬ Professorꎬ major in wheat disease researchꎻ Tel: +86-10-62815946ꎬ E ̄mail: xyduan@ippcaas.cn.
doi:10.13926 / j.cnki.apps.2014.05.012
研究简报
First Report of SNP Primers of Three House ̄keeping
Genes of Puccinia striiformis f. sp. tritici
LI Ming ̄ju 1ꎬ2ꎬ CHEN Wan ̄quan2ꎬ DUAN Xia ̄yu2ꎬ
LIU Tai ̄guo2ꎬ GAO Li2ꎬ LIU Bo2
( 1 Institute of Agricultural Environment and Resourcesꎬ Yunnan Academy of Agricultural Sciencesꎬ Kunming 650205ꎬ Chinaꎻ
2State Key Laboratory for Biology of Plant Diseases and Insect Pestsꎬ Institute of Plant Protectionꎬ
Chinese Academy of Agricultural Sciencesꎬ Beijing 100193ꎬ China)
小麦条锈菌 3 个看家基因 SNP 引物的首次报道 李明菊1ꎬ 2ꎬ 陈万权2∗ꎬ 段霞瑜2∗ꎬ 刘太国2ꎬ
高 利2ꎬ 刘 博2 ( 1 云南省农业科学院农业环境资源研究所ꎬ昆明 650205ꎻ 2中国农业科学院植物保护研究所ꎬ植物病虫害
生物学国家重点实验室ꎬ北京 100193)
Abstract: In order to analyze the population structure of Puccinia striiformis f. sp. tritici (Pst)ꎬ SNPs primers
of Pst were developed from DNA sequences of nine house ̄keeping genes Chsꎬ Actꎬ Mapk1ꎬ Tubꎬ Cdc2ꎬ Rdꎬ
Smꎬ Ls and Ef ̄1α obtained from GenBank. Eight of which were from Pst and one from P. graminis f. sp. tritici
(Pgt) . Thirteen pairs of primers were designed and screened based on at least 30 isolates of Pst obtained from di ̄
verse locations. Three of them were polymorphic namely Map1351S / Map1683Aꎬ Cd28S / Cd352A and Ef137S /
Ef531A. Polymorphic loci analysis based on 149 Pst isolates from 5 provinces indicated that the three primers had
good polymorphism. Cd28S / Cd352A had 8 polymorphic lociꎬ three of them were phylogenetically informative.
Ef137S / Ef531A had 6 polymorphic loci and 4 of them were informative. Map1351S / Map1683A had 8 polymor ̄
phic loci and 4 were informative. The 3 primers were used for analyzing Pst population and revealed the ancestral
originꎬ phylogeny relation of haplotypesꎬ genetic differentiation of the populationꎬ gene flowꎬ and the evolution
and migration relation between Pst populations thereby. The findings indicate that the 3 genes SNP primers can
be used for Pst population genetic structure analysis.
Key words: house ̄keeping geneꎻ single nucleotide polymorphisms ( SNPs)ꎻ primerꎻ puccinia striiformis
f. sp. triticiꎻ population genetic structure
文章编号: 0412 ̄0914(2014)05 ̄0536 ̄06
Wheat stripe rustꎬ caused by Puccinia striifor ̄
mis f. sp. tritici (Pst)ꎬ is one of the most destruc ̄
tive wheat diseases in China as well as all over the
world[1ꎬ2] . Knowledge of population genetic struc ̄
ture is helpful in revealing the originꎬ evolution
and migration of the pathogen and can provide
5期 LI Ming ̄ju ꎬet al:First Report of SNP Primers of Three House ̄keeping Genes of Puccinia striiformis f. sp. tritici
information for the strategy of the disease manage ̄
ment[3] . Various molecular markers based on DNA
fingerprinting technology have been used to study
Pst population structure[4-7] and promoted the study
on molecular population genetics of Pst. With the
development of sequencing technology and tools of
Bioinformaticsꎬ it brings a revolution in molecular
markers. SSR and AFLP have been gradually
replaced by SNPs. This shift has been evolved from
the initial application in human and other commercial
important species to a wide range of non ̄model
species [8] . SNP is a sort of sequence polymorphism
based on single nucleotide’ s change. It exists in
higher frequency in organism genomesꎬ can provide
much information for population structure and be
detected easily. Sequence results are stableꎬ reliable
and have good repeatability and can be used for
complex population models than DNA fingerprint.
The statistical conclusions obtained are more accurate
[9] . It has been applied to study originꎬ evolutionꎬ
migrationꎬ identification and pathogenic mechanism
of plant pathogen in recent years [10-12] . Parks et
al.[13] analyzed wheat powdery mildew populations
using SNPs and found 25 haplotypes from the four
concatenated genes of the U. S. population. The
pathogen isolates were subdivided into four groups
corresponding to distinct regions according to
Hudson’s tests and analysis of molecular variance.
Genotypic diversity analysis indicated that cooler
locations with greater conduciveness to regular
powdery mildew epidemics had the greatest
haplotype richness. The results suggested that
Blumeria graminis f. sp. tritici population was young
in eastern U. S.ꎬ might come from old world
populations with isolation and genetic drift and was
subdivided into northern and southern subpopula ̄
tions. No report was publishedꎬ to our knowledgeꎬ
for Pst population. To fulfill the population analysis
on its originꎬ evolution and migrationꎬ the important
features for the study of the dynamics of the
pathogen populationꎬ the development of SNPs
primer is the prerequisite. We report here the result
of the development of SNPs primers for Pst to enable
the use of SNPs in the researches in above mentioned
areas.
1 MATERIALS AND METHODS
1.1 Primer design and synthesis
The sequences of Pst and Puccinia graminis f.
sp. tritici (Pgt) house ̄keeping genes were searched
in GenBank. Nine protein ̄coding genes as candidate
genes for developing SNP primer of Pst were
identified namelyꎬ Class II chitin synthase (Chs)ꎬ
Actin (Act)ꎬ Map kinase 1 (Mapk1)ꎬ Beta ̄tubu ̄
lin1 ( Tub)ꎬ Cyclin ̄dependent kinase 2 (Cdc2)ꎬ
RNase III dicer ̄like protein (Rd)ꎬ Sterol 4 ̄C ̄me ̄
thyltransferase (Sm)ꎬ Lanosterol synthase (Ls) and
Elongation factor 1α (Ef ̄1α) . All of the sequences
were from Pst except Ef-1α. The sequence of Ef ̄1α
from Pgtꎬ the closest relative species of Pst are used
to design the primer because the Ef ̄1α gene
sequence of Pst has not been submitted to Genbank
by now. Selected regions contained one or more
introns of the nine gene sequences were used for
primer design and 13 primer pairs obtained by using
Primer Premier 5.0 software. Designed primer pairs
were synthesized by Shanghai SANGON Biological
Engineering Company Limited. The primers informa ̄
tion was listed in Table 1.
1.2 PCR reaction and sequencing
Trials were conducted for the amplification and
the optimal reaction conditions were as follows PCR
was carried out in 20 μL volume. The amplification
conditions and the reagent concentrations were the
same except for annealing temperature for the 3
primers (Table 2) . Each reaction contained 10 μL
of TIANGEN 2×Taq PCR MasterMix [0. 1 U Taq
Polymerase μL-1ꎬ 500 μM dNTPs eachꎬ 20 mM
Tris ̄HCl ( pH8. 3)ꎬ 100 mM KClꎬ 3 mM MgCl2ꎬ
and other stablizing and reinforcing agent]ꎬ 10 μM
each primer 1 μLꎬ 20 ng of genomic DNA 1 μLꎬ
and ddH2O 7 μL. The amplification cycling condi ̄
tions followed the method reported by Parks
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植物病理学报 44卷
et al.[13]ꎬ which included one cycle of 96° C for 5
minꎬ 34 cycles of 96°C for 25 sꎬ (annealing tempera ̄
ture)
for 25 sꎬ and 72°C for 45 s and followed by a final
extension step of 72°C for 5 min.
Table 1 Sequenceꎬ product length and polymorphism of 13 primers of 9 genes
Gene Gene product
Organ ̄
ism
GenBank
accession no.
Primer sequence / 5 ̄ 3
Product
length
/ bp
Polymorphism
Mapk1 Map kinase 1 Pst HM535614.1 Map1351S:GTCGGTCGGGTGTATCCT
Map1683A:GGTTCATCTTCGGGGTCA
332 Polymorphic
Map22S:GCTTCGTTGAATGTAATG
Map572A:GGTAAGAGGGGTAGAAAT
551 Monomorphic
Cdc2 Cyclin ̄
dependent
kinase 2
Pst GQ911579.1
Cd28S:AAATCATCCACATCTGCTCCAC
Cd352A:AAATCATCCACATCTGCTCCAC
325 Polymorphic
Cd28S:AAATCATCCACATCTGCTCCAC
Cd970A:TCCCAAATGCTCTTGCTAATCC
943 Polymorphic
Ef ̄1α Elongation
factor
Pgt X73529.1 Ef137S:AAGCCGCATCCTTCGTTG
Ef531A:TTGCCATCCGTCTTCTCG
395 Polymorphic
Chs Class II chitin
synthase
Pst GQ329851 Chs1271S:AGACGGCTGGAAGAAAGT
Chs1658A:GACCAAACGCATTGAAAA
388 Monomorphic
Chs137S: CTTTTGGCTTATGGTTGG
Chs613A:TCTGTCATCTTGGTTTCG
477 Monomorphic
Act Actin Pst JF313350.1 Act127S:TATCATCATCCAACCCAA
Act331A:CGTCACCCACATAAGAAT
205 Monomorphic
Act262S:ATCTGTGGTCGTCCTCGTC
Act756A:GTAACCCCGCTCCATCAA
495 Monomorphic
Tub Beta ̄tubulin 1 Pst FJ612006.1
Tub87S:TCGGTGGTGGAACTGGTG
Tub461A:GCAATCGAGGGAAAGGAA
375 Monomorphic
Tub226S:CACCTTGTCGGTTCATCA
Tub688A:GTTGGAGTTCTTGCTTTG
463 Monomorphic
Rd RNase III
dicer ̄like protein
Pst JN033211.1 Rd85S:GCTTCTACTTCTCCCACC
Rd559A:GCTATTTACTCTTCCCCAC
475 Monomorphic
Sm Sterol 4 ̄C ̄
methyltran ̄
sferase
Pst JN033205.1 Sm27S:AAGAACTCCAGCCCATCC
Sm608A:GTCGCCTTTGACTACCTC 582 Monomorphic
Ls Lanosterol
synthase
Pst JN033206.1 Ls259S:GAACTCGTTTCCCTCTTG
Ls906A:ATTGTTTGTGGGCTTTGC
648 Monomorphic
835
5期 LI Ming ̄ju ꎬet al:First Report of SNP Primers of Three House ̄keeping Genes of Puccinia striiformis f. sp. tritici
Table 2 Annealing temperatureꎬ polymorphic lociꎬ and
max identity with template of the 3 primers
Gene Primer
Annealing
temperature
/ ℃
Product position at template
gene from 5 beginning
Polymorphic
loci
Informative
loci
Maximal identity
with the published
/ %
Cdc2 Cd28S / 352A 62 28 ̄352 8 3 99
Ef ̄1α Ef137S / 531A 51 137 ̄531 6 4 83
Mapk1 Map1351S / 1683A 55 1351 ̄1683 8 4 99
Table 3 Variable locus and positionꎬ informative locus
Primer
Locusꎬ character type
Cd28S / 352A Ef137S / 531A Map1351S / 1683A
Standard locus A A G C A G A G G C T C T C C C C G G C G G
Variable locus position
from 5 beginning
55 65 99 108 174 247 303 316 37 39 62 154 331 343 61 100 102 103 109 202 214 323
Variable locus C C T T T T G T A A C T C T T A A C A T T A
Phylogenetically
informative locus
n n n n i n i i n n i i i i i n n n n I i I
Note: Each variable position is phylogenetically informative ( i) or noninformative (n) . To be informativeꎬ a position must have
mutations that appear in at least two haplotypes. Primer Ef137S / Ef531A is designed by using Pgt gene. Soꎬ polymorphic loci
position used the first sample sequence as template.
The PCR products were resolved by 0.8% aga ̄
rose electrophoresis to see if the band was unique
and bright. Finallyꎬ good PCR productsꎬ from at
least 30 Pst isolates collected in diverse locationsꎬ
were sequenced to detect their polymorphism. Mono ̄
morphic primers were discarded and the polymorphic
primers based on 30 Pst isolates were further detec ̄
ted by more than 100 Pst isolates to confirm the pol ̄
ymorphism. Sequencing was carried out by Shanghai
SANGON Biological Engineering Company Limited.
1.3 Analysis of primer polymorphism and
population structure of Pst
SNP analysis was carried out by multiple evolu ̄
tionary analysis software. Sequence alignmentꎬ
shearꎬ connectingꎬ population mutation rate and
haplotype phylogeny tree were analyzed by MEGA
4. 0. Haplotype analysis was conducted by SNAP
Workbench 2.0 and multiple sequences alignment by
BioEdit. AMOVA (Analysis of molecular variance)
was done by Arlequin 3.1. Haplotype diversityꎬ nu ̄
cleotide diversityꎬ Tajima’s D valueꎬ recombination
eventꎬ Coefficient of genetic differentiation and gene
flow intensity analysis were calculated by DnaSP
v.5.10.
2 RESULTS
2.1 Polymorphism of the SNP primers
Sequences of the 30 isolates and the standard
sequence were aligned for each primer. The standard
sequences for Cdc2 and Mapk1 genes followed Gen ̄
Bank. The Ef ̄1α sequence of the first Pst sample
from Yunnan Province in 2008 (No. B1) was de ̄
fined as the standard sequence for Ef ̄1α because the
primer was designed from Pgt. If the 30 sequences
were the same as the standardꎬ or there were fewer
than two phylogenetically informative lociꎬ we took
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植物病理学报 44卷
the corresponding primer as monomorphic. Other ̄
wiseꎬ the primer was polymorphic. Thus three poly ̄
morphic primers were obtained from the 13 primers
of 9 housekeeping genes of Pst or Pgt. Sequence a ̄
lignment results indicated that the 3 primers showed
good polymorphism based on 149 Pst isolates col ̄
lected from 5 provinces in Chinaꎬ namely Yunnanꎬ
Guizhouꎬ Sichuanꎬ Shaanxi and Gansu in 2008.
The 3 primers annealing temperatureꎬ product
position at the standard sequenceꎬ polymorphic loci
and maximal identity with the standard were listed in
Table 2. Variable locus information and position
were listed in Table 3. Primer Cd28S / Cd352A had 8
polymorphic loci and 3 of them were phylogenetical ̄
ly informative loci. Ef137S / Ef531A had 6 polymor ̄
phic loci and 4 were informative. Map1351S /
Map1683A had 8 polymorphic loci and 4 were in ̄
formative.
Maximal identity were 99% between the sam ̄
ples colleted and the published for Cdc2 and Mapk1
while it was 83% for Ef ̄1α. This indicated the maxi ̄
mal identity between Pst and Pgt for Ef ̄1α as the
primer was developed from Pgt.
2.2 Population genetic structure of Pst re ̄
vealed by SNP
The sequences of 149 isolates were aligned for
the 3 genesꎬ respectively. After cutting off the unre ̄
liable parts from the both ends of the sequences and
making sure all sequences of the 149 isolates were
the same length for analysis convenienceꎬ Cdc2 was
sheared to 265 bp longꎬ Ef ̄1α to 331 bpꎬ and
Mapk1 to 259 bp. Then the 3 gene fragments were
connected to a concatenated sequence of 855 bp long
in the order of Cdc2ꎬ Ef ̄1α and Mapk1ꎬ and was
used to analyses the population genetic of 149 iso ̄
lates of 2008. The results indicated that there were
26 haplotypes and 18 polymorphic sites after remo ̄
ving 5 indels and 7 of which were phylogenetically
informative. The ancestral haplotype originated from
Yunnan. Three haplotypes shared between Yunnan
and other provinces had a higher frequency than all
the other haplotypes and added up to 75% of all the
isolates sampled. Yunnan and Gansu populations had
its privacy haplotypesꎬ respectively. The genetic di ̄
versity and recombination events of Yunnan popula ̄
tion were lower than Gansu populationꎬ but its popu ̄
lation mutation rate was higher than Gansu. The mu ̄
tation originated from inner population and accoun ̄
ted for 79% of total variance.
Coefficient of genetic differentiation Gst was
0.127 40 among sub ̄populations in Yunnan
Provinceꎬ and gene flow intensity Nm was 1.71. It
indicated that the Yunnan population had high
heterogenecity and the pathogen migration was not
frequent within Yunnan Province.
Yunnan population had older and younger hap ̄
lotypesꎬ longer evolutionary historyꎬ higher hetero ̄
genecity and evolutionary efficiency than Gansu
population. Coefficient of genetic differentiation Gst
was 0.047 39 between Yunnan and Gansuꎬgene flow
intensity Nm was 5.03. Low Gstꎬ high Nm and high
frequency of shared haplotypesꎬ gave evidence that a
large scale of pathogen migration exists between the
two regions.
The above results indicate that the 3 SNP
primers of Pst can be used for Pst population genetic
structure analysisꎬ and can reveal evolution and
migration among populations of Pst. The detailed
results will be reported in a separate article.
3 DISCUSSION AND CONCLUSION
SNP primers of the three house ̄keeping genes
Cdc2ꎬ Ef ̄1α and Mapk1 of Pst were developed for
the first time in the world. The primer pairs Cd28S /
Cd352Aꎬ Ef137S / Ef531A and Map1351S /
Map1683A showed good polymorphism based on the
analysis over a population of 149 Pst isolates from
Yunnanꎬ Guizhouꎬ Sichuanꎬ Shaanxi and Gansu.
Each gene has 6 or 8 polymorphic lociꎬ including 3
or 4 phylogenetically informative loci. The concate ̄
nated 3 genes can reveal the evolution and migration
among Pst populationsꎬ and can be used for Pst
population genetic structure analysis. SNPs are
045
5期 LI Ming ̄ju ꎬet al:First Report of SNP Primers of Three House ̄keeping Genes of Puccinia striiformis f. sp. tritici
proved suitable for the analyses of population.
Howeverꎬ the number of genes and polymorphic loci
are not sufficient for more detailed studies. More
gene primers need to be developed further. Another
primer pairꎬ Cd28S / Cd970Aꎬ of Cdc2 gene was
also developed in the lab. The product length is 943
bp. Howeverꎬ the two ̄way sequencing and spliting
joint is needed and its polymorphism to be confirmed
in bigger populations.
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