Population diversity of “Candidatus Liberibacter asiaticus” in Guangdong Province based on different gene loci
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摘要:目的
探究广东省不同地区柑橘黄龙病菌“Candidatus Liberibacter asiaticus” (CLas)的种内遗传结构及遗传多样性情况；评价用多基因位点分析柑橘黄龙病菌遗传多样性的科学性。
方法基于原噬菌体区域(SC1、SC2和PJXGC)、转座子位点(CLIBASIA_05620~CLIBASIA_05625)和短串联重复序列STR1(CLIBASIA_03080)、STR12(CLIBASIA_01215) 6个基因位点的多态性，对来自广东省10个市(县)的176个黄龙病样品进行病原菌遗传多样性分析。
结果基于噬菌体类型鉴定出6组菌株，其中携带Type II类型原噬菌体的菌株为优势种群(85.23%)；基于转座子位点鉴定出4种类型菌株，含有缺失MCLas-A类型转座子的B350片段的菌株为优势种群(76.70%)；基于短串联重复序列STR1和STR12分别鉴定出9和10类菌株，且STR1位点上含有3个“CAGT”串联重复的菌株为优势种群(56.82%)；STR12位点上多态性条带分布不集中，没有优势条带类型。聚类分析结果表明湛江、茂名和深圳的种群与其他地区种群的差异较大，而清远和梅州的黄龙病菌种群关系较近。
结论基于不同位点和基于所有位点所得的聚类结果不尽相同，说明利用单个或单种基因位点对黄龙病菌进行种群遗传结构分析具有较大的局限性。
Abstract:ObjectiveTo explore the intraspecific genetic structure and genetic diversity of “Candidatus Liberibacter asiaticus” (CLas) in different regions of Guangdong Province, and evaluate the feasibility of using multiple loci in studying genetic diversity of CLas.
MethodSix polymorphic gene loci, including three phage regions (SC1, SC2 and PJXGC), transposon region (CLIBASIA_05620 − CLIBASIA_05625) and short tandem repeat (STR) genes, STR1 (CLIBASIA_03080) and STR12 (CLIBASIA_01215), were used to evaluate genetic diversity of 176 CLas samples from 10 cities in Guangdong Province.
ResultBased on the prophage types, the CLas isolates were classified into six groups, among which the strains with Type II prophage were predominate, accounting for 85.23% of the population. Four types of isolates were identified based on transposon sites, and the Clas strains containing the B350 fragment (non-MCLas-A type) were the dominant group, accounting for 76.70% of the population. Based on the two STR loci, nine and ten band types were identified respectively, and the isolates with three CAGT tandem repeats at the STR1 locus were predominate accounting for 56.82% of the population. The distribution of polymorphic bands at the STR12 locus was scattered, and no predominate band type was identified. Cluster analysis showed that CLas populations from Zhanjiang, Maoming and Shenzhen were different from populations of other regions, while the CLas populations collected from Qingyuan and Meizhou were similar.
ConclusionCluster results based on individual locus and based on all six loci are different, indicating the limitation of using single gene locus to analyze the genetic structure of CLas population.
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图 1 LapPF1-f/LapPF1-r引物对广东部分柑橘黄龙病菌株的PCR扩增条带
M：DL2000 DNA marker；CK：健康样品；1~8为不同的柑橘黄龙病菌株；719、731 bp的条带为B720；303、291、292 bp的条带为B350
Figure 1. PCR amplification bands of partial “Candidatus Liberibacter asiaticus” strains in Guangdong Province with primers of LapPF1-f/LapPF1-r
M: DL2000 DNA marker; CK: DNA from healthy citrus; 1−8 indicate different “Candidatus Liberibacter asiaticus” strains; Bands of 719 or 731 bp are defined as B720; Bands of 303, 291 or 292 bp are defined as B350
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图 2 STR1/STR12引物对广东部分柑橘黄龙病菌株的PCR扩增条带PAGE电泳结果
M: 10 bp DNA ladder marker；图a为基于短串联重复序列STR1(CLIBASIA_03080)的扩增产物，1~21分别表示不同的柑橘黄龙病菌株；图b为基于短串联重复序列STR12(CLIBASIA_01215)的扩增产物，1~25分别表示不同的柑橘黄龙病菌株
Figure 2. PAGE electrophoresis of PCR amplified bands of partial “Candidatus Liberibacter asiaticus” strains in Guangdong Province with primers of STR1/STR12
M: 10 bp DNA ladder marker; STR1 are PCR amplicons of STR1 (CLIBASIA_03080) in figure a, and numbers 1−21 indicate different “Candidatus Liberibacter asiaticus” strains; STR12 are PCR amplicons of STR12 (CLIBASIA_01215) in figure b, and numbers 1−25 indicate different “Candidatus Liberibacter asiaticus” strains
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图 3 基于不同位点的广东地区黄龙病菌种群的聚类分析
A是基于柑橘黄龙病种群的噬菌体类型(SC1、SC2和PJXGC)；B是基于转座子位点(CLIBASIA_05620~CLIBASIA_05625)；C是基于短串联重复序列STR1(CLIBASIA_03080)；D是基于短串联重复序列STR12(CLIBASIA_01215)
Figure 3. The dendrogram analysis of “Candidatus Liberibacter asiaticus” populations in Guangdong Province based on different gene loci
A is generated according to phage types (SC1, SC2 and PJXGC) in “Candidatus Liberibacter asiaticus” populations; B is generated according to transposon loci of CLIBASIA_05620-CLIBASIA_05625; C is generated according to short tandem repeat STR1 (CLIBASIA_03080); D is generated according to short tandem repeat STR12 (CLIBASIA_01215)
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图 4 基于6个位点的广东省柑橘黄龙病种群的聚类
Figure 4. The dendrogram of “Candidatus Liberibacter asiaticus” populations in Guangdong Province based on six gene loci
表 1 特异性引物信息
Table 1 Information of specific primers
基因类型1)
Gene type引物
Primer引物序列
Primer sequence产物大小/bp
Product length短串联重复序列
Short tandem repeat基因位点
Gene locus引物来源
Primer origin原噬菌体
ProphageSC1-045F CCGTTCGTCTTTTGCCCATA 87 SC1_gp045 本研究
This studySC1-045R GCATTCTTCGCATCATCGGA SC2-035F AGGTCACAAGGATTTAGCCCA 86 SC2_gp040 本研究
This studySC2-035R CTCCTAATCCCGCACCGATA PJXGC-8F CGGCGCTGAACTCTTGTATT 85 PJXGC_08 本研究
This studyPJXGC-8R AAGGGCGTTGTTCTTGTCAC MITE LapPF1-f GCCACTTTGGGGTAGCAGTA 350~
731CLIBASIA_05620~
CLIBASIA_05625文献[23]
Reference [23]LapPF1-r AAAACTTTCGTCACGGCTTT STR STR1F TCGCTTGAAAGTTATTATTGGG 181 CAGT CLIBASIA_03080 文献[22]
Reference [22]STR1R GCATAAGAATAGAAGAACCTA STR12F GTTGCTTCGTTTATCC 239 TACAGAA CLIBASIA_01215 文献[22]
Reference [22]STR12R GCATTCTGTGCCTCTT 1) MITE为微型反向重复转座元件；STR为短串联重复序列
1)MITE: Miniature inverted-repeat transposable element; STR: Short tandem repeat
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表 2 广东省柑橘黄龙病菌株携带噬菌体类型的情况
Table 2 Phage types carried by the “Candidatus Liberibacter asiaticus” strains in Guangdong Province
类型
Type来源及数目 Origin and number 清远
Qingyuan韶关
Shaoguan肇庆
Zhaoqing梅州
Meizhou惠州
Huizhou深圳
Shenzhen江门
Jiangmen茂名
Maoming湛江
Zhanjiang广州
Guangzhou总计
TotalType II 22 12 34 12 21 3 5 17 21 3 150 Type III 1 1 Type I+ Type II + Type III 1 1 2 Type I + Type III 14 1 15 Type I + Type II 1 6 7 Type II+ Type III 1 1 总计 Total 22 12 36 12 22 4 6 31 27 4 176
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表 3 基于转座子CLIBASIA_05620~CLIBASIA_05625位点对广东省柑橘黄龙病菌株的扩增条带分析
Table 3 Analysis of amplified bands of “Candidatus Liberibacter asiaticus” strains in Guangdong Province based on transposon sites in CLIBASIA_05620-CLIBASIA_05625 gene
带型
Amplicon type1)来源与数目 Origin and number 清远
Qingyuan韶关
Shaoguan肇庆
Zhaoqing梅州
Meizhou惠州
Huizhou深圳
Shenzhen江门
Jiangmen茂名
Maoming湛江
Zhanjiang广州
Guangzhou总计
TotalB350 22 12 34 11 21 2 5 14 11 3 135 B720 1 11 12 B720+B350 1 1 2 1 17 1 23 无条带 No amplicon 1 5 6 总计 Total 22 12 36 12 22 4 6 31 27 4 176 1)B720是指测序结果为719 或731 bp的条带；B350是指测序结果为303、291或292 bp的其中一种条带
1)B720 are sequenced bands of 719 or 731 bp；B350 are sequenced bands of 303, 291 or 731 bp
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表 4 基于短串联重复序列CLIBASIA_03080位点对广东省柑橘黄龙病菌株的扩增条带分析
Table 4 Analysis of amplified bands of “Candidatus Liberibacter asiaticus” strains in Guangdong Province based on short tandem repeats in CLIBASIA_03080 gene
条带大小/bp
Sequence length清远
Qingyuan韶关
Shaoguan肇庆
Zhaoqing梅州
Meizhou惠州
Huizhou深圳
Shenzhen江门
Jiangmen茂名
Maoming湛江
Zhanjiang广州
Guangzhou总计
Total140~150 3 3 150~160 17 12 22 10 21 1 3 6 6 2 100 160~170 5 1 1 1 1 10 19 170~180 10 1 11 150~160、200~220 2 2 140~150、150~160 1 2 2 2 1 18 1 1 28 150~160、160~170 1 1 150~160、170~180 2 2 1 3 8 无条带 No amplicon 2 2 4 总计 Total 22 12 36 12 22 4 6 31 27 4 176
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表 5 基于短串联重复序列CLIBASIA_01215位点对广东省柑橘黄龙病菌株的扩增条带分析
Table 5 Analysis of amplified bands of “Candidatus Liberibacter asiaticus” strains in Guangdong Province based on short tandem repeats in CLIBASIA_01215 gene
条带大小/bp
Sequence length清远
Qingyuan韶关
Shaoguan肇庆
Zhaoqing梅州
Meizhou惠州
Huizhou深圳
Shenzhen江门
Jiangmen茂名
Maoming湛江
Zhanjiang广州
Guangzhou总计
Total200~240 1 1 2 240~260 2 2 2 5 1 4 6 2 24 260~280 2 2 6 1 1 3 12 27 280~300 1 3 4 1 2 1 12 1 25 300~320 2 1 2 1 1 1 2 1 1 12 320~340 5 4 6 4 3 1 1 24 340~360 2 3 3 1 3 1 2 15 >360 1 7 1 1 2 12 双条带 Double amplicons 4 5 1 1 4 1 16 无条带 No amplicon 4 4 3 2 6 19 总计 Total 22 12 36 12 22 4 6 31 27 4 176
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