[논문]RAPD 지문을 통한 우리나라에서 분리된 Pseudomonas syringae pv. actinidiae biovar 3 균주의 유전적 다양성 평가

이영선; 김경희; 고영진; 정재성

doi:10.5352/jls.2020.30.1.1

RAPD 지문을 통한 우리나라에서 분리된 Pseudomonas syringae pv. actinidiae biovar 3 균주의 유전적 다양성 평가
Evaluation of the Genetic Diversity of Biovar 3 Strains of Pseudomonas syringae pv. actinidiae Isolated in Korea 원문보기

생명과학회지 = Journal of life science, v.30 no.1, 2020년, pp.1 - 9

이영선 (순천대학교 생물학과) , 김경희 (순천대학교 식물의학과) , 고영진 (순천대학교 식물의학과) , 정재성 (순천대학교 생물학과)

초록
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키위 세균성 궤양병의 원인균인 Pseudomonas syringae pv. actinidiae는 유전적으로 구별되는 다섯 개의 biovar (1, 2, 3, 5, 6)로 나뉘어 진다. 그 중 biovar 3에 속하는 균주가 2008년 이래 전세계적으로 대유행을 일으키고 있다. 본 연구의 목표는 우리나라에서 분리된 biovar 3균주들의 집단 구조를 밝히고 그들의 기원을 추적하는데 있다. 13개의 우리나라 대표 균주와 2개의 중국 균주를 포함하는 15개 biovar 3 균주의 유전적 다양성을 RAPD-PCR로 평가하였다. RAPD 결과 연구에 사용된 균주들은 8개로 나누어져 이들을 subgroup I - VIII로 명명하였다. Subgroups II와 III은 중국 균주로 우리나라에서 발견되지 않았다. 따라서 우리나라 biovar 3 균주는 유전적으로 구별되는 6개의 subgroup (I, IV, V, VI, VII, VIII)으로 구성되어 있음을 알 수 있었다. New Zealand와 Europe균주에 특이적인 각각의 프라이머를 사용하여 PCR을 수행했을 때 subgroups V와 VI에 속하는 균주가 예상했던 DNA 절편을 증폭시켜 이들이 각각 두 지역에서 유입된 균주임을 시사하였다. 우리나라에서 처음 발견된 biovar 3 균주인 subgroup VIII에 특이적인 프라이머를 개발하여 조사한 결과 이 subgroup 균주는 처음 출현한 이후 더 이상 발견되지 않았다.

Abstract ▼ AI-Helper

Pseudomonas syringae pv. actinidiae, the causal agent of a bacterial canker disease in kiwifruit, is subdivided into five genetically distinct populations, namely biovars 1, 2, 3, 5, and 6. Of these, strains belonging to biovar 3 are responsible for a pandemic bacterial canker of kiwifruits since 2008. This study aimed to characterize the structure of the biovar 3 population and investigate the origin of biovar 3 strains isolated in Korea. The genetic variability of fifteen biovar 3 strains, thirteen Korean and two Chinese, were evaluated through random amplified polymorphic DNA (RAPD)-PCR. The RAPD results revealed the presence of eight lineages, designated as subgroups I-VIII, across the biovar 3 strains used in this study. As the strains in subgroups II and III from China were not found in the Korean examples, we concluded that six genetically different biovar 3 subgroups (I, IV, V, VI, VII, and VIII) are present in Korea. In PCR analysis using primers specific to the strains of New Zealand and Europe, Korean strains in subgroups V and VI amplified the relevant DNA bands, suggesting that these were introduced from these two origins, respectively. PCR primers specific to subgroup VIII were developed to monitor the spread of the first biovar 3 strain in Korea, and investigations revealed that this strain was not found in Korea after its first occurrence.

주제어

표/그림 (5)

표 Table 1. Pseudomonas syringae pv. actinidiae strains used in this study
그림 Fig. 1. Example of RAPD polymorphisms of Pseudomonas syringae pv. actinidiae strains using random 10-mer primers.
그림 Fig. 2. Dendrogram generated by GelCompar II software showing the relationship between Pseudomonas syringae pv. actinidiae strains.
그림 Fig. 3. PCR amplification with specific primers of New Zealand (A) and European strains (B).
그림 Fig. 4. Agarose gel electrophoresis of PCR products amplified using the PKN-F2/4 primer pair and genomic DNA of Pseudomonas syringae pv. actinidiae strains.

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문제 정의

In this study, we aimed to determine the population structure of biovar 3, which was propagated rapidly between 2014 and 2016 in Korea.

대상 데이터

The genetic diversity of biovar 3strains of PSA isolated in Korea was assessed by RAPD. In a first trial, sixty eight 10-mer primers with arbitrary sequences were tested on five PSA strains in order to select suitable primers for RAPD. Thirty primers produced reliable fingerprints for the bacterial strains.
Thirty primers produced reliable fingerprints for the bacterial strains. The selected 30 primers were used to analyze genomic DNAs of 17 representative strains of PSA, including 13 biovar 3 strains isolated in Korea, two biovar 3 from China, one biovar 1 and one biovar 2 strains. Examples of RAPD-PCR fingerprints obtained using random primers are shown in Fig 1.

이론/모형

Bands with the same mobility among individual PSA strains were considered as identical fragments, regardless of the staining intensity. The RAPD data obtained using the 30 primers were combined and similarity values of DNA profiles were computed on the basis of band positions using the Dice similarity index [35]. The similarity values were then used to construct a dendrogram by the unweighted pair group method using arithmetic averages (UPGMA).
actinidiae strains. The analysis of the bands was performed using the Dice coefficient and unweighted pair group method with arithmetic averages (UPGMA).
The RAPD data obtained using the 30 primers were combined and similarity values of DNA profiles were computed on the basis of band positions using the Dice similarity index [35]. The similarity values were then used to construct a dendrogram by the unweighted pair group method using arithmetic averages (UPGMA).

성능/효과

The approximate range of band size was 250-2,500 bp. A total of 236 bands were obtained from 30 primers, and the number of polymorphic amplicons in RAPD was 153, accounting for 64.8%.
To determine if any of the biovar 3 subgroups isolated in Korea include strains belonging to New Zealand or European populations, PCR amplifications were carried out against 53 biovar 3 strains using two specific primer sets. As shown in Fig. 3, a 609 bp fragment was amplified using China F/R primers in DNAs from five strains including HYH1471, subgroup V strain, and strain ICMP18708 isolated from New Zealand, while the Europe F/R primer set produced a 733 bp fragment with DNAs of subgroup VI strain, KTS1471, and four strains including the European reference strain CFBP7286. These results indicate that the subgroup V and VI strains might originate in New Zealand and Europe, respectively.

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저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

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