Obihiro University of Agriculture and Veterinary Medicine
OAK Obihiro University Archives of KnowledgeNot a member yet
5082 research outputs found
Sort by
アズキの品質と収量に関与する量的形質遺伝子座の検出
Get PDF帯広畜産大学博士（農学）2022application/pdf博士学位論文
大学院畜産学研究科畜産科学専攻
Doctoral Program of Animal Science and Agriculturedoctoral thesi
Epidemiological study on tick-borne parasitic diseases in livestock from Tanzania and Kenya
Get PDF帯広畜産大学博士（獣医学）2022application/pdfTick-borne diseases (TBDs) pose a major challenge to the livestock industry in many tropical and sub-tropical countries. In East Africa, Tanzania and Kenya are among the countries located just south of the horn of Africa. This region is popularly known for a large population of livestock. However, TBDs in the region present a serious threat to the livestock sector particularly cattle and small ruminants. The most important TBDs in the region are theileriosis, anaplasmosis, babesiosis and ehrlichiosis. Despite the damage incurred by these diseases in Tanzania and Kenya, limited epidemiological data on the occurrence and distribution of TBDs in the two countries is available. Therefore, several molecular studies were carried out in different locations of the two countries to address the problem.
In chapter 1, a total of 245 blood samples from different breeds of cattle were randomly collected on Pemba Island, in Tanzania. Polymerase chain reaction (PCR) and sequencing was used to detect and identify the tick-borne pathogens (TBPs). The assays were performed using primers based on Theileria spp. (18S rRNA), Babesia bovis (SBP-2), B. bigemina (RAP-1a), Anaplasma marginale (MSP-5 and groEL), Ehrlichia ruminantium (pCS20), T. parva (p104), T. mutans (18S rRNA), and T. taurotragi (18S rRNA). PCR screening of cattle samples collected on Pemba Island revealed overall infection rates for Theileria spp. (62.4%), B. bigemina (17.6%), A. marginale (15.9%), E. ruminantium (7.4%) and B. bovis (4.5%). Further analysis using sequences of Theileria spp. (18S rRNA) revealed infection of cattle with T. mutans (68.6%), T. taurotragi (48.4%), T. parva (41.2%), and T. ovis (1.9%). Co-infections of cattle, with up to six TBPs, were revealed in 46.9% of the samples. Sequence analysis indicated that T. parva (p104), E. ruminantium (pCS20) and A. marginale (MSP-5) genes are conserved among cattle blood samples in Pemba, with 99.3% - 100%, 99.6% - 100% and 100% sequence identity values, respectively. In contrast, the B. bigemina (RAP-1a) and B. bovis (SBP-2) gene sequences were relatively diverse with 99.5% - 99.9% and 66.4% - 98.7% sequence identity values, respectively. The phylogenetic analyses revealed that T. parva (p104), E. ruminantium (pCS20) and A. marginale (MSP-5) gene sequences clustered in the same clade with other isolates from other countries. In contrast, the B. bigemina (RAP-1a) and B. bovis (SBP-2) gene sequences showed significant differences in the genotypes, as they appeared in separate clades. The data provided should improve the understanding of the epidemiology of tick-borne diseases, and is expected to improve the approach for diagnosis and control of tick-borne diseases in Tanzania.
In chapter 2, blood samples were collected randomly in 236 cattle of different breeds from Zanzibar Island, Tanzania. The PCR and sequencing were used to screen the samples for detection of TBPs. The assays were performed using primers described in chapter 1. The PCR screening revealed that 64.5% of animals were infected by TBPs, including T. mutans (38.1%), T. parva (34.3%), T. taurotragi (30.9%), A. marginale (10.2%), B. bigemina (5.1%), T. velifera (3.4%) and B. bovis (2.1%). Overall, a total of 86 animals (36.4%) were co-infected with up to five pathogens concomitantly. The pathogens mostly involved in the co-infection were T. parva, T. taurotragi and T. mutans. Sequence analysis indicated that T. parva (p104) and B. bigemina (RAP-1a) genes are diverse among the sampled animals on Zanzibar Island, with 99.64%–100% and 99.51%–100% nucleotide sequence identity value, respectively. In contrast, the A. marginale (MSP-5) and B. bovis (SBP-2) genes are conserved, with 100% and 99.66%-100% nucleotide sequence identity values respectively. The phylogenetic analyses revealed that T. parva (p104) and B. bigemina (RAP-1a) gene sequences showed significant differences of genotypes, as they appear in different clades. Meanwhile, A. marginale (MSP-5) and B. bovis (SBP-2) gene sequences appear in the same clade with other sequences extracted from the NCBI GenBank. The epidemiological findings revealed in this study will provide important information on tick-borne diseases in Tanzania and will be used as scientific basis for planning future control strategies.
In chapter 3, a total of 250 blood samples were randomly collected from indigenous cattle of Tanga region, Tanzania. The assays performed were based on primers described in chapter 1. The results show an overall infection rate for T. mutans (48%), A. marginale (32.4%), T. parva (25.6%), T. taurotragi (20.8%) and B. bigemina (13.2%). Co-infections of up to four pathogens were revealed in 44.8% of the cattle samples. Sequence analysis indicated that T. parva (p104) and A. marginale (groEL) genes were conserved among the sampled animals with sequence identity values of 98.92 – 100% and 99.88% – 100%, respectively. On the other hand, the B. bigemina (RAP-1a) gene and the (V4 region of the 18S rRNA of T. mutans genes were diverse among the sampled cattle, indicating the sequence identity values of 99.27% - 100% and 22.45% - 60.77%, respectively. The phylogenetic analyses revealed that T. parva (p104) and A. marginale (groEL) gene sequences of this study were clustered in a clade. In contrast, the B. bigemina (RAP-1a) and the T. mutans (V4 region of the 18S rRNA) gene sequences appeared in the different clades. The findings revealed in this work shows that indigenous breed of cattle reared in free range system in Tanzania can be the source of tick-borne infections in other naïve breeds of cattle in the country. Therefore, this information is useful in the control of tick-borne diseases.
In chapter 4, blood samples were randomly collected from 76 apparently healthy sheep in Machakos and Homa-bay counties in Kenya. The assays were performed using primers based on Theileria spp. (18S rRNA), Anaplasma spp. (16S rRNA), Babesia ovis (18S rRNA), A. ovis (AoMSP-4) and E. ruminantium (pCS20). The overall infection rates of sheep samples collected in Machakos and Homabay counties in Kenya for Theileria spp. (51.3%), Anaplasma spp. (40.8%), A. ovis (34.2%) and E. ruminantium (7.9%). The overall co-infection was (61.8%). All Theileria spp. positive samples were confirmed to be T. ovis on sequencing. A phylogenetic analysis of (18S rRNA) gene sequences revealed that isolates of this study clustered with T. ovis sequences from other regions suggesting this gene is highly conserved. The E. ruminantium (pCS20) sequences were in the same clade on the phylogenetic tree. However, three (AoMSP-4) sequences appeared in the same clade while one sequence formed a separate branch revealing genetic divergence to the other sequences. The 16S rRNA sequencing revealed uncultured Anaplasma spp. and A. ovis. The phylogenetic analyses of uncultured Anaplasma spp. revealed that the two sequences formed a divergent clade signifying genetic differences to other isolates. This study provides important information regarding tick-borne pathogens occurrence and their degree of genetic diversity among sheep in Kenya.
In conclusion, the studies reveal that TBDs are well distributed in Tanzania and Kenya. Theileria spp. were the most prevalent TBPs in the region led by Theileria mutans, T. parva and T. taurotragi. Moreover, mixed infection in cattle and sheep were the prominent findings in the study areas. These data provide basement epidemiological background which will contribute to the future control strategies of tick-borne diseases in Tanzania and Kenya.マダニ媒介性感染症は、多くの熱帯および亜熱帯地域の畜産業に対し大きな脅威となっている。タンザニアとケニアは、共に東アフリカに位置しており、畜産業が盛んな地域として知られているが、家畜におけるマダニ媒介性寄生虫症の被害は甚大なものとされている。この地域で最も重要な家畜のマダニ媒介性寄生虫症は、一般的にタイレリア症、アナプラズマ症、バベシア症およびエーリキア症とされている。しかしながら、当該地域の家畜におけるマダニ媒介性寄生虫症の発生と分布に関する詳細な疫学的データは限られている。そこで、本研究ではタンザニアとケニヤの主要家畜である牛と羊におけるマダニ媒介性寄生虫症の流行実態の一端を解明するために分子疫学的調査を実施した。
第1章では、タンザニアのペンバ島で採集した245頭の牛の血液サンプル中のマダニ媒介性病原体についてPCR法によるスクリーニングとシークエンス解析を行った。その結果、Theileria spp.（62.4%）、B. bigemina（17.6%）、A. marginale（15.9%）、E. ruminantium（7.4%）およびB. bovis (4.5%)が検出された。Theileria属に属する種の特定を行ったところ、 T. mutans (68.6%)、T. taurotragi (48.4%)、T. parva (41.2%)、およびT. ovis (1.9%) が検出された。PCR産物の塩基配列に基づいて系統解析を行ったところ、T. parva (p104)、 E. ruminantium (pCS20)、および A. marginale (MSP-5) の標的遺伝子塩基配列が、他のアフリカの国と地域の分離株と近縁のクラスターに位置していることが判明した。対照的に、B. bigemina (RAP-1a) と B. bovis (SBP-2) の標的遺伝子配列は、他のアフリカの国と地域で分離された株とは独自のクラスターを形成した。これらのデータは、タンザニアにおけるマダニ媒介性寄生虫症の流行実態の一端を解明し、さらにコントロール対策を構築する上で一助となると考えられる。
第2章では、タンザニアのザンジバル島で採集した236頭の牛の血液サンプル中のマダニ媒介性病原体についてPCR法によるスクリーニングとシークエンス解析を行った。その結果、T. mutans (38.1%)、T. parva (34.3%)、T. taurotragi (30.9%)、A. marginale (10.2%)、B. bigemina (5.1%)、T. velifera (3.4%)およびB. bovis (2.1%)が検出された。系統解析では、T. parva (p104) 、B. bigemina (RAP-1a) およびB. bovis (SBP-2)の標的遺伝子配列は、タンザニア独自のクラスターを形成した。一方、A. marginale (MSP-5)の標的遺伝子配列は、地理的に離れた国及び地域の分離株と近縁のクラスタを形成した。これらの結果は、タンザニアにおけるマダニ媒介性寄生虫症に関する重要な情報を提供し、将来の制御戦略を構築する上で有用な科学的根拠として活用できると考えられる。
第3章では、タンザニアの陸地であるタンガ地域で採集した250頭の牛の血液サンプル中のマダニ媒介性病原体についてPCR法によるスクリーニングとシークエンス解析を行った。その結果、T. mutans (48%)、T. parva (25.6%)、T. taurotragi (20.8%)、B. bigemina (13.2%)およびA. marginale (32.4%)が検出された。系統解析では、T. parva (p104) および A. marginale (groEL) の標的遺伝子配列がタンザニア独自のクラスターを形成した。対照的に、B. bigemina (RAP-1a) および T. mutans (18S rRNA のV4領域) の標的遺伝子配列は、アフリカと世界中で分離された株と近縁関係にあることが判明した。これらの結果は、タンザニアの放し飼いシステムで飼育された土着の牛の品種が、他のナイーブ品種への伝播の感染源になり得ることが示唆された。したがって、これらの情報はタンザニアの内陸地域におけるマダニ媒介性寄生虫症の制御戦略を構築する上有効に活用できると考えられる。
第4章では、ケニアのマチャコスとホマベイ地域の羊76 頭から採集した血液サンプル中のマダニ媒介性病原体についてPCR法によるスクリーニングとシークエンス解析を行った。その結果、T. ovis（51.3％）、A. ovis（34.2％）、およびE. ruminantium（7.9％）が検出された。系統解析では、T. ovis (18S rRNA)の標的遺伝子配列は、ケニア分離株において高度に保存され、アフリカの他の地域やトルコで分離された株と近縁関係にあった。また、E. ruminantium (pCS20)塩基配列は東アフリカで分離された株で独自のクラスターを形成した。A. ovis (AoMSP-4) の標的塩基配列では、３つのケニア分離株が同一クラスターに属し、また、一つの分離株は他の３株と分岐を形成した。これらの結果から、ケニアの羊の間でマダニ媒介性病原体の発生とその遺伝的多様性の関する有用な情報を提供し、小型反芻動物におけるマダニ媒介性寄生虫症の診断と制御戦略の構築に有用であると考えられる。
以上のように、この研究ではタンザニアとケニアの主要家畜である牛と羊におけるマダニ媒介性寄生虫症の流行実態の一端を明らかにした。そのなかでもT. mutans、 T. parvaおよび T. taurotragi 最も広く分布しているマダニ媒介性病原体であることが明らかとなった。さらに、多くの牛と羊が複数のマダニ媒介病原体に同時感染していることが示唆された。これらのデータは、タンザニアとケニアにおける家畜のマダニ媒介性寄生虫症の制御戦略構築に役立つ基礎データを提供するものと考えられる。博士学位論文
大学院畜産学研究科 獣医学専攻
Doctoral Program of Veterinary Sciencedoctoral thesi
Comparison of the relative tolerance of Colpoda resting cysts and vegetative cells to electrostatic exposure
Get PDFapplication/pdfThe soil protist ciliate Colpoda cucullus forms resting cysts that have extreme tolerance to many environmental stresses. In this study, we show that Colpoda resting cysts, unlike vegetative cells, can survive electrostatic exposure. The viability of vegetative cells fell with the number of electrostatic exposures; cyst viability did not show this response. The morphology of exposed cysts appeared to be essentially normal at the optical microscope level; excysted cells from exposed cysts retained a similar proliferative ability to those of non-exposed cysts, whereas most vegetative cells were fatally damaged by electrostatic exposure.journal articl
Nationwide Survey about the Occurrence of Aspergillosis in Captive Penguins in Zoos and Aquariums in Japan
Get PDFapplication/pdfWe surveyed the facilities that were members of the Japan Association of Zoos and Aquariums to clarify the incidence of aspergillosis, which is a major cause of death in captive penguins, and to discern effective preventive measures. Responses were obtained for 2910 penguins in 64 facilities; 73 penguins (2.5%) in 35 facilities had died from aspergillosis during the past 5 years from April 2016 to March 2021. Answers to questions about the rearing environment indicated that aspergillosis occurred significantly more often in facilities where penguins were reared outdoors, were in contact with soil, or were moved outside of the rearing enclosure. Answers to questions about their dead penguins indicated that 76% may have been at individual risk (e.g., young age, old age, molting period, and breeding season) and 54% were thought to be reared in uncomfortable environments (e.g., high temperature, high humidity). Aspergillosis may occur when individual risk factors and uncomfortable environmental factors are added to the risk factors of exposure to Aspergillus, such as the presence of soil. These conditions must be recognized as risk factors for aspergillosis, and appropriate preventive measures, such as avoiding penguin contact with the soil where Aspergillus is expected to be present, can minimize aspergillosis-related deaths.journal articl
An epidemiological survey of vector-borne pathogens infecting cattle in Kyrgyzstan
Get PDFapplication/pdfCattle production is a major contributor to the national economy of Kyrgyzstan. Most cattle in Kyrgyzstan are managed via extensive systems and graze in communal pastures. As a result, infestations with ectoparasites are widespread, implying that various vector-borne diseases might be common in cattle. However, methods to control such infectious diseases are not available in Kyrgyzstan because the epidemiology of vector-borne pathogens (VBPs) infecting cattle remains unclear. The present study was therefore designed to survey Kyrgyz cattle for VBPs. We prepared blood DNA samples from 319 cattle in Kyrgyzstan and screened them with specific PCR assays for detecting Babesia bovis, Babesia bigemina, Babesia naoakii, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, and Anaplasma marginale infections. Our findings indicated that the surveyed cattle were infected with six of the eight pathogens targeted, with the exceptions being B. naoakii and Try. evansi. The most common pathogen was T. orientalis (84.3%), followed by B. bigemina (47.6%), T. annulata (16.6%), A. marginale (11.6%), Try. theileri (7.2%), and B. bovis (2.5%). Additional screening of the B. bovis- and B. bigemina-negative samples with a Babesia genus-specific 18S rRNA PCR identified two positive samples, and sequencing analysis confirmed that each of them was infected with either Babesia major or Babesia occultans. To the best of our knowledge, this is the first report of B. bovis, B. bigemina, B. occultans, Try. theileri, and A. marginale infections in cattle in Kyrgyzstan. Our findings suggest that cattle in Kyrgyzstan are at high risk of infectious diseases caused by VBPs. © 2023 Elsevier B.V.journal articl
バレイショにおける青枯病抵抗性に関する研究
Get PDF帯広畜産大学博士（農学）Doctor of Agriculture2023application/pdfナス科ナス属であるバレイショ類は栽培種7種と野生種226種に分類される多様な遺伝的背景を持ち，このうち現在世界中で栽培されているのはSolanum tuberosum subsp. tuberosumである。このように多様な遺伝的背景を持つにも関わらず，日本を含めた欧米のバレイショ育種における遺伝子プールの脆弱性が指摘されている。一方で，バレイショは世界中の多様な環境で栽培されており，近縁野生種を育種に利用することで多様な形質を栽培種に導入し各地域の気候や病虫害に対応してきた。しかし，地球温暖化による気候変動が進行するにつれ，バレイショ栽培においてもいくつかの問題が生じることが予測される。その一つとして高温で多発する青枯病による被害がある。これまで，日本を含めた世界各国で青枯病抵抗性育種は行われてきたが，バレイショが4倍体であること，青枯病抵抗性が量的形質遺伝子座(Quantitative trait locus, QTL) により支配されていること，そして青枯病の原因細菌が種複合体(Ralstonia solanacearum species complex, RSSC)でありバレイショヘの病原力が多様であることから，普及性の高い抵抗性品種の育成が難しかった。国内における抵抗性個体の選抜はRSSCに汚染された圃場を利用した生物検定に頼っており，青枯病の発病には環境要因が大きく影響を及ぼすため，抵抗性評価に最低3年は必要となっている点が抵抗性育種の大きな制限要因となっている。このような問題に対応するためには，抵抗性検定方法の改善，RSSCの病原力および抵抗性に対する理解，そして育種における効率的選抜を可能にするDNAマーカーの開発が必要である。
第1章では，バレイショの青枯病抵抗性に関するin vitro検定法を開発するため，抵抗性系統‘西海35号’および罹病性品種‘Kennebec’をin vitro条件下で培養し，R. pseudosolanacearumのphylotype I/biovar 4の菌株を接種株として両系統の発病度を明瞭に識別できる条件を検討した。その結果，最適な検定条件は，6～8葉期の培養植物に対し，接種する菌濃度は10 2 CFu ml -1，接種後の培養温度は28°Cであった。発病程度の評価部位については，葉よりも茎を評価対象とした方の信頼性が高かった。この方法を用いて，圃場での抵抗性程度が異なる9つの品種を評価した。その結果，圃場での抵抗性が高い品種ほど発病指数が低く，本検定法は制御された環境での青枯病抵抗性の評価に有効であると考えられた。
第2章では，RSSCの病原力を明らかにするため，国内で採取されたバレイショに被害を及ぼす26菌株(phylotype IおよびIV)について，接種後の培養温度を24°Cないし28°Cとし，抵抗性系統‘西海35号’および罹病性品種‘アイユタカ’に対する発病度をin vitro検定法で評価した。多くの菌株で，‘西海35号’の発病指数が‘アイユタカ’より有意に低かった。病原力は菌株によっても異なり，また接種後の温度によっても大きく異なっていた。また，それぞれの菌株の品種および培養温度に対する発病度を基に，階層および非階層クラスター分析を行ったところ，26菌株は5つの病原力型（病原型A, B, C, D, およびE)に分類された。これはphylotypeを含むこれまでの分類方法とは一致しなかった。したがって，対象地域で安定した抵抗性品種を育成するためには，その地域の主要病原型菌株に対する選抜を行う必要がある。
第3章では，バレイショにおける青枯病に対する抵抗性のQTL解析を行った。抵抗性2 倍体系統‘10-03-30’(RP) と罹病性2倍体系統‘F1-1’(SP)を交配し，94系統からなる凡集団を育成した。この集団をtwo-way pseudotestcrossとみなし，一塩基多型(Single nucleotide polymorphism, SNP) マーカーを用いて，RPに対しては1,476 SNPsが422座にマップされ，SPに対しては2,663 SNPsが475座にマップされた高密度連鎖地図を構築した。F1集団の抵抗性評価は，R.pseudosolanacearum (phylotype I/biovar 4/race 1/病原型A) を用いたin vitro 検定法により行われた。バレイショの第1, 3, 7, 10,および11番染色体上に5つのQTLs(qBWR-1~-5) が同定され，各QTLの寄与率は9.3~18.4％を示した。抵抗性親はqBWR-2, qBWR-3, およびqBWR-4に抵抗性アレルを持ち，qBWR-1とqBWR-5に罹病性アレルを持っていた。5つのQTLsに抵抗性アレルが集積されることで，抵抗性親と比較して抵抗性程度が向上した。また，第1, 7, および11番染色体におけるQTLsの相互作用による抵抗t生程度の向上効果も確認された。本研究は，ゲノムワイドマーカーを用いてバレイショの青枯病抵抗性に関する新規QTLを同定した初めての研究である。
第4章では，第3章の結果を拡張するため，同じ2倍体凡集団およびSNPマーカーによるRPとSPの連鎖地図を用いた。In vitro 検定を用いて3種の異なる菌株(R.pseudosolanacearum のphylotype I/biovar 4/病原型A株とphylotype I/biovar 3/病原型C株，およびR.syzygiiのphylotype IV/biovar N2/病原型A株）を接種し，24°cまたは28°Cで培養した後抵抗性を評価した。その結果， 第1, 3, 5, 6, 7, 10, および11番染色体上に5つの主働QTLを含む合計10 QTLsを同定した。主働QTLであるPBWR-3 とPBWR-7 はR.pseudosolanacearum (phylotype I) とR.syzygii (phylotype IV) に対して安定した抵抗性を示したが，同じく主働QTLであるPBWR-6b はR.pseudosolanacearum (phylotype I/biovar 3) に対する菌株特異性を示し，低温でより高い抵抗性を示した。したがって，広範な抵抗性を示すQTL と菌株特異的抵抗性QTLを組み合わせることで，有効な青枯病抵抗性品種を開発できることが示唆された。
第5章では，前章において同定された菌株特異性を示し，低温でより高い抵抗性を示すQTLであるPBWR-6bのマーカー開発を行った。QTL領域内に位置する候補遺伝子の1つNucellin-like aspartic protease遺伝子の塩基配列を，RPとSPで比較した。その結果，RPの抵抗性アレルに固有の配列を持つAl アレルを同定し，これを特異的に検出する2つの分子マーカー (Rbw6-1とRbw6-2) を開発した。マーカーを用いてRPの系譜を辿ったところ，青枯病抵抗性QTLであるPBWR-6bは，‘インカのめざめ’の交配親であるS.tuberosum subsp. andigena に由来すると推定された。また，107のバレイショ品種系統を調査したところ，‘インカのめざめ’の後代系統でのみRbw6-1 とRbw6-2 が検出されその多くで抵抗性が確認された。2倍体および4倍体の交配集団においてマーカーの有無と抵抗性の程度は一致し，PBWR-6b は顕性遺伝により後代に伝わることが明らかとなった。さらに，Rbw6-2 マーカーを既存のマルチプレックスポリメラーゼ連鎖反応(PCR)法に組み込み，青枯病(PBWR-6b) ，ジャガイモシストセンチュウ(Hl)，ジャガイモY ウイルス(Rychc) ，ジャガイモXウイルス(Rxl) ，および疫病(Rl， 品種‘さやあかね’由来のR2) に対する抵抗性遺伝子が同時検出できるようになったため，青枯病抵抗性系統の迅速な選抜が可能となった。
以上述べたように，本研究では，新たな青枯病抵抗性の評価方法が開発され，より効率的に抵抗性育種を進めることが可能となった。加えて，国内の青枯病菌が5つの病原力型に分類されたことから，今後は，これら病原力型別に抵抗性を評価することで，国内の幅広い地域で安定した抵抗性を示す品種育成が可能になると考えられる。また，青枯病抵抗性の主働QTLを同定し，その一つであるPBWR-6bの抵抗性アレルの有無を判定するDNAマーカーを開発した。このマーカーを従来のマルチプレックスPCRに組み込んだことで，主要な6つの病虫害抵抗性遺伝子の迅速な選抜技術を開発した。これらの得られた知見と開発した技術は，バレイショにおける青枯病抵抗性育種の効率化に大いに寄与するものと期待される。Potato and its relatives are classified into 7 cultivated and 226 wild species in the Solanaceae family. Among them, Solanum tuberosum subsp. tuberosum is only species currently cultivated worldwide. The fragility of the genetic diversity is concerned for the gene pools in Western and Japanese potato breeding programs, despite its diverse genetic variability existing in closely related wild species. On the other hand, to grow potatoes under diverse environments, useful traits such as tolerance to biotic and abiotic stresses have been introduced from primitive cultivated and wild potato species. However, climatic changes due to global warming are progressing, which make challenges to continue stable and safe supply of potatoes. One of the significant challenges is against increasing damages caused by bacterial wilt, which occur frequently at high temperatures. However, potatoes are mainly tetraploid, resistance to bacterial wilt is controlled quantitatively, bacterial wilt is caused by a complicated species complex (the Ralstonia solanacearum species complex, RSSC), and the virulence of RSSC varies widely depending on strains and potato varieties, altogether making it difficult to rapidly breed resistant cultivars. Since selection of resistant genotypes to bacterial wilt is conducted in the heavily infested field and affected largely by environmental conditions, the resistance assay takes at least three years in Japan, which is considered a major limiting factor for resistance breeding. To overcome these problems, it is desired to develop a simple and reliable resistance assay method, to understand virulence types of the various RSSC strains and resistance types against different strains, and to develop DNA markers for rapid selection of resistant genotypes.
In Chapter 1, to develop an in vitro assay for resistance to bacterial wilt of potatoes, the resistant breeding line 'Saikai 35' and the susceptible variety 'Kennebec' were cultured under in vitro conditions. The inoculation conditions were examined by culturing the phylotype I/biovar 4 strain of R. pseudosolanacearum as inoculum. The optimal conditions were; inoculating plants at the 6-8 leaf stage at a bacterial concentration of 10 2 CFU ml -1 and an incubation temperature of 28 °C. Stems were more reliable as the site for evaluation of disease severity than leaves. This method evaluated nine cultivars with different degrees of resistance in the field. The results showed that varieties with higher resistance in the field had lower disease indices, suggesting that this test method is effective for evaluating bacterial wilt resistance in a controlled environment.
In Chapter 2, virulence of various RSSC strains was investigated. Twentysix strains (phylotypes I and IV) of RSSC collected in Japanese potato fields were evaluated by in vitro assays for virulence against the resistant line 'Saikai 35' and the susceptible variety 'Aiyutaka'. The effect of environmental temperature on the virulence of RSSC strains was very diverse, and virulence varied greatly among strains. Hierarchical and non-hierarchical cluster analyses classified the 26 strains into five virulence types (pathotypes A~E), inconsistent with previous classification methods, including phylotypes. Therefore, selection for breeding stable resistant varieties in the target region should be based on virulence types.
In Chapter 3, QTL analysis was performed for resistance to potato bacterial wilt. The resistant diploid line'10-03-30'was crossed with the susceptible diploid line'F1-1'to generate an F1 population of 94 genotypes. Resistance evaluation of the F1 population was performed using an inoculation strain of R.pseudosolanacearum (phylotype I/biovar 4/race 1/pathotype A) in an in vitro assay. Five QTLs (qBWR-1 to qBWR-5) were identified on potato chromosomes 1, 3, 7, 10, and 11, with each QTL contributing 9.3 to 18.4%. The resistant parent had resistance alleles at qBWR-2, qBWR-3, and qBWR-4 and diseased alleles at qBWR-1 and qBWR-5. The accumulation of resistance alleles at all five QTLs resulted in increased resistance compared to the resistant parent. The effect of interaction among QTLs at chromosomes 1, 7, and 11 on the degree of resistance was also observed. This is the first study to identify novel QTLs for resistance to bacterial wilt in potatoes using genome-wide markers.
In Chapter 4, a further expansion of the QTL analysis performed in Chapter 3 was conducted using the same F1 population and the linkage maps with different RSSC strains and temperature conditions. An in vitro assay was used to inoculate different strains and species (phylotype I/biovar 3, phylotype I/biovar 4, and phylotype IV /biovar N2) and evaluate resistance under controlled conditions at 24 or 28°C. Ten QTLs were identified, including five major QTLs on chromosomes 1, 3, 5, 6, 7, 10, and 11. PBWR-3 and PBWR-7, the major QTLs, showed stable resistance to R.pseudosolanacearum (phylotype I) and R. syzygii (phylotype IV), whereas PBWR-6b, also a major QTL, showed strain-specific and more effective properties at low temperatures against R.pseudosolanacearum (phylotype I/biovar 3). Therefore, it is suggested that effective bacterial wilt resistant cultivars can be developed by combining broad resistance QTL and strain-specific resistance QTL.
In Chapter 5, to develop DNA markers for PBWR-6b, the nucleotide sequence of one of the candidate genes located within the QTL region of PBWR-6b was compared between the parents used for QTL analysis. The resistance allele was identified, and resistance allele-specific molecular markers Rbw6-1 and Rbw6-2 were developed for PBWR-6b. PBWR-6b is thought to be derived from S. tuberosum subsp. andigena, the hybrid parent of 'Inca-no-mezame'. Both markers were detected only in the 'Inca-no-mezame' progeny among 107 potato cultivar and breeding lines. The developed resistance allele-specific DNA markers demonstrated that it is possible to select resistant individuals from diploid and tetraploid populations and inferred that the mode of inheritance of PBWR-6b is dominance. Furthermore, Rbw6-2 was incorporated into an existing multiplex polymerase chain reaction (PCR) method to select for resistance to bacterial wilt (PBWR-6b), golden cyst nematode (Hl), Potato virus Y (RYchc), Potato virus X (Rxl), and late blight (R1 and 'Saya-akane' -derived R2). This method is expected to improve the efficiency of breeding for resistance to bacterial wilt in potato.
In conclusion, the in vitro assay method developed in this study is reliable and enable to evaluate bacterial wilt resistance more efficiently. Since the RSSC strains in Japan were classified into five virulence types, varieties with stable resistance in a wide range of regions in Japan could be bred by considering these virulence types. Major resistance QTLs were identified, which made possible to develop DNA markers. One of markers diagnostic to a major resistance QTL, PBWR-6b, was incorporated into the existing multiplex PCR, which enabled to simultaneously detect six major disease and pest resistance genes. Based on these findings and the developed technologies, it is expected to breed more efficiently bacterial wilt resistant varieties.博士学位論文大学院畜産学研究科doctoral thesi
The molecular epidemiology of bovine Babesia species in livestock animals in Mongolia
Get PDF帯広畜産大学博士（獣医学）2022application/pdf博士学位論文
大学院畜産学研究科 獣医学専攻
Doctoral Program of Veterinary Sciencedoctoral thesi