稻瘟病菌株的DNA指纹分析及小种鉴别

基于对稻瘟病菌(Pyricularia oryzae)基因文库的分析,我们找到了一套含重复顺序的克隆。其中POR6和POR7被证实具有高度的多态性并随机散布于稻瘟病菌生理小种的致病性时,可以获得可分辨的基因组特异的杂交带型。我们还分析了致病性与8个稻瘟病菌株DNA指纹图谱之间的关系,结果表明各个小种组合间的百分相似率S_xy,值与该小种组合间共同侵染的鉴别品种数目有正相关性。     

稻瘟病菌重复顺序PoR6和稻瘟病菌的分型

POR6是一具有高度多态性的稻瘟病菌(Pyricularia oryzae)重复顺序。利用脉冲电泳技术和Southern分析,表明它是非均匀地散布于基因组中的。经测定POR6的拷贝数约为30—40,序列测定未发现在内部有更小的重复单位。用POR6作探针对44株稻瘟病菌进行DNA指纹分析,分析的中国北方地区的22个菌株可根据相似率归并成8个谱系。对一些转管培养中致病型发生变化的菌株用POR6进行指纹分析,发现这些菌株在转管过程中基因组DNA是有变化的。     

肺囊康定产生菌Glarea lozoyensis线粒体基因组序列的再分析

[目的] Glarea lozoyensis是抗真菌药物卡泊芬净的产生菌,其突变菌株ATCC 74030的线粒体基因组已被报道。我们此前的研究发现诱变剂能引起该菌某些细胞核基因的突变,但诱变剂是否也能引起线粒体DNA序列的改变并不清楚。[方法] 组装野生型菌株ATCC 20868的线粒体基因组,并与发表的突变型菌株ATCC 74030的线粒体基因组进行比较。通过PCR验证野生和突变菌株线粒体基因组间表现差异之处,并利用正确的线粒体基因组序列进行新的分析。[结果] 我们成功组装出野生型菌株ATCC 20868的线粒体基因组,通过比较其与发表的ATCC 74030的线粒体基因组序列,发现存在6处单核苷酸变异位点和2处具有长度差异的区域。然而,随后的PCR验证和序列比较并没有发现2个菌株间存在这些差异。最初观察到的碱基差异是因为发表的ATCC 74030线粒体基因组存在序列错误。有趣的是,在Glarea lozoyensis的线粒体基因组中,我们发现存在3个具有内含子的tRNA基因和1个rnpB基因。同时,该菌线粒体基因组中存在多种重复序列,在其线粒体和细胞核基因组间也存在明显的DNA片段重复事件。[结论] 诱变剂没有引起G. lozoyensis线粒体DNA的任何改变;发表的ATCC 74030的线粒体基因组存在序列错误。我们报道G. lozoyensis正确的线粒体基因组序列,并且发现该菌线粒体和细胞核基因组间频繁的基因交流。     

一株嗜酸硫杆菌M4-422-6的分离及其全基因组测序和比较基因组学分析

【目的】开展具有硫氧化能力的嗜酸硫杆菌属(Acidithiobacillus)的分离及其比较基因组学分析,不仅可以丰富硫氧化细菌菌种资源,而且有助于加深理解嗜酸硫杆菌的分子进化与生态适应机制。【方法】利用以硫代硫酸钠为唯一能源的培养基分离具有硫氧化能力的细菌;利用Illumina HiSeq X和Oxford Nanopore测序平台对一株嗜酸硫杆菌M4-422-6进行全基因组测序;利用相关生物信息学分析软件对原始数据进行组装和基因组注释,并与一株亲缘关系最近的菌株Igneacidithiobacillus copahuensis VAN18-1进行比较基因组学分析。【结果】分离获得一株具有硫氧化能力的嗜酸硫杆菌M4-422-6。基因组注释结果显示,菌株M4-422-6基因组由1个染色体和2个质粒组成,基因组大小为2 917 823 bp,G+C含量为58.54%,共编码2 925个蛋白。16S rRNA基因和基因组系统发育树显示,菌株M4-422-6代表嗜酸硫杆菌属的一个潜在新种。功能基因注释结果显示,菌株Acidithiobacillus sp. M4-422-6拥有与菌株特性相关的众多基因,包括硫氧化相关基因、CO₂固定相关基因和耐酸相关基因。比较基因组学分析发现,虽然菌株M4-422-6与VAN18-1的亲缘关系最近,但两者仍拥有众多的差异基因,主要包括噬菌体抗性相关基因和移动元件编码基因。【结论】菌株M4-422-6代表嗜酸硫杆菌属的一个潜在新种,该菌株具有同种内菌株所不具有的特有基因,并据此推测嗜酸硫杆菌种内分化可归因于对特定生态位的适应。     

一株羊源A型多杀性巴氏杆菌的全基因组测序及生物学特性分析

[背景] 多杀性巴氏杆菌（Pasteurella multocida,Pm）是一种革兰氏阴性菌,可引起动物和人类的呼吸道疾病和败血症等。本实验室前期分离鉴定一株A型Pm HN02菌株。[目的] 通过对HN02菌株的全基因组测序及生物信息学分析,扩充多杀性巴氏杆菌的基因组数据库信息;通过毒力基因鉴定和系统进化树分析,明确该菌株含有的毒力基因和遗传进化关系,为临床预防和诊断提供理论依据。[方法] 使用单分子实时测序（Single Molecule Real Time Sequencing,SMRT）技术对Pm HN02菌株进行全基因组测序,利用Illumina测序校正后进行基因功能注释和生物信息学分析。使用PCR鉴定菌株毒力基因,并构建进化树进行分析。[结果] Pm HN02菌株全基因组大小为2 333 292 bp,GC含量为40.15mol%,预测到的编码基因有2 389个,包含19个rRNA （6个23S rRNA、6个16S rRNA、7个5S rRNA）、62个tRNA基因、5个sRNA;含84个串联重复序列、66个小卫星DNA、2个微卫星DNA、9个基因岛、9个前噬菌体;分别有1 648、2 190和1 917个基因注释在GO、KEGG和COG数据库中,而且大部分富集于Pm的代谢过程;还有85个III型分泌系统效应蛋白、191个表型突变基因、165个毒力因子相关基因。根据分析结果绘制该菌株的全基因组圈图,并将基因组信息提交至NCBI后获得登录号cp037865。PCR鉴定发现该菌株含有fimA、toxA等14个毒力基因,缺失了tadD等毒力基因。系统进化树分析发现该菌株同北京的Pm3菌株（MH150895.1）进化关系最接近。[结论] 研究完成了A型Pm HN02株的全基因组测序和生物学特性鉴定,揭示了其同国内外Pm分离株的进化关系,为预防Pm疾病流行和探索Pm致病机制提供了参考。     

一株糖降解噬糖菌FZY0027的多糖水解活性及基因组分析

【目的】潮间带海水中分离获得一株具有水解多糖能力的菌株FZY0027,分析其对不同多糖的水解能力和基因组特征。【方法】通过形态观察、16S rRNA基因测序和基于Illumina NovaSeq和OxfordNanopore PromethION测序技术全基因组测序对菌株FZY0027进行鉴定。使用dbCAN、EasyCGTree、BRIG和Easyfig等生物信息学软件将菌株FZY0027和降解糖噬糖菌(Saccharophagus degradans) 2-40^T进行比较。使用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid, DNS)法测定多糖水解活性。【结果】菌株FZY0027与S. degradans 2-40^T的16S rRNA基因序列相似度达到99.9%,初步鉴定为降解糖噬糖菌(S. degradans) FZY0027。该菌株在水解淀粉、木聚糖和甘露聚糖时产生的还原糖浓度最高,分别为2.28、1.75和1.10 mg/mL。菌株FZY0027基因组全长5 178 381 bp,共编码4 156个基因,G+C含量为45.8%。菌株FZY0027与S. degradans 2-40^T的平均核苷酸一致性(average nucleotide identity, ANI)、平均氨基酸一致性(average amino acid identity, AAI)和DNA-DNA分子杂交(digital DNA-DNA hybridization, dDDH)值分别为96.5%、96.7%和70.0%。经碳水化合物活性酶数据库注释获得303个基因,其中,菌株FZY0027和S. degradans 2-40^T分别有糖苷水解酶(glycoside hydrolases, GHs)结构域的基因137个和130个。菌株FZY0027具有多个参与淀粉、木聚糖等多糖水解的基因,这与菌株FZY0027对淀粉和木聚糖的水解能力强的结果一致。然而,与S. degradans 2-40^T相比,菌株FZY0027在实验条件下只能水解少数多糖,这可能需要特定的诱导条件才能充分发挥其多糖水解能力。【结论】菌株FZY0027是一株多能型多糖水解菌,具有潜在开发价值。     

一株太平洋表层海水来源阿拉伯海假交替单胞菌N1230-9的全基因组测序及比较基因组学分析

【目的】阐述阿拉伯海假交替单胞菌(Pseudoalteromonas arabiensis)的分子进化与生态适应策略。【方法】借助Illumina HiSeq X Ten和Oxford Nanopore PromethION测序平台对一株分离自太平洋表层海水的菌株Pseudoalteromonas arabiensis N1230-9进行全基因组测序,利用相关生物信息学分析软件对原始数据进行组装和基因组注释,并与一株分离自深海沉积物环境的模式菌株Pseudoalteromonas arabiensis JCM 17292进行比较基因组分析。【结果】菌株N1230-9基因组由2条染色体组成,基因组大小为4 627 470 bp,G+C含量为40.85%,共编码4 202个蛋白。功能基因注释结果显示,菌株N1230-9具有适应海洋环境的多种类型功能基因,包括重金属抗性基因、多种铁离子摄取系统编码基因、多种噬菌体防御系统编码基因、种类丰富的水解酶编码基因、多种碳水化合物代谢相关基因,以及数量众多的二元信号传导系统编码基因。通过比较基因组分析发现,菌株N1230-9和菌株JCM 17292拥有适应不同生态位的特有基因,这些基因主要涉及血红素摄取、重金属抗性、噬菌体防御、二元信号系统传导和横向基因水平转移。【结论】来自表层海水的菌株P. arabiensis N1230-9已演化出了适应其生态位的特有基因。     

Bacillus sp.ZJS3基因组测序及砷胁迫下相关基因表达分析

【背景】环境中高毒性As³⁺的微生物氧化在砷的生物地球化学循环中起重要作用,具有潜在的应用价值。【目的】Bacillus sp.ZJS3菌株是本实验室前期分离鉴定的一株As³⁺耐受菌株,而且对多种重金属具有耐受性,期望进一步明确该菌株在As³⁺胁迫下菌体形态变化及应对砷胁迫的遗传基础,为As³⁺耐受细菌的研究提供基础数据。【方法】使用单分子实时测序（single-molecule real-time sequencing,SMRT）及Illumina测序技术对Bacillus sp.ZJS3菌株进行全基因组测序,对其基因进行功能注释和生物信息学分析,并结合绝对定量PCR技术对砷抗性及砷代谢相关基因进行分析。【结果】Bacillus sp.ZJS3菌株基因组大小为5.82 Mb,GC含量为35.9%,包含染色体1个、质粒3个、CDS数量为5 981个、tRNA 104个、sRNA 136个、rRNA 42个、串联重复序列173个、基因岛13个、转运蛋白1 023个、跨膜蛋白1 717个和双组分调控基因160个。NR、Swiss-Prot、Pfam、COG、GO和KEGG数据库分别可注释Bacillus sp.ZJS3菌株基因组中97.66%、69.30%、78.52%、65.49%、67.65%和43.87%的基因。绝对定量PCR结果表明,arsC基因在砷处理条件下显著高于对照组,而arsB基因在砷处理条件下显著低于对照组。【结论】Bacillus sp.ZJS3菌株在As³⁺胁迫下可能导致细胞分裂无法正常进行,进而影响细胞形态。基因组中aqpZ、arsA、arsB、arsC等基因的存在表明该菌株具有As³⁺外排和还原As⁵⁺的能力,phoUpstBACS的存在表明菌株可以吸收As⁵⁺,但菌株受到外界环境As³⁺胁迫时arsB表达水平降低。     

苯胺双加氧酶基因的克隆与序列分析*

通过设计苯胺双加氧酶基因特异引物,以苯胺降解菌株ANA5基因组DNA为模板,PCR扩增出目的基因片断。然后利用粘粒pLAFR3作为载体,以E.coliEPI100作为受体,构建了菌株ANA5的基因组粘粒文库。以PCR扩增产物作为探针,通过菌落原位杂交筛选得到两个阳性克隆,经Southern杂交及亚克隆测序分析,初步确认克隆到苯胺双加氧酶基因。同时完成了苯胺双加氧酶基因atdA3A4A5序列的测定,并对其核苷酸及其推导的氨基酸序列进行分析,结果表明克隆到的苯胺双加氧酶基因与GenBank报道的     

Genetic Diversity of Magnaporthe grisea in China as Revealed by DNA Fingerprint Haplotypes and Pathotypes

We determined DNA fingerprint haplotypes and pathotypes of the rice‐blast fungus Magnaporthe grisea collected from 13 areas in China. This DNA fingerprinting analysis, using rep‐PCR, of 381 haplotypes (482 isolates) from China indicated that the M. grisea populations cannot be delineated into region‐specific groups. Analyses of the number of alleles (na), Nei's gene diversity, unbiased genetic distance, and Shannon's Information index among 13 populations showed that clusters were not related to the geographic distance between populations with the exception of the Ningxia (NX) and Jilin (JL) cluster. Among northern populations, NX and JL were more similar to one another than to other populations. Pathogen populations consisting of 121 isolates from China were grouped into 53 pathotypes on the basis of disease reaction in differential rice lines. Isolates assayed for pathotypes were detected based on disease reactions. No correlation was observed between fingerprint groups and pathotypes of the pathogen. High frequency of virulence was found on the rice line Shin2 (Pi‐k^s and Pi‐sh) followed by PiNo.4 (Pi‐ta² and Pi‐sh) and K1 (Pi‐ta), while it was low on Kanto 51 (Pi‐k + ?), K3 (Pi‐k^h), and Fujisaka (Pi‐i and Pi‐sh). Virulence was rare on Toride 1 (Pi‐z^t and Pi‐sh). Tetep (Pi‐k^h + ?) was predicted to be a highly effective, as none of the isolates infected this line. These blast‐resistant rice lines can be used in resistance breeding for the effective management of rice blast in the respective regions of China.     

Characterization of Rice Blast Isolates by the Differential System and their Application for Mapping a Resistance Gene,Pi19(t)

To facilitate resistance gene characterization in the present study, the pathogenicities of newly collected blast isolates from rice fields in the Philippines were characterized using international blast differential varieties consisting of 31 monogenic lines that target 24 resistance genes. To classify and designate the blast isolates, we used a new international blast designation system, which has been proposed as a suitable naming system for comparing blast races among different studies. A total of 23 rice blast isolates collected from the Philippines were classified into 16 pathotypes, which showed reaction patterns different from those seen in the standard isolates. Among the blast pathotypes, 11 had differentiating ability for four Pik alleles (Pik, Pik‐m, Pik‐h, and Pik‐p) and Pi1, whereas the standard blast isolates from the Philippines were not able to differentiate these genes. In addition, several blast isolates were avirulent to IRBLt‐K59, IRBL19‐A, and Lijiangxintuanheigu, although the standard differential blast isolates were virulent to these lines. Moreover, two blast isolates were virulent to a monogenic line, IRBL9‐W, which harbours Pi9 and was resistant to all standard differential blast isolates. By using the isolates avirulent to IRBL19‐A, Pi19(t) was successfully mapped in the centromeric region on chromosome 12 with simple sequence repeat markers RM27937 and RM1337. These markers are useful for marker‐assisted Pi19(t) introgression worldwide.     

DNA Fingerprinting with a Dispersed Repeated Sequence Resolves Pathotype Diversity in the Rice Blast Fungus

The poor definition of pathotype variation in the rice blast fungus has historically handicapped strategies for reducing blast disease damage to the world's rice crop. We have employed a probe for a dispersed repeated DNA sequence called MGR [Hamer et al. (1989). Proc. Natl. Acad. Sci. USA 86, 9981-9985] to construct genotype-specific, EcoRl restriction fragment length profiles (MGR-DNA fingerprints) from United States field isolates of this fungus. By using a blind-test design, we demonstrated that MGR-DNA fingerprints distinguished the major pathotypes in the United States, accurately identified the pathotypes of isolates collected over a 30-year period, and defined the organization of clonal lineages within and among pathotype groups. These results resolved a lingering controversy regarding rice blast pathotype stability and illustrated new opportunities for tracking the population dynamics and evolution of this important crop pathogen.     

Comparative Analysis of Pathogenicity and Phylogenetic Relationship in Magnaporthe grisea Species Complex

Outbreaks of rice blast have been a threat to the global production of rice. Members of the Magnaporthe grisea species complex cause blast disease on a wide range of gramineous hosts, including cultivated rice and other grass species. Recently, based on phylogenetic analyses and mating tests, isolates from crabgrass were separated from the species complex and named M. grisea. Then other isolates from grasses including rice were named as M. oryzae. Here, we collected 103 isolates from 11 different species of grasses in Korea and analyzed their phylogenetic relationships and pathogenicity. Phylogenetic analyses of multilocus sequences and DNA fingerprinting revealed that the haplotypes of most isolates were associated with their hosts. However, six isolates had different haplotypes from the expectation, suggesting potential host shift in nature. Results of pathogenicity tests demonstrated that 42 isolates from crabgrass and 19 isolates from rice and other grasses showed cross-infectivity on rice and crabgrass, respectively. Interestingly, we also found that the isolates from rice had a distinct deletion in the calmodulin that can be used as a probe.     

Assessment of Genetic Diversity in Thai Isolates of Pyricularia grisea by Random Amplification of Polymorphic DNA

One hundred and seventy‐four isolates of Pyricularia grisea were collected from various hosts such as barley, rice, weed and wild rice in Thailand. Seven arbitrary decamer primers from the set of University of British Columbia were employed and nine lineages were classified. Lineages B, C and H were predominant, contributing up to 70% of total pathogens in this study. Analysis showed that the distribution of each lineage differs from the predominant lineages across Thailand in such that other lineages were restricted in particular area. For instance, lineage A was limited only in southern Thailand, whereas wide distribution of lineages B and C reflected an influence of both biological and physical effects on pathogen variation. Principal component analysis resulted in a total of four groups of blast pathogen with small distinctions between barley‐, rice‐, weed‐ and wild rice‐infected blast. Bridging relationships occurred among border isolates of weed and rice blast suggesting a chance of migrations between hosts. Higher diversity was observed in northern, north‐eastern and central Thailand while eastern and southern parts were rather low. Genetic diversity indices elucidated an abundance of pathogen lineages existing in northern Thailand suggesting that it should be the centre of diversity.     

Analysis of Pathotypic and Genotypic Diversity of Xanthomonas oryzae pv. oryzae in China

Virulence analysis and restriction fragment length polymorphism (RFLP) were used to evaluated the population structure of Xanthomonas oryzae pv . oryzae ( Xoo ) from the main rice-growing region in China. The pathotype of Xoo was determined for 103 strains by inoculating 13 near-isogenic rice lines using IR24 as the recurrent parent. Sixty-one pathotypes was shared by these strains, on the basis of the consensus of three clustering statistics, and four clusters for pathotype were formed. Cluster 2 consists of strains with high molecular polymorphorism and many pathotypes that are either virulent to a majority of the 13 major resistance ( R ) genes or avirulent only to Xa21 , and is geographically dispersed. The resistance gene Xa21 has broader resistance than others to the strains tested. A probe from a member of the avrBs3/pthA type III effector family, 1376 bp Sph I-digested fragment, was used to screen the genomes of 52 strains tested. Four common bands were found in the DNA fingerprint pattern of Xoo , suggesting basic patterns of evolutionary relationship for members of avrBs3/pthA family and/or the pathogen. Each distinct RFLP banding pattern of each strain was considered as a haplotype; 42 haplotypes were revealed by the probe and divided into four lineages by the same statistics method. It was observed that some isolates with different pathotypes shared the same haplotype and others with different haplotypes harboured identical pathotype. There was a weak correlation between virulent pathotypes and molecular haplotypes.     

Identification and fine mapping of a resistance gene to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in a space-induced rice mutant

Finding novel sources of resistance (R) to rice blast disease should facilitate breeding for improved resistance. The objectives of the present study were to evaluate reactions to blast and identify in a space-induced mutant an R gene to a representative isolate of rice blast pathogen. The mutant H4, its parent and twelve monogenic lines were evaluated for their responses to 35 isolates collected from Guangdong Province, China. H4 was found to be resistant to more isolates than its parent and the twelve monogenic lines, suggesting newly acquired resistance may be a function of one or more R genes. A representative isolate GD0193 was used to identify and map the R gene from H4. Genetic analysis revealed that resistance to the isolate GD0193 was controlled by a single dominant gene, designated Pi46(t). Linkage analysis using susceptible F2 individuals showed that Pi46(t) was mapped between the markers RM224 and RM27360 within 1.04 and 1.2 cM on the long arm of chromosome 11. Subsequently, Pi46(t) was delimited to an interval of approximately 183.7 kb flanked by the markers K67 and T94. These results provide essential information for the cloning of the Pi46(t) gene and will facilitate marker-assisted selection in rice breeding.     

Sequence variation of avirulence gene AVR-Pita1 in rice blast fungus, Magnaporthe oryzae

The interaction between rice, Oryza sativa, and rice blast fungus, Magnaporthe oryzae, is triggered by an interaction between the protein products of the host resistant gene, and the pathogen avirulence gene. This interaction follows the ‘gene-for-gene' concept. The resistant gene has effectively protected rice plants from rice blast infection. However, the resistant genes usually break down several years after the release of the resistant rice varieties because the fungus has evolved to new races. The objective of this study is to investigate the nucleotide sequence variation of the AVR-Pita1 gene that influences the adaption of rice blast fungus to overcome the resistant gene, Pi-ta. Thirty rice blast fungus isolates were collected in 2005 and 2010 from infected rice plants in northern and northeastern Thailand. The nucleotide sequences of AVR-Pita1 were amplified and analyzed. Phylogenetic analysis was conducted using the MEGA 5.0 program. The results showed a high level of nucleotide sequence polymorphisms and the positive genetic selection pressure in Thai rice blast isolates. The details of sequence variation analysis were described in this article. The information from this study can be used for rice blast resistant breeding program in the future.