四川安岳柑橘衰退病病原鉴定及序列分析

[目的]探明引起四川安岳柑橘衰退病的病原。[方法]利用RT-PCR、克隆、测序等技术对四川安岳地区的柑橘类果树进行柑橘衰退病毒(CTV)检测,并对其外壳蛋白(CP)全序列进行了分析。[结果]81个疑似CTV侵染的样品中共有18个检测呈阳性,检出率为22.2%,表明安岳地区柑橘类果树衰退病由CTV引起。基于CP全序列同源性比较及进化分析表明,危害该地区柑橘类果树的CTV分属5个组群,亲缘关系较复杂。[结论]为安岳地区CTV的防控与防治提供了理论依据。     

Genetic Evolution Analysis on Wild Isolates of Citrus Tristeza Virus Originated in China Based on Coat Protein Genes Sequences

The coat protein （CP） genes were cloned and sequenced from viral particles of 11 isolates of citrus tristeza virus （CTV） collected from wild citrus plants in China and 4 Chinese isolates from cultivated sweet orange and pummelo varieties, respectively. By analyzing and comparing the nucleotide and amino acid sequences of CP genes, the 11 wild CTV isolates were found over 92% identical with 4 Chinese CTV isolates and 21 exotic CTV isolates from cultivated citrus. From 91 to 100% of the CTV CP gene sequences in wild type citrus plants were generally well conserved. Genetic evolution analysis indicated that the GC% of the CP gene was less than AT%, and more transition were found in the CP genes than transversion with the transition/transversion ratio ranging from 6.3 to 7.0 among species. The substitution frequency was the highest at the third codon, followed by the first and second codon. The ratio of non-synonymous mutations （du） to synonymous mutations （ds） was far lower than 1, suggesting that the CP gene might have experienced purifying selection in the evolution. Phylogenetic analysis revealed that the 11 CTV isolates in Chinese wild type citrus belonged to different phylogenetic clusters, and shared higher homology and closer relationships with other cultivated citrus CTV isolates from different countries, which indicated complicated genetic relationships among the CTV isolates. In addition, CTV isolates with similar biological characteristics usually located into the same clusters. Therefore, the conclusion was drawn that pathogenicity was critical to evolution and origin of CTV.     

中国野生柑橘上衰退病毒分离株分子特征分析

 【目的】了解中国野生柑橘上携带柑橘衰退病毒（CTV）分离株的CP/HinfⅠRFLP组群构成和分子特征。【方法】运用RT-PCR对采自中国云南、广西、四川、湖南、江西等省11个野生柑橘上CTV分离株的病毒衣壳蛋白基因(CPG)进行扩增，利用RFLP和SSCP对CPG进行分析，并将测定的CPG序列进行同源性比较和聚类分析。【结果】发现野生柑橘上11个CTV分离株以单一组群感染为主；这些CTV分离株CPG的核苷酸序列和相应的氨基酸序列同源性分别为92.5％～99.5％和95.0％～100％；与GenBank收录的9个全基因组CTV分离株的相应基因序列进行聚类分析，其核苷酸序列和氨基酸序列同源性分别为92.1％～98.8％和94.6％～100％。【结论】序列同源性比较说明CTV CPG具有高度保守性，同时系统进化分析表明收集到的野生CTV分离株与国外分离株分属5大类群，具有较复杂的亲缘关系。     

ISSR-Based Molecular Characterization of an Elite Germplasm Collection of Sweet Potato （Ipomoea batatas L.） in China

To determine the genetic diversity and population structure of sweet potato accessions cultivated in China, and to establish the genetic relationships among their germplasm types, a representative collection of 240 accessions was analyzed using inter-simple sequence repeat （ISSR） markers. The mean genetic similarity coefifcient, Nei’s gene diversity, and shared allele distance of tested sweet potato accessions were 0.7302, 0.3167 and 0.2698, respectively. The 240 accessions could be divided into six subgroups and ifve subpopulations based on neighbor-joining （NJ） clustering and STRUCTURE results, and obvious genetic relationships among the tested sweet potato accessions were identiifed. The marker-based NJ clustering and population structure showed no distinct assignment pattern corresponding to lfesh color or geographical ecotype of the tested sweet potato germplasm. Analysis of molecular variance （AMOVA） revealed small but signiifcant difference between white and orange-lfeshed sweet potato accessions. Small but signiifcant difference were also observed among sweet potato accessions from the Southern summer-autumn sweet potato region, the Yellow River Basin spring and summer sweet potato region and the Yangtze River Basin summer sweet potato region. This study demonstrates that genetic diversity in the tested sweet potato germplasm collection in China is lower than that in some reported sweet potato germplasm collections from other regions. Pedigree investigations suggest that more diverse Chinese sweet potato varieties should be formed by broadening the selection scope of breeding parents and incorporating the introduced varieties into future breeding programs.     

广东梅州柑橘黄龙病和病毒病的发生调查及其黄龙病菌原噬菌体多样性

【目的】调查柑橘黄龙病（Huanglongbing,HLB）和病毒病在广东省梅州市柑橘主产区的危害情况,分析该地区柑橘黄龙病菌（CLas）的原噬菌体多样性。【方法】以16S rDNA为模板设计引物,利用实时荧光定量PCR（qPCR)检测梅州市不同抽检地在2017—2019年3年间柑橘黄龙病的发病情况;同时分别采用已报道的柑橘衰退病毒（Citrus tristeza virus,CTV）、柑橘裂皮病毒（Citrus exocortis viroid,CEVd）、柑橘褪绿矮缩病毒（Citrus chlorotic dwarf-associated virus,CCDaV）、柑橘碎叶病毒（Citrus tatter leaf virus,CTLV）和柑橘黄脉病毒（Citrus yellow vein clearing virus,CYVCV）的特异性检测引物,提取样品的RNA,反转后以样品的cDNA为模板,通过普通PCR的方法分析梅州市抽检地区柑橘病毒病的发生情况。此外,以基于2种原噬菌体类型（SC1和SC2）对应的超变异基因区域设计的2对引物（Lap-TJ-F/Lap-TJ-R1和Lap-TJ-F/Lap-TJ-R2）,运用普通PCR方法分析该地区CLas菌株原噬菌体的遗传多样性。【结果】除2018年9月梅州大浦顺兴公司蜜柚基地的抽检样品外,梅州市其他被检地区均发现CLas。总体来说,梅州市的脐橙CLas检出率为55.3%,蜜柚为61.5%,沙田柚为61.7%。其中2017年采集自平原县大拓镇的蜜柚和沙田柚样品CLas检出率为100%,兴宁市白马镇的蜜柚为100%,沙田柚为80%;其他抽检地区的样品CLas检出率在16.7%—83.3%。虽然梅州市部分地区的检出率较高,但其病原菌的含量并不高,大部分处于柑橘黄龙病发病初、中期,属可防可控阶段;此外,梅州市柑橘病毒的总检出率不高,CTV、CTLV和CYVCV为梅州的主要病毒,CEVd和CCDaV没有检测到。CTV、CTLV和CYVCV在脐橙上的检出率依次为78.9%、7.9%和21.1%,蜜柚为15.4%、25.0%和9.6%,沙田柚为6.4%、2.1%和4.3%。基于Lap-TJ-F/Lap-TJ-R1和Lap-TJ-F/Lap-TJ-R2来研究CLas原噬菌体多样性的PCR扩增条带共有4种类型（SC1-1、SC1-2、SC2-1和SC2-2）,经过PCR电泳和测序得出,来自梅州市脐橙和沙田柚的CLas菌株以SC2-1型为主,蜜柚上以SC1-1型为主。【结论】梅州柑橘产区的柑橘黄龙病目前属于可防可控阶段,其病原菌菌株的原噬菌体在不同品种上具有其独特性;由于在抽检时发现有柑橘病毒病的存在,因此在种植过程中需加强种苗监管,同时应采取有效措施对柑橘黄龙病和柑橘木虱（Diaphorina citri）进行绿色防控。     

Genetic Diversity and Global Distribution of Citrus tristeza virus （CTV） Strains

Citrus tristeza virus （CTV）, the most devastating viral pathogen in citrus, causes tremendous economic losses to citrus industry worldwide. The CTV isolates exhibit variable pathogenicities on their ho...     

抗柑橘衰退病毒基因工程研究进展

本文从选育和利用抗（耐）柑橘衰退病毒（Citrus tristeza virus,CTV）的柑橘品种和砧木、弱毒株交叉保护、转基因诱导的CTV抗性等方面综述抗柑橘衰退病毒基因工程研究进展,并就人工miRNA（artifical microRNA,amiRNA）介导抗性加以展望,旨在为更好地控制CTV危害提供借鉴。     

柑桔衰退病毒基因组学研究进展

柑桔衰退病毒(CTV)是柑桔普遍存在的一种重要病毒病害,因其自身基因变异和寄主的不同而产生不同的症状,给防治CTV带来一定困难.为防治柑桔衰退病,需要研究CTV本身的相关特征.从CTV的基因组学上的特征综述了国内外在CTV基因组学最新的科研进展.     

胡柚上柑橘衰退病毒的分子检测

胡柚是我国东部地区重要的柑桔栽培品种.近年来,一种以叶片黄化为主要症状的新病害在胡柚种植区暴发流行.通过血清学和分子生物学检测,研究首次从黄化的胡柚叶片中检测到柑橘衰退病毒.系统进化分析表明,分离的CTV CS-7分离物与P09.18、NZRB-TH30等分离物具有较近的进化关系,而与T30等强毒株系有较大的遗传距离,...     

江西柑橘地方品种资源及野生近缘种SSR分子标记

利用SSR分子标记方法研究了63个江西柑橘地方品种资源及野生近缘种的遗传多样性,用30对SSR引物,检测到374条等位基因,平均每对引物检测到等位基因3～28个,平均12．47个,平均信息量为0．805。用UPGMA方法可将63个试材划分为8个类群．聚类结果可以推断出崇义野生橘可能是江西柑橘的始祖,井冈野生橙可能是橘类和橙类的杂交种。     

Production of Transgenic Anliucheng Sweet Orange （Citrus sinensis Osbeck） with Xa21 Gene for Potential Canker Resistance

Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR （pattern recognition receptor） gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene （HPT） was introduced into Anliucheng sweet orange （Citrus sinensis Osbeck） via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin （HYG） and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identiifed by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was brielfy discussed.     

柑桔特殊种质武夷橙遗传背景的研究

福建柑桔特殊种质武夷橙，俗称“四不象”．其性状特别，如果实扁圆，囊瓣隐约可辨，果皮紧，不易剥离，实心，叶片无翼叶，其亲缘关系不明．采用传统的外部形态与内部结构的观测、花粉形态、同工酶和核型分析相结合的方法进行研究，结果认为武夷橙不是雪柑与福桔或其它柑桔类的天然杂种，而是雪柑珠心胚的变异株系．对武夷橙遗传背景及其亲缘关系的研究，在柑桔遗传育种上具有重要理论与实践的意义     

广西柑桔种质资源的AFLP分析

广西是柑桔原产地之一,蕴藏着丰富的柑桔种质资源.选用8对AFLP引物组合,对42份广西地方柑桔资源和广西柑桔主栽品种进行遗传多样性分析及分类研究.共获得清晰可见标记377条,其中多态性标记245条,占65.0％.在遗传相似系数0.92处,可以将柑桔品种分为柚、橙、柑、桔、金柑5个类群.供试材料两两间遗传相似系数在0.75～1之间.根据遗传相似系数( UPGMA,N-J)得到的分类树状图与传统方法的分类结果相似.研究结果表明广西柑桔种质资源具有较高的遗传多样性,广西柚类品种两两间遗传相似系数在0.93 ～0.97之间,广西酸桔品种间亲缘关系较近.     

基于cpInDel标记和cpSSR标记的柑橘属及近缘属植物亲缘关系

【目的】通过对柑橘叶绿体基因组变异位点的研究,从胞质层面揭示柑橘类果树的遗传进化,为柑橘种质资源的收集、评价和利用提供参考。【方法】以甜橙叶绿体高变区序列为参考,利用已发表的二代基因组测序数据对代表性的柑橘属及其近缘属材料进行De Novo组装,获得叶绿体基因组高变区组装序列,通过序列比对,发掘代表性材料的差异位点,针对差异位点设计cpInDel引物。对甜橙叶绿体基因组两个及以上核苷酸重复构成的SSR位点进行定位分析,找出代表性材料间有差异的位点,针对差异位点设计cpSSR引物。选择在分类学和/或起源研究上有代表性的柑橘属及近缘属材料48份,利用新开发的cpInDel、cpSSR标记和已报道的cpSSR标记进行扩增检测、电泳谱带分析和聚类分析。【结果】本研究新开发4个cpInDel标记和13个cpSSR标记。扩增检测显示,这些标记在48份试验材料中均能扩增出较为清晰的多态性条带;但cpInDel标记扩增出的条带更为单一,类群区分时,仅通过单个标记便能实现对某一类群或几个类群的区分,具有更好的区分效果。聚类分析显示,cpInDel和cpSSR标记得到了比较类似的结果,但在小类群关系以及部分材料的归类上存在差异。二者均揭示枸橼类柠檬-藜檬-来檬杂种的胞质来源并非香橼,枳杂种的胞质也并非枳,显示宽皮柑橘类黄柑的代表性品种‘旺苍皱皮柑’有不同于宽皮柑橘类其他品种的胞质来源,‘资阳香橙’和‘韩国香橙’胞质差异明显。针对宽皮柑橘类、甜橙-酸橙类和宜昌橙-大翼橙类3个小类群的相互关系,cpInDel显示它们关系较近,而cpSSR则将它们归为独立的类群;cpInDel标记将印度野橘归于真正的枸橼类,cpSSR将其单独聚为一类。【结论】叶绿体基因组分子标记分析能从胞质角度揭示不同于核基因组的柑橘属及近缘属植物的亲缘关系,但若与核基因组分子标记分析相结合,能更全面地揭示材料间的亲缘关系,更准确地鉴定柑橘种质资源。     

The Mining of Citrus EST-SNP and Its Application in Cultivar Discrimination

Single nucleotide polymorphisms （SNPs） are the most abundant sequence variations found in plant genomes and are widely used as molecular genetic markers in cultivar identification and genetic diversity studies. The objective of this study was to identify SNP markers useful for discrimination of citrus cultivars, since large numbers of expressed sequence tags （ESTs） of sweet orange are available from the National Center for Biotechnology Information （NCBI）. We now have the opportunity to discover SNP markers suitable for determining the haplotypes with which to distinguish very closely related cultivars and to assess genetic diversity within or between related species of citrus. SNPs and small insertions/deletions （Indels） from ESTs of sweet orange and satsuma were identified by the in silico SNP discovery strategy. 55 296 EST sequences of sweet orange and 2 575 of satsuma retrieved from the NCBI repository were mined for potential SNPs. Cleaved amplified polymorphic sequences （CAPS） and sequencing approaches were used to validate putative SNPs in a sample of 30 citrus accessions. A total of 3 348 putative SNPs were identified based on the abundance of sequences and haplotype cosegregation. Of these 3 348 SNPs, the transitions, transversions and Indels ratios were 47.9, 36.1 and 16.0%, respectively. The SNPs occurred on average at a frequency of 1 per 164 bp in the coding region of citrus. 14 SNPs were randomly selected and genotyped according to 30 citrus accessions including 23 accessions of sweet orange; 11 SNPs displayed polymorphism with an average polymorphism information content （PIC） of 0.20 among 30 citrus accessions. The genetic diversity present in sweet orange was low, so the 14 SNP markers failed to discriminate different cultivars of sweet orange, but they did succeed in distinguishing accessions of inter-species of citrus. In this study, SNPs were mined from EST sequences of sweet orange and satsuma, which displayed potential capability as molecular markers to discriminate inter-species accessions of citrus. It is anticipated that these putative SNPs could be applied in citrus genetics research and breeding.     

野生‘龙门香橙’的植物学特征观察及起源研究

【目的】广东省龙门县境内南昆山主峰天堂顶海拔1 000 m处有当地称之为‘龙门香橙’的野生柑橘,论文拟从形态学和DNA分子证据上探讨其与已知柑橘资源的异同,为合理利用该资源奠定基础。【方法】实地调查‘龙门香橙’的生长环境,观察记录叶、花、果实的形态特征。分析果实的可溶性固形物、糖、酸和抗坏血酸含量。在电子显微镜下观察花粉的显微特征。对比分析包括‘龙门香橙’在内的24个柑橘资源的AFLP图谱以及10个资源的8个SNP位点的基因型。克隆、测序分析‘龙门香橙’的β-胡萝卜素羟化酶（β-carotene hydroxylase,CHX）基因的2个等位基因,并与其他柑橘材料的CHX基因序列进行比对。【结果】生境调查结果表明‘龙门香橙’非人工种植,是真正的野生柑橘。其果实、种子的形状和单胚等特征与柚类相似,但果实与柚相比明显偏小、出汁率与可食率很低、味苦等性状提示其比较原始。叶片形状、线形翼叶、花丝联合等特征类似宽皮柑橘类,比较进化;但花丝和新叶染有紫红色等性状则比较原始。基于AFLP图谱的聚类分析显示其不与其他研究材料聚类,但介乎橘柚之间。8个SNP分子标记分析结果显示,‘龙门香橙’的3个SNP位点为宽皮柑橘纯合基因型,1个位点为橘柚杂合基因型,其余4个位点为柚子纯合基因型,这种基因型组合不同于其他任何所分析的材料。‘龙门香橙’的CHX基因的核苷酸序列显示其2个等位基因间相互差异较小,且含有其他已知柑橘CHX基因上未发现的SNP。CHX基因序列进化分析结果表明其与其他柑橘的遗传距离较远、差异似达种间水平。【结论】‘龙门香橙’是一个真正的野生柑橘资源。其形态性状兼具原始与进化特征。在DNA分子水平上,‘龙门香橙’与已知柑橘不同,甚为独特。     

Virulence and Diversity of Blumeria graminis f. sp. tritici Populations in China

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important disease in China. To characterize the virulence and diversity of the pathogen, 1 082 isolates were obtained from 8 major wheat-growing regions during the spring growing season in 2011. The virulence test was performed by inoculation on detached leaves of 22 differential lines with known Pm genes. Frequencies of virulence on these genotypes ranged from 0 to 97.4%. None of the 1 082 isolates was compatible to Pm21 and less than 20.0%were virulent to the genotype carrying Pm13. In contrast, the virulence frequencies of each population was more than 50.0%to differentials carrying Pm1a, Pm3b, Pm3c, Pm3f, Pm5a, Pm6 and Pm8. In total, 1 028 pathotypes were detected, of which 984 were unique. Phenotypic diversity indices revealed a high level of diversity within populations. Genetic distance between different populations correlated signiifcantly with geographical distance （R2=0.494, P 0.001）. In addition, isolates from Xinjiang appear to form a separate group. Signiifcant positive or negative associations between alleles at pairs of virulence loci were detected in 57 allele pairs to Pm genes. Virulence and diversity of the 8 populations suggested that varieties with effective resistance gene combinations should be developed at a regional level.     

柑橘类植物SOD基因片段的克隆和SNP分析

[目的]对柑橘类植物遗传多样性进行精确分析研究。[方法]根据已报道的各种植物Mn/Fe-SOD基因的氨基酸序列上前后2个保守区设计1对简并引物,采用RT-PCR技术,分别从岭南沙田柚、柠檬等6种柑橘类植物中克隆得到长426bp的Mn/Fe-SOD基因片段,并对所得序列进行了SNP分析。[结果]柑橘类植物核苷酸同源性高达97.2%,与其他植物Mn/Fe-SOD基因核苷酸序列的同源性都在77.7%以上;经过氨基酸序列推导,每个基因片段分别编码142个氨基酸,6个基因片段的氨基酸同源性为97.9%,与其他植物Mn/Fe-SOD基因的氨基酸序列同源性在79.0%以上。在Mn/Fe-SOD基因片段中有18个核苷酸位点存在SNPs,导致3个氨基酸位点发生变异,多态性频率为1SNP/23.7bp,核苷酸变异度为4.23%,氨基酸变异度为2.38%。[结论]该研究为丰富构建柑橘类植物基因图谱标记和抗逆性状的遗传标记奠定了基础。