Screening and biochemical characterisation of resistant and susceptible citrus species against dry root rot disease (Fusarium solani)

Fourteen citrus species were screened for their resistance against dry root rot under artificial inoculation conditions and classified as resistant (RHRL-122, RHRL-124, Australian sour, Sour dig, Balaji, Rangpurlime), moderately resistant (PKM-1, AL-Srirampur, Rough lemon), Susceptible (TAL 95/1, TAL 95/2, TAL 95/3, Nalgonda selection) and highly susceptible (TAL 94/13). The higher polyphenol oxidase (PPO) activity was observed in all infected leaves and roots of citrus species when compared to healthy leaves and roots at 15 and 30 days after inoculation (DAI). Higher PPO activity was observed in Balaji, Australian sour and Rangpurlime whereas lowest PPO activity was observed in TAL 94/13. In the case of leaf Peroxidase (PEO) isozyme profile an additional band which was darker and thicker was observed at an Em value of 0.24 in the case of Australia sour, Balaji and AL-Srirampur. In roots the PEO isozyme profile has the induction of single thick and darker additional band with an Em value of 0.47 was observed in Australian sour and Balaji. The banding profiles of estarase in leaves showed the induction of an extra band in the ase of Australian sour at Em 0.1, and at Em 0.53 in the case of Rangpurlime and Sourdig when compared to other species. The banding profile of esterase in roots was well expressed in Australian sour, PKM-1, Rough lemon, TAL-95/3, Rangpurlime and Sour dig. However, a minor band at Em 0.27 in Australian sour, TAL 95/1 and at Em 0.33 in Balaji was observed.     

分子标记鉴定常山胡柚优良基因型的初步研究

本研究利用RAPD和ISSR分子标记对常山胡柚的优良基因型进行鉴定,并探讨常山胡柚的起源。从100个RAPD引物中筛选出12个多态性引物用于正式扩增,共得到117条DNA带,其中多态性DNA带64条,占扩增片段的54．7%;从105个ISSR引物中筛选出11个多态性引物用于正式扩增,共得到94条DNA带,其中多态性DNA带58条,占扩增片段的61．7%。RAPD和ISSR分析揭示了常山胡柚及其近缘种的一些特异性条带。ISSR共产生了15条特异条带,RAPD共产生12特异性条带。实验数据用AMOVA软件计算遗传距离,用NTSYS-pc软件构建UPGMA聚类树状图。结果显示,所有的基因型及不同种之间均能够彼此区分,分析得到的指纹图谱对常山胡柚种和基因型的鉴定具有潜在的应用价值,可用于优良基因型的鉴定。聚类分析结果显示常山胡柚和甜柚聚为一枝,确定了甜柚是杂交亲本之一,但是常山胡柚和柚的遗传距离较远,说明常山胡柚可能是甜橙、柚和柑桔属其他种的多重自然杂交的结果。     

采用RAPD和ISSR标记探讨中国疣粒野生稻的遗传多样性

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) methods were applied to detect genetic variation of 20 bulking samples and two individually sampled populations of Oryza granulata (Nees et Arn. ex Watt.) from China (M5 from Hainan Province and M27 from Yunnan Province). For the bulking sampled populations, 20 RAPD and 12 ISSR primers generated 209 and 122 bands, of which 134 (64.11%) and 89 (72.95%) were polymorphic respectively. For population M5, 146 and 95 bands were generated with 21.48% and 34.78% being polymorphic (PPB). For population M27, 151 (PPB = 17.22%) and 94 (PPB = 19.15%) bands of RAPD and ISSR were obtained. The results indicated that the level of genetic variation of O． granulata was lower than other species detected in the genus Oryza. UPGMA cluster based on genetic similarity and principle component analyses (PCA) based on band patterns divided the populations into two groups corresponding to their sources, which revealed that the majority of genetic variation of O. granulata occurred between Hainan and Yunnan. Consequently, more populations of the species should be considered for a reasonable conservation management. Comparison between the two marker systems showed that ISSR was better than RAPD in terms of reproducibility and ability of detecting genetic polymorphism. Mantel test revealed that the goodness of fit between them was significant (r=0.917, t=12.718) when detecting genetic diversity at species level, but was poor at population level (r<0.200). The discrepancy was considered as the facts that different fragments were targeted by RAPD and ISSR, and that evolutionary process of O. granulata varied at different hierarchical levels.     

Intraspecific variability of Bipolaris sorokiniana isolates determined by random-amplified polymorphic DNA (RAPD)

Isolates of Bipolaris sorokiniana were analyzed by random-amplified polymorphic DNA (RAPD) techniques to determine the amount of intraspecific genetic variability and to study host-pathogen interactions. Ten isolates originated from different regions of Brazil were examined. Plants of the wheat cultivars BR8, BH1146 (original host) and IAC-5 Maringá, classified as resistant, moderately resistant or susceptible to B. sorokiniana, respectively, were inoculated with these 10 isolates. Twenty-seven isolates were recovered from these cultivars and were analyzed by RAPD assay and compared to the RAPD of the original 10 isolates. According to the RAPD profiles there was a high level of genetic variability among the isolates. We detected 69 polymorphic fragments, ranging from 1.6 to 0.54 kb, in the original 10 isolates; 57 fragments with sizes between 1.98 and 0.38 kb from the isolates recovered from BH1146; 47 polymorphic bands, ranging from 1.96-0.54 kb, were detected in the isolates from BR8 and 32 fragments between 1.98 and 0.42 kb in isolates were recovered from IAC-5 Maringá. The number of polymorphic fragments varied, even for the same isolate, when the isolates were recovered from different cultivar hosts.     

Analysis of genetic heterogeneity among five gynogenetic clones of silver crucian carp, Carassius auratus gibelio Bloch, based on detection of RAPD molecular markers

The gynogenetic silver crucian carp, Carassius auratus gibelio, is a unique model system for studying evolutionary genetics and selective breeding, owing to its specific genetic background and reproductive modes. Five gynogenetic clones were analyzed by the random amplified polymorphic DNA (RAPD) technique, using 30 10-nucleotide-long primers. Twenty-six primers produced well-amplified DNA fragments with reproducible banding patterns, and 24 primers were polymorphic. Nearly identical banding patterns were observed among individuals within each clone, suggesting that each clone might possess a specific pattern owing to its gynogenesis. In contrast, the RAPD patterns of the five clones differed from each other. A phylogenetic tree was constructed using UPGMA cluster analysis based on a total of 3,744 distinguishable fragments (156 per individual). Average genetic distances within and among the five clones clearly indicated their intraclonal homogeneity, interclonal heterogeneity, and phylogenetic relationships. Clones A and P were the most closely related, whereas the most divergence was seen between clone D and clone E or F. A total of 88 polymorphic fragments were scored from 24 primers after excluding bands that were monomorphic for the five clones. Most primers corresponding to the polymorphic fragments amplified reproducible markers specific for one clone or that were shared by two, three, or four clones. Several primers (e.g., Opj-1, Opj-7, and Opp-10) produced abundant banding patterns that could be used to discriminate between the five clones. Markers specific for one or two clones were also identified. The RAPD markers identified in this study will likely benefit evolutionary genetics and selective breeding studies.     

用RAPD和ISSR检测的橡胶树野生种质和栽培品种的遗传多样性

用19个RAPD引物和12个ISSR引物对14份野牛橡胶树种质和我国的37份栽培品种进行了遗传多样性分析。RAPD引物共产生132条带,多态性带占88．6％,相似系数变化范围在0．432—0．947。ISSR引物其产生101条带,多态性带占87．1％,相似系数为0．505—0．941。平均基因杂合度分析表明野生种质比栽培品种具有较高的遗传多样性。根据UPGMA法对51份材料进行聚类分析,结果表明,ISSR分析中所有材料可分为2类：第一类为野生种质,第二类为栽培品种：而RAPD分析中野牛种质和栽培品种不能被分为明显的两人类。虽然ISSR和RAPD的聚类分析结果存在差异,但对两种方法进行的相关分析表明,他们之间仍存在极显著相关性,相关系数为0．574。品种PR107、热研217等一些栽培品种可以通过特异带在51份供试材料中被区分开。这些结果可以对橡胶树的育种上作起到一定的指导作用,同时RAPD和ISSR技术也是进行橡胶树品种鉴定和遗传多样性研究的有效手段。     

Chemical and genetic variability of selected Turkish oregano (Origanum onites L.) clones

Turkish oregano (Origanum onites L.) is a major aromatic plant that belongs to Labiatae family. In this work fourteen Turkish oregano clones have been characterized chemically and genetically. Essential oil obtained by hydro-distillation was characterized by gas chromatography. Genetic variation was determined by use of random amplified polymorphic DNA (RAPD) markers. Variation of essential oils in the selected clones was subjected to cluster analysis, and two chemotypes, carvacrol and thymol, were identified. Carvacrol was found to be the main component in all clones except clone-661, the main component of which was thymol. The oregano clones were divided into three main groups by clustering on the basis of RAPD markers. Genetic similarity values among the oregano clones ranged between 0.49 and 0.73 which was indicative of a low level of genetic variation. Clones originating from locations close to each other had similar RAPD markers. Correlation analysis of the genetic distance matrix and the Euclidian distance matrix revealed no significant correlation between them. The results also indicated that there is no relationship between genetic structure of the selected clones and essential oil composition.     

Molecular Analysis of Rhynchosporium secalis Population Collected from Barley Cultivars having Different Resistance Specificities

Molecular analysis was performed to detect genetic diversity in 106 Rhynchosporium secalis isolates collected from different regions of Canada using random amplified polymorphic DNA (RAPD) markers. The isolates collected from barley cultivars having different resistance specificity to R. secalis and grown in geographically distinct regions, exhibited reproducible variation for 2–3 polymorphic PCR products per decamer primer. Analysis of 1960 RAPD markers data obtained with five primers formed 5 groups with different genetic similarity. High genetic variation was observed in R. secalis isolates obtained from resistant and susceptible cultivars of barley. Isolates collected from susceptible cultivars showed a tendency to group together, whereas isolates from resistant cultivars were divergent. R. secalis isolates infecting different barley cultivars released as resistant to the barley scald formed a specific group with UPGMA, even though all these isolates were collected from the same epidemiological region. Analysis of 15 isolates collected from one resistant cultivar Duke formed three clusters with low bootstrap values indicating high genetic diversity among the isolates present on a single host cultivar.     

羊肚菌栽培菌株遗传多样性分析及种特异性RAPD-SCAR标记开发

近年来中国的羊肚菌Morchella spp.栽培技术取得了长足进步,但基础研究薄弱影响其稳产和高产,国内外尚无羊肚菌栽培菌株种质资源遗传多样性的研究报道。本文对来自全国12省份的36个羊肚菌栽培菌株进行了ITS系统发育分析,并采用RAPD进行了遗传多样性评价。结果表明,结合有效的参考菌株序列,通过ITS序列分析可以将供试菌株进行区分和鉴定,在36个菌株中,26个菌株属于梯棱羊肚菌Morchella importuna,其他10个菌株属于六妹羊肚菌M. sextelata;将自40条RAPD引物中筛选出的14条用于供试菌株遗传多样性分析,共扩增出124条多态性条带;UPGMA聚类可将供试菌株分为两大类群,分别对应于ITS系统发育分析中的梯棱羊肚菌和六妹羊肚菌两个物种,梯棱羊肚菌种内菌株多态性高于六妹羊肚菌。OPA17引物和OPA18引物分别在AA02和AA15菌株中扩增出具有唯一性的特征条带,对两个特征条带进行回收测序后,设计出两个特异性SCAR的引物,它们能有效地从36个供试菌株群体中将菌株AA02和AA15鉴别出来。本文首次全面系统地采用ITS分析鉴别了我国羊肚菌栽培菌株的种性,采用RAPD分子标记系统地评价了羊肚菌栽培菌株的遗传多样性,并验证了RAPD分子标记转化为菌株特征性SCAR标记的可行性。     

RAPD-based genetic linkage maps of Tribolium castaneum.

A genetic map of the red flour beetle (Tribolium castaneum) integrating molecular with morphological markers was constructed using a backcross population of 147 siblings. The map defines 10 linkage groups (LGs), presumably corresponding to the 10 chromosomes, and consists of 122 randomly amplified polymorphic DNA (RAPD) markers, six molecular markers representing identified genes, and five morphological markers. The total map length is 570 cM, giving an average marker resolution of 4.3 cM. The average physical distance per genetic distance was estimated at 350 kb/cM. A cluster of loci showing distorted segregation was detected on LG9. The process of converting RAPD markers to sequence-tagged site markers was initiated: 18 RAPD markers were cloned and sequenced, and single-strand conformational polymorphisms were identified for 4 of the 18. The map positions of all 4 coincided with those of the parent RAPD markers.     

辣椒种质遗传多样性的RAPD和ISSR及其表型数据分析

用RAPDI、SSR分子标记及28个表型性状数据对辣椒属5个栽培种的13份材料进行了分析,结果表明:23条RAPD引物共扩增出209条带,平均每个引物扩增出9.09条,多态性位点比率为83.73%;16条ISSR引物共扩增出94条带,平均每个引物扩增出5.88条,多态性位点比率为79.79%.与RAPD相比,ISSR标记检测到的有效等位基因数(Ne)及Shannon多样性指数(I)、遗传离散度(Ht)和遗传分化系数(Gst)等遗传多样性参数都较大,多态性位点比例在亲缘关系较近的一年生辣椒(Capsicum annuum)种内较高,说明ISSR有更高的多态性检测效率,并且适合亲缘关系较近的种群间遗传多样性分析.基于RAPDI、SSR的聚类与基于表型数据的聚类之间存在极显著正相关,且都能将C.annuum与其它栽培种区分开来.     

WHBE 兔、日本大耳白兔和新西兰兔遗传多样性的 RAPD 分析

目的：应用随机引物扩增多态性DNA技术（ random amplified polymorphic DNA , RAPD）对大耳白黑眼兔（ white hair black eyes rabbit , WHBE rabbit ）、日本大耳白兔（ Japanese white rabbit , JW rabbit ）和新西兰兔（New Zealand white rabbit, NZW rabbit）3个实验兔品系进行遗传分析。方法选用90只实验兔的皮肤组织样品提取基因组DNA,用60个随机引物对实验兔基因组DNA进行PCR扩增,根据电泳结果筛选出多态性较高的引物进行RAPD-PCR分析,再利用Popgene 3．2统计软件对3个品系的扩增条带进行遗传分析,获得实验数据。结果分析结果表明：（1）60个随机引物中筛选出25个多态性较高的引物,3个品系实验兔共检测到493个扩增片段,长度在100～1800 bp之间,筛选的25个引物中,其中16个引物既可扩增出3个品系共同的DNA条带,也可扩增出WHBE兔特有的特征条带;（2） WHBE兔位点数为234个,其中多态位点数166个,多态位点比为70．94％,JW兔位点数为228个,其中多态位点数122个,多态位点比为53．51％,NZW兔位点数为231个,其中多态位点数94个,多态位点比为40．69％;（3）三个群体的Shannon多样性指数分别为0．3385,0．2222和0．1905;（4） JW兔和NZW兔的遗传相似系数最高,为0．8443,其次为WHBE兔和JW兔的遗传相似系数,为0．8204,WHBE兔和NZW兔的遗传相似系数最低,为0．7862。结论结果表明WHBE兔与JW兔和NZW兔之间有遗传的相似性,也存在着遗传差异,应用RAPD技术可以很好地检测实验兔不同品系之间以及同一品系不同个体之间的亲缘关系。     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

Phylogenetic analysis of the polymorphic 4× species complex Lonicera caerulea (Caprifoliaceae) using RAPD markers and noncoding chloroplast DNA sequences

The blue-berried honeysuckle (Lonicera caerulea L.) is one of the most representative species of the genus Lonicera L. in horticulture. This article presents the results of research on the taxonomy of blue-fruited honeysuckles, which is quite complicated due to the phenotypic plasticity, ability to hybridize and distribution across different ecological zones. We used the random amplified polymorphic DNA (RAPD) markers and sequencing of seven chloroplast DNA (cpDNA) regions (trnH-psbA, rpS12-rpL20, trnL-trnF, trnS-trnG, trnG, rpS16 and trnS-psbZ) to assess the phylogenetic relationships among the taxa within the polymorphic 4 × species complex L. caerulea and to determine the position of Lonicera boczkarnikowae Plekh. and Lonicera venulosa Maxim. within this complex. Lonicera chrysantha Turcz. ex Ledeb., L. orientalis Lam. and L. xylosteum L. were used as the outgroup species. The RAPD and cpDNA analyses both indicated that all of the studied taxa of the blue-fruited honeysuckle form a single cluster consisting of two subclusters. A second cluster includes the outgroup species. According to the cpDNA analysis, L. boczkarnikowae and L. venulosa belong to the subcluster that includes the taxa of the polymorphic tetraploid complex L. caerulea. A separate subcluster within the cluster of blue-fruited honeysuckles contains L. altaica and L. edulis.     

应用RAPD标记研究野生荔枝种质资源

应用RAPD方法对海南60份野生荔枝进行基因组多态性分析,20个随机引物共产生165条RAPD带,DNA片段大小在200～1500bp之间,其中121条为多态性带,占总带数的73．3％,并利用遗传距离进行聚类分析。结果表明,60份野生荔枝可归为6类,表明种群存在一定的遗传变异,也为分析野生荔枝群体间及群体内的遗传多样性探索了有效方法。     

Evaluation of the genetic fidelity of in vitro-propagated gerbera (<Emphasis Type="Italic">Gerbera jamesonii</Emphasis> Bolus) using DNA-based markers

The genetic fidelity of in vitro-raised gerbera clones was assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 35 RAPD and 32 ISSR primers screened, only 12 RAPD and 10 ISSR primers produced clear, reproducible and scorable bands. The 12 RAPD primers produced 54 distinct and scorable bands, with an average of 4.5 bands per primer. The number of scorable bands for ISSR primers varied from 3 (ISSR-14) to 9 (ISSR-07), with an average of 5.5 bands per primer. The number of bands generated per primer was greater in ISSR than RAPD. All banding profiles from micropropagated plants were monomorphic and similar to those of the mother plant. A similarity matrix based on Jaccard’s coefficient revealed that the pair-wise value between the mother and the in vitro-raised plantlets was 1, indicating 100% similarity. This confirmed the true-to-type nature of the in vitro-raised clones.     

Characterization of fusarium wilt-resistant and fusarium wilt-susceptible somaclones of banana cultivar rastali (Musa AAB) by random amplified polymorphic DNA and retrotransposon markers

Two DNA fingerprinting techniques, random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP), were used to characterize somaclonal variants of banana. IRAP primers were designed on the basis of repetitive and genome-wide dispersed long terminal repeat (LTR) retrotransposon families for assessing the somaclonal variation in 2Musa clones resistant and susceptible toFusarium oxysporum f. sp.cubense race 4. RAPD markers successfully detected genetic variation within and between individuals of the clones. IRAP makers amplified either by a single primer or a combination of primers based on LTR orientation successfully amplified different retrotransposons dispersed in theMusa genome and detected new events of insertions. RAPD markers proved more polymorphic than IRAP markers. Somaclonal variation seems to be the result of numerous indels occurring genome-wide accompanied by the activation of retroelements, as a result of stress caused by micropropagation. It is concluded that characterization of the somaclonal variants requires more than one DNA marker system to detect variation in diverse components of the genome.     

与小麦白粉病抗性基因Pm2紧密连锁RAPD标记的筛选研究

以２５６个随机引物对含小麦抗白粉病基因Ｐｍ２近等基因系进行ＲＡＰＤ分析,发现１７个随机引物的扩增产物在抗、感ＮＩＬｓ材料间表现多态性,且其中５个引物经４次以上重复,均获相同结果,其多态性标记分别为ＯＰＭ０８(１６００)、ＯＰＩ０４(１７００)、ＯＰＨ１９(１１００、ＯＰＥ０９(９００)及ＯＰＭ１６(８５０)。当以这５个随机引物对１４个已知含Ｐｍ２基因的抗病材料及９个不含Ｐｍ２基因的感病材料进行检测时,只有标记ＯＰＩ０４(１７００)在１２个抗病材料中出现（另两个抗病材料中未检测到）,而在９个感病材料中均未出现。进一步用 ＯＰI(０４)对１０２株（Ｃｈａｎｃｅｌｌｏｒｘ Ｕｋａ／８＊Ｃｃ）Ｆ２分离群体进行分析,估算出标记ＯＰＩ０４(１７００)与Ｐｍ２基因间的遗传距离为１２．２±３．３ｃＭ。     

Correspondence of ISSR and RAPD markers for comparative analysis of genetic diversity among different apricot genotypes from cold arid deserts of trans-Himalayas

The phylogenetic relationships of 36 locally grown Prunus armeniaca genotypes which are collected from nine sampling sites from two valleys viz. Nubra (9,600 ft) and Leh (11,500 ft) of trans-Himalayan region were analyzed using 31 PCR markers (20 RAPDs and 11 ISSRs). This is the first report of molecular genetic diversity studies in apricot from this region of the world. RAPD analysis yielded 139 fragments, of which 136 were polymorphic, with an average of 6.8 polymorphic fragments per primer. ISSR analysis produced 58 bands, of which 56 were polymorphic, with an average of 5.09 polymorphic fragments per primer. The primers based on (CT)n produced maximum number of bands (nine) while, (AT)n and many other motifs gave no amplification. RAPD markers were found more efficient with regards to polymorphism detection, as they detected 97.84 % as compared to 96.5 % for ISSR markers. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in RAPD and combined data of RAPD + ISSR. The results of PCA analysis were comparable to the cluster analysis. These analyses, allowed us to identify the groups corresponding to the two apricot collection sites.Key words: Prunus armeniaca, Apricot, Genetic Diversity, RAPD, ISSR, AMOVA