RAPD polymorphisms among variant and phenotypically normal rice (Oryza sativa var.indica) somaclonal progenies

Summary RAPD analysis was performed among eight rice somaclonal families known to vary for specific characters and four somaclonal families which were phenotypically normal. The parental cultivar,indica rice cv. FR13A, was found to be homogeneous and homozygous at all but one of the 45 RAPD loci. Polymorphisms were found at 28 of the 45 bands among the somaclonal families, including both loss of parental bands, and the appearance of novel non-parental bands. Segregation data revealed both heterozygous and homozygous mutation events, with recessive mutations more prevalent than dominant. All somaclonal families differed significantly from the parental material, indicating that genomic alterations occurred in all families regardless of phenotype. None of the variant families could be regarded as isogenic lines of FR13A at the DNA level. However, some of the DNA level variation may be in highly repeated sequences with no phenotypic effects. The implications for somaclonal breeding and genetic engineering programs are discussed.     

Comparative assessment of variation in the USA Arachis pintoi (Krap. and Greg.) germplasm collection using RAPD profiling and tissue culture regeneration ability

Arachis pintoi accessions were used to study genetic diversity using RAPD markers. Concurrently, two tissue culture protocols were evaluated for organogenesis and the capacity to generate somaclonal variation. Data were collected on callus growth, callus weight gain, and number of regenerated plants. Robust RAPD profiles were obtained and eight primers amplified 100 different bands with 98% polymorphisms. The proportion of polymorphic RAPD loci was 89%. Average genetic distance was 0.36 and indicated that a large amount of genetic diversity exists within the germplasm evaluated. Genetic distances were used to prepare a dendogram for the A. pintoi accessions that separated them into four groups. A large degree of variability for callus induction and callus weight gain was observed among the accessions. Shoot regeneration was achieved for several accessions on both media with no structures indicative of somatic embryogenesis detected. Root induction was difficult to obtain, and many shoots died during this process. RAPD band profiles of regenerated tissue culture plants were similar to their parent plants, and therefore no somaclonal variation was evident using these methods.     

Analyzing somaclonal variation in micropropagated bananas (<Emphasis Type="Italic">Musa</Emphasis> spp.)

Summary In a micropropagation program, where it is of paramount importance to produce true-to-type planting material, somaclonal variation of any kind is undesirable. Variation among plants regenerated from tissue culture is termed ‘somaclonal variation’. In banana, somaclonal variants of different type have been reported with regard to plant morphology. This article discusses various factors due to which somaclonal variations may arise. Somaclonal variation may be detected by visual screening or by using molecular markers such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and by cytological studies. Although somaclonal variation is undesirable in the context of micropropagation, it can be used to advantage for genetic improvement of banana, as has been described.     

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.     

Selection and Analysis of Polymorphisms in Somaclonal Variants of <i>Agave americana</i> Resistant to <i>Fusarium oxysporum</i> via an Ethyl Methanesulphonate Treatment

Agave americana L. callus were exposed to different concentrations of ethyl methanesulphonate (EMS) 0, 15, 30, 45 and 60 mM and to different times of exposure (2 and 4 h). The viability and capacity of shoot formation were shown to be affected when the callus were exposed to high concentrations (30–60 mM). Only the callus exposed to 15 mM EMS presented shoot formation; the exposure time of two hours produced the largest quantity of shoots regenerated per callus (21 shoots/callus). In order to generate somaclonal variants resistant to Fusarium oxysporum, a selection pressure was applied through of a culture filtrate (CF) of 100 ppm of the fungus. This was made in callus obtained in the treatment with 15 mM EMS during 2 h of exposure. The CF caused oxidation and necrosis in 71.25% of the callus; however, they were capable of generating shoots (3.5 shoots/callus). Molecular markers type RAPD, ISSR and DAMD were used to evaluate the genetic variation arising from the mutations caused by EMS on control plants and 16-month-old somaclonal variants. The polymorphic information content (PIC) for each one of the initiating groups was: 0.28 (DAMD), 0.09 (ISSR) and 0.14 (RAPD). DAMD revealed a greater percentage of polymorphism than RAPD and ISSR. Polymorphic bands were detected in the somaclonal variants. This indicated that the EMS caused genetic variation in the regenerated plants conferring resistance to them against Fusarium oxysporum.     

水稻体细胞无性系变异

水稻体细胞无性系变异研究取得了很大进展,获得了大量抗病、抗逆、优质、矮杆等突变体。对这些突变体遗传分析表明,大多数突变性状由1对或2对基因控制。水稻体细胞无性系变异的发生与基因型、性状、继代时间、培养方式等有关,并具有内在的机制,点突变和反转录转座子插入可能是引起水稻无性系变异的两个重要原因。     

Somaclonal variation at the nucleotide sequence level in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) as revealed by RAPD and ISSR markers,and by pairwise sequence analysis

The nature of somaclonal variation at the nucleotide sequence level was studied in rice cv. Nipponbare. First, we investigated genomic variations by using 2 molecular marker systems: RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This was followed by sequencing of selected bands that represented genomic variations, and pairwise sequence analysis taking advantage of the whole genome sequence of rice. In addition, transpositional activity of the active MITE, mPing, was analysed by locus-specific PCR amplifications. The 2-year-old calli and their regenerated plants, analysed with 24 RAPD and 20 ISSR primers, showed moderate levels of genomic variation (20.83% and 17.04%, respectively). To test whether DNA methylation plays a role in somaclonal variation, the calli were treated with 5-azacytidine, a chemical agent that reduces cytosine methylation by blocking the activity of DNA methyltransferase. Though dwarfism occurred in regenerants from treated calli (a hallmark of the drug treatment), there was only a slight increase in the frequency of somaclonal variation detected in the treated calli and their regenerated plants relative to untreated controls. The transposon mPing also remained immobile in both treated and untreated calli. Nevertheless, dendrograms constructed according to the Jaccard coefficient calculated by UPGMA of the ISSR bands revealed that the 5-azacytidine-treated and untreated somaclones were grouped into 2 distinct clusters, suggesting a possible indirect effect of the treatment on the genomic changes, depending on the marker used. Sequence analysis indicated a low level of variation (0.31%), with single-base-pair substitutions predominating.     

Genetic stability of micropropagated almond plantlets,as assessed by RAPD and ISSR markers

Almond shoots produced by axillary branching from clone VII derived from a seedling of cultivar Boa Casta were evaluated for somaclonal variation using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) analysis. To verify genetic stability we compared RAPD and ISSR patterns of plantlets obtained after 4 and 6 years of in vitro multiplication. A total of 64 RAPD and 10 ISSR primers gave 326 distinct and reproducible band classes, monomorphic across all 22 plantlets analysed. Thus, a total of 7,172 bands were generated, exhibiting homogeneous RAPD and ISSR patterns for the plantlets tested. These results suggest that the culture conditions used for axillary branching proliferation are appropriate for clonal propagation of almond clone VII, as they do not seem to interfere with the integrity of the regenerated plantlets. These results allowed us to establish the use of axillary branching plantlets (mother-plants) as internal controls for the analysis of somaclonal variation of shoots regenerated from other in vitro culture processes performed with clone VII (adventitious regeneration, regeneration from meristem culture, virus sanitation programs and genetic engineering).M. Martins and D. Sarmento contributed equally to this paper     

水稻体细胞无性系变异

水稻体细胞无性系变异研究取得了很大进展 ,获得了大量抗病、抗逆、优质、矮杆等突变体。对这些突变体遗传分析表明 ,大多数突变性状由 1对或 2对基因控制。水稻体细胞无性系变异的发生与基因型、性状、继代时间、培养方式等有关 ,并具有内在的机制 ,点突变和反转录转座子插入可能是引起水稻无性系变异的两个重要原因。     

转基因白桦的遗传变异分析

应用细胞学方法分析了由农杆菌介导法获得的转基因白桦的细胞学变异情况,结果表明转基因白桦的染色体变异频率为78.5％,远远高于非转基因白桦的变异频率(15.3％),且变异以非整倍体占多数。同时用RAPD标记方法研究了转基因白桦在DNA水平的变异情况,结果显示DNA多态性指数为31.67,并与其它转基因植物的变异情况作了比较研究。最后分析、讨论了产生变异的原因:(1)组织培养过程中产生突变;(2)外源基因的整合及重排时宿主基因组的插入位点及相邻基因转录表达的干扰;(3)应用抗生素和除草剂等筛选转基因植株时促进了转基因植株的变异程度。并提出减少转基因植物体细胞克隆变异的建议。     

Studies on somaclonal variation in Phalaenopsis

The morphological and genetic variations in somaclones of Phalaenopsis True Lady “B79-19” derived from tissue culture were evaluated. In 1360 flowering somaclones, no apparent difference was found in the shape of the leaves, whereas flowers in some somaclones were deformed. We have demonstrated that 38 selected random primers can be used to generate amplified segments of genomic DNA and to differentiate polymorphisms of somaclonal variations in Phalaenopsis. The random amplified polymorphic DNA (RAPD) data indicated that normal and variant somaclones are not genetically identical. We also studied the banding patterns of aspartate aminotransferase (AAT) and phosphoglucomutase (PGM) in young leaves of variant and normal somaclones of Phalaenopsis. With respect to AAT, three distinct banding patterns were found in normal somaclones and only two-banded phenotypes were detected in variant somaclones. In a comparison of the banding patterns of PGM isozymes, three to four bands were detected in normal somaclones and two to three bands in variant ones. Received: 15 August 1997 / Revision received: 16 February 1998 / Accepted: 1 May 1998     

Comparison between RAPD and SSR molecular markers in detecting genetic variation in kiwifruit (Actinidia deliciosa A. Chev)

Two different DNA-based techniques, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers, were used for fingerprinting kiwifruit genotypes and for detecting undesirable genetic variation in micropropagated plants. The fragments were scored as present (1) or absent (0), and those readings were entered in a computer file as a binary matrix (one for each marker). Two cluster analyses were performed to express - in the form of dendrograms - the relationships among the genotypes and the genetic variability detected. Both DNA-based techniques were able to amplify all of the genotypes, but only SSR markers could detect genetic variation induced in micropropagated plants of cv. Tomuri. Two hypotheses were formulated to explain these results, both of them are in agreement with the results obtained using these two types of molecular markers. We conclude that when the tissue culture technique is used, the analysis of somaclonal variability could require more than one DNA-based technique; in fact, the genetic variation present in different sources could interfere or combine with the more or less polymorphic ability, as our results showed for SSR and RAPD markers.     

Somaclonal Variation in Rice after Two Successive Cycles of Mature Embryo Derived Callus Culture in the Presence of NaCl

Two successive cycles of mature embryo-derived callus culture separated by one cycle of sexual reproduction of R₀ regenerated plants were performed using two rice (Oryza sativa L.) cultivars in order to gain information upon the nature of somaclonal variation in this species. Plants regenerated after one cycle of tissue culture exhibited higher variability and lower performances than those of initial cultivar. A second cycle performed using R₁ embryos as explants showed that the cellular component of salt resistance in terms of growth and regenerating abilities selected during the first cycle could be transmitted to the progenies. The extent and the nature of somaclonal variation depended on the identity of R₀ mother plant and culture conditions, somaclonal variation being strongly reduced in some families obtained from salt-treated calli.     

PCR-based molecular markers for assessment of somaclonal variation in <Emphasis Type="Italic">Pinus pinea</Emphasis> clones micropropagated <Emphasis Type="Italic">in vitro</Emphasis>

Four different markers [random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and selective amplified microsatellite polymorphism length (SAMPL)] were applied for evaluating somaclonal variation of micropropagated genotypes of stone pine (Pinus pinea L.). The total number of primers tested was 130, with 223 combinations assayed. A high number of them amplified successfully (178), representing 79.82 % of the total, and the average number of amplified fragments ranged from 2.47 (ISSR) to 65.76 (SAMPL). Based on internal controls, no problem of reproducibility was detected. Almost no somaclonal variation was detected within the clones. Of the tested markers, ISSR, AFLP, and SAMPL showed monomorphic amplification profiles, with only RAPD markers showing some interclonal variation.     

Use of Random Amplified Polymorphic DNA Analysis for Economically Important Food Crops

The objective of this review is to summarize numerous studies on the use of the random amplified polymorphic DNA （RAPD） technique on rice, corn, wheat, sorghum, barley, rye, and oats to examine its feasibility and validity for assessment of genetic variation, population genetics, mapping, linkage and marker assisted selection, phylogenetic analysis, and the detection of somaclonal variation. Also we discuss the advantages and limitations of RAPD. Molecular markers have entered the scene of genetic improvement in different fields of agricultural research. The simplicity of the RAPD technique made it ideal for genetic mapping, plant and animal breeding programs, and DNA fingerprinting, with particular utility in the field of population genetics.     

Somaclonal variation in cryopreserved embryogenic clones of white spruce [Picea glauca (Moench) Voss.]

 Trees were regenerated from six white spruce embryogenic clones after cryopreservation for 3 and 4 years, respectively. Genetic stability was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprints. Somaclonal variation was detected in some in vitro embryogenic cultures 2 and 12 months after they were re-established following cryopreservation but not in the corresponding regenerated trees. These results suggest that trees regenerated from cryopreserved cultures in subsequent years are primarily genetically stable in the genomic regions tested and that variation observed due to the in vitro culture process infrequently affects trees regenerated from normally maturing and germinating somatic embryos. However, trees regenerated from somatic embryos that matured or germinated abnormally in in vitro culture exhibited altered RAPD fragment patterns. Received: 20 July 1998 / Revision received: 15 October 1998 / Accepted: 14 December 1998     

Population Structure of Magnaporthe grisea from North-western Himalayas and its Implications for Blast Resistance Breeding of Rice

Neutral and pathogenicity markers were used to analyse the population structure of Magnaporthe grisea rice isolates from the north‐western Himalayan region of India. Random amplified polymorphic DNA (RAPD)‐based DNA fingerprinting of 48 rice isolates of M. grisea with five primers (OPA‐04, OPA‐10, OPA‐13, OPJ‐06 and OPJ‐19) showed a total of 65 RAPD bands, of which 54 were polymorphic. Cluster analysis of 48 rice isolates of M. grisea on the basis of these 65 RAPD bands revealed the presence of high genotypic diversity and continuous DNA fingerprint variation in the pathogen population. No correlation was observed between RAPD patterns and virulence characteristics of the pathogen. The observed population structure contrasted with presumed clonal reproductive behaviour of the pathogen and indicated the possibility of ongoing genetic recombination in the pathogen population. Analysis of the virulence organization of five RAPD groups (RG1–RG5) using 20 rice genotypes comprising at least 15 resistance genes revealed that no combination of resistance genes would confer resistance against all RAPD fingerprint groups present in the M. grisea rice population. The possible implications of the observed population structure of M. grisea for blast resistance breeding have been discussed.     

Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers

Two protocols of plant regeneration for cotton were adopted in this study, namely, 2, 4-D and kinetin hormone combination and IBA and kinetin hormone combination. Twenty-eight embryogenic cell lines via somatic embryogenesis and 67 regenerated plants from these embryogenic calli were selected and used for random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), chromosomal number counting, and flow cytometric analysis. The roles of RAPD and SSR markers in detecting somaclonal variation of cotton (Gossypium hirsutum L.) were evaluated. Two cluster analyses were performed to express, in the form of dendrograms, the relationships among the hormone combinations and the genetic variability. Both DNA-based techniques were able to amplify all of the cell clones and regenerated plantlets genomes and relative higher genetic variation could be detected in the culture type with 2, 4-D and kinetin hormone combination. The result suggested that 2, 4-D and kinetin hormone combination could induce relative high somaclonal variation and RAPD and SSR markers are useful in detecting somaclonal variation of regenerated cotton plants via somatic embryogenesis. Chromosome number counting and flow cytometry analysis revealed that the number of chromosomes and ploidy levels were nearly stable in all regenerated plants except two regenerated plantlets (lost 4 and 5 chromosomes, respectively) which meant that cytological changes were not correlated with the frequency of RAPD and SSR polymorphisms. This result also might mean that the cell lines with variation of chromosome numbers were difficult to regenerate plants.     

Identification of a STS marker linked to the Aegilops speltoides-derived leaf rust resistance gene Lr28 in wheat

 A sequence-tagged-site (STS) marker is reported linked to Lr28, a leaf rust resistance gene in wheat. RAPD (random amplified polymorphic DNA) analysis of near-isogenic lines (NILs) of Lr28 in eight varietal backgrounds was carried out using random primers. Genomic DNA enriched for low-copy sequences was used for RAPD analysis to overcome the lack of reproducibility due to the highly repetitive DNA sequences present in wheat. Of 80 random primers tested on the enriched DNA, one RAPD marker distinguished the NILs and the donor parent from the susceptible recurrent parents. The additional band present in resistant lines was cloned, sequenced, and STS primers specific for Lr28 were designed. The STS marker (Indian patent pending: 380 Del98) was further confirmed by bulk segregation analysis of F₃ families. It was consistently present in the NILs, the resistant F₃ bulk and the resistant F₃ lines, but was absent in recurrent parents, the susceptible F₃ bulk and the susceptible F₃ lines. Received: 20 February 1998 / Accepted: 4 March 1998     

RFLP- and physical mapping of resistance gene homologues in rice (O. sativa) and Barley (H. vulgare)

 The deduced peptide sequences of 25 gene fragments of NBS-LRR resistance (R) gene homologues from rice and barley and of characterized R genes were compared, revealing a string of six conserved motifs. Mapping of the R-gene candidates in rice showed linkage to genes conferring race-specific resistance to rice blast (Pi-k, Pi-f and Pi-1) and bacterial blight disease (Xa-1, Xa-3 and Xa-4), in barley to powdery mildew (Mla) and the rust fungus (Rpg1). In rice four mixed clusters were detected, each harboring at least two highly dissimilar NBS-LRR genes. A YAC-contig was established for one of these mixed clusters. YAC fragmentation experiments revealed the presence of at least five NBS-LRR genes within 200 kb in head-to-tail orientation. Received: 24 July 1998 / Accepted: 14 August 1998