RAPD在三色堇(Viola wittrockiana)自交系遗传关系研究及杂种优势预测中的应用

用RAPD技术分析了18个三色堇(Viola wittrockiana)自交系的遗传多样性。21个随机引物扩增了167条带,其中127条具多态性,显示自交系间存在较大的遗传变异。用UPGMA法可将自交系聚为五大类,其分类结果与花径和材料来源地基本一致。以其中的5个自交系进行双列杂交试验,研究了RAPD遗传距离与三色堇杂交后代10个性状杂种优势的关系,实验结果表明:RAPD遗传距离仅与花数达到0.1的显著水平,而与其它8个性状杂种优势的相关性不显著;用RPAD遗传距离预测三色堇的花数杂种优势具有一定的可靠性,但用于对其它性状杂种优势的预测目前是不可行的。     

RAPD在三色堇（Viola wittrockiana）自交系遗传关系研究及杂种优势预测中的应用

用RAPD技术分析了18个三色堇(Viola wittrockiana)自交系的遗传多样性。21个随机引物扩增了167条带,其中127条具多态性,显示自交系间存在较大的遗传变异。用UPGMA法可将自交系聚为五大类,其分类结果与花径和材料来源地基本一致。以其中的5个自交系进行双列杂交试验,研究了RAPD遗传距离与三色堇杂交后代10个性状杂种优势的关系,实验结果表明:RAPD遗传距离仅与花数达到0.1的显著水平,而与其它8个性状杂种优势的相关性不显著;用RPAD遗传距离预测三色堇的花数杂种优势具有一定的可靠性,但用于对其它性状杂种优势的预测目前是不可行的。     

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.     

Genetic diversity in North American ginseng (Panax quinquefolius L.) grown in Ontario detected by RAPD analysis

Genetic diversity within North American ginseng (Panax quinquefolius L.) grown in Ontario was investigated at the DNA level using the randomly amplified polymorphic DNA (RAPD) method via the polymerase chain reaction (PCR). A total of 420 random decamers were initially screened against DNA from four ginseng plants and 78.8% of them generated RAPD fragments. Thirty-six of the decamers that generated highly repeatable polymorphic RAPD markers were selected for further RAPD analysis of the ginseng population. With these primers, 352 discernible DNA fragments were produced from DNA of 48 ginseng plants, corresponding to an average of 9.8 fragments per primer, of which over 45% were polymorphic. The similarity coefficients among the DNA of ginseng plants analyzed were low, ranging from 0.149 to 0.605 with a mean of 0.412, indicating that a high degree of genetic diversity exists in the ginseng population. Lower levels of genetic diversity were detected among 3-year-old ginseng plants selected on the basis of greater plant height than among the plants randomly selected from the same subpopulation or over the whole population, suggesting that genetic factors at least partly contribute to morphological variation within the ginseng population and that visual selection can be effective in identifying the genetic differences. The significance of a high degree of genetic variation in the ginseng population on its potential for improvement by breeding is also discussed.     

Evaluation of genetic diversity and uniformity of head cabbage DH lines by the use of RAPD markers

DH lines derived from cabbage cvs. Kamienna G?owa, S?awa z Enkhuizen and Langendijker, representing R1 generation, were analysed by the use of RAPD markers for their diversity and uniformity. For the evaluation of genetic diversity, eight primers yielding informative bands were used. Of the total of 83 RAPD bands scored in this study, 16.9% were polymorphic between a set of 13 DH lines. The similarity of the DH lines, estimated by Jaccard's coefficient, was depicted in the UPGMA dendrogram. Fourteen generated informative RAPD bands allowed the identification of DH lines developed from each cultivar. The evaluation of the uniformity for six closely related DH lines was possible by the use of three primers which generate one or two polymorphic bands. The lack of differences among ten plants of the five investigated DH lines manifested their uniformity. One line showed intraline polymorphism with two RAPD primers. The occurrence of the differences at the molecular level among ten plants indicated that their parental R0 plant was probably obtained from somatic cells, not by androgenesis.     

DNA polymorphism in various goose lines by RAPD-PCR

RAPD markers often primers were used to assess the polymorphism among pooled DNA of eight goose lines. The number of bands amplified by each primer ranged from 1 to 8, within a mean of 2.86. Some bands appeared specific for the line or genetic background. RAPD technique is an effective method for generating the polymorphic DNA marker in the goose. RAPD patterns from mixed DNA samples can reflect the genetic information of populations. The present study indicated that 10 generations selected for egg production and body weight under various pressure, resulted in genetic variation among goose lines as detected by RAPD. Selection for meat traits caused greater genetic diversity than selection for egg production.     

Efficiency of phenotypic and DNA markers for a genetic diversity study of alfalfa

Information on genetic diversity and germplasm characterization is essential for successful crop improvement. Diverse data sets (pedigree, morphological, biochemical, DNA based-markers) are employed in various aspects of plant analysis. The objective of this study was to determine the efficiency of phenotypic and RAPD markers in diversity assessment of ten alfalfa (Medicago spp.) accessions from Europe, North America and Australia. Field experiment was designed as a randomised complete block with three replications over two consecutive years (2004, 2005) at one location. Twelve morpho-agronomic traits were recorded on 50 plants per each accession. Genomic DNA’s from 16–20 randomly selected individual plants per accession were used for RAPD analysis. Six primers selected in this study generated a total of 93 polymorphic RAPD bands. The number of polymorphic bands detected per primer ranged from 11 to 20. Genetic distances (GD) among investigated accessions and two-dimensional principal coordinate analysis (2D PCoA) based on phenotypic and molecular data were obtained. The average GD between (0.283–0.416) and within (0.247–0.332) accessions based on RAPD data was higher than GD values obtained by morpho-agronomic traits (0.171–0.354 and 0.157–0.261, respectively). 2D PCoA based on GD from RAPD data grouped most of the studied individual plants to four clusters according to their geographical or taxonomy origin. 2D PCoA based only on morpho-agronomic data did not group plants congruently to their origin, probably due to a strong environmental influence on studied traits. Our results indicated that the RAPD markers were effective in assessing genetic diversity within and between studied alfalfa accessions. In addition, the obtained results suggested that the RAPD markers might be useful for grouping of germplasm with similar genetic background and for pre-screening of potential heterotic groups in our breeding programme.     

RAPD profile based genetic characterization of chemotypic variants of Artemisia annua L.

The annual herbaceous plant, Artemisia annua L., belonging to family Asteraceae, is the natural source of the highly potent antimalarial compound, artemisinin, besides producing valuable essential oil. The plant is at present the sole commercial source for artemisinin production since all the chemical syntheses are non-viable. Therefore, economic and practical considerations dictate that plants with maximum content of artemisinin be found and/or ways to increase their artemisinin content be sought. The key to this selection and breeding is a comprehension of chemical and genetic variability and suitable selection(s) of elites from within the available population. In the present study, RAPD analyses of selected chemotypes from a decade old introduced population in India were carried out using arbitrary primers. The RAPD data clearly indicate the distinction amongst these plants. Further, the detection of highly polymorphic profiles (97 polymorphic markers out of a total of 101 markers) suggests the existence of very high levels of genetic variation in the Indian population despite geographical isolation and opens out a strong possibility of further genetic improvement for superior artemisinin content. UPGMA analyses of RAPD and phytochemical trait data indicate that the wide phytochemical diversity is included within the genetic diversity. These results further support the prospects for selection and breeding of superior artemisinin containing lines.     

Genetic structure of the annual weed Senecio vulgaris in relation to habitat type and population size

Throughout the world, the highly selfing annual common groundsel, Senecio vulgaris (Asteraceae) is a common weed. Recently, it has also colonized ecological compensation areas in agro-ecosystems. We investigated the genetic structure of S. vulgaris using random amplified polymorphic DNA (RAPD) profiles of 80 plants from nine populations representing three habitat types in two regions in Switzerland. RAPD variation among regions (19.8%), among populations within regions (19.2%) and within populations (61.1%) was highly significant (ANOVA; P < 0.001). Gene flow estimated from the observed differentiation among populations (PhiST = 0.382) was low (assuming Wright's island model, Nem = 0.404). Genetic distances between pairs of populations were significantly correlated with geographical distances (Mantel test; r = 0.37, P < 0.03). Molecular variance obtained with AMOVA was lowest in the small populations in compensation areas (1.13), intermediate in vineyard populations (2.49), all located in northern Switzerland and highest in the larger vegetable field populations from western Switzerland (3.41; P < 0.05). Overall, there was a positive correlation of molecular variance and population size (P < 0.05), as expected under genetic drift. However, molecular variance was negatively correlated with population size among populations in ecological compensation areas, suggesting that selection was also important. We also applied triazine herbicide to leaves of three offspring of each of the 80 plants. Plants from populations of compensation areas showed higher mean levels and reduced variation in the resistance to triazine herbicide than plants from vineyards and vegetable fields. This suggests that compensation areas were colonized from adjacent corn fields, in which there has been selection for herbicide resistance. We discuss the implications of our results for the biological control of S. vulgaris.     

香菇空间诱变突变体的分子生物学鉴定研究*

香菇Cr04菌株经空间诱变后从中筛选出了农艺性状明显改善的突变菌株Cr04DZ。本研究用RAPD和AFLP技术对Cr04DZ与其地面对照菌株Cr04进行了DNA指纹分析,找出了它们之间的DNA多态性,从而从分子水平上证实了香菇经空间诱变后遗传物质发生了变化。此外,还比较了AFLP与RAPD检测香菇DNA多态性的效率,优化了对食用菌进行RAPD与AFLP分析的反应条件。通过对Cr04DZ及其对照株Cr04的RAPD及AFLP分析,筛选出了突变体的3个RAPD及29个AFLP多态性产物,已完成了3个AFLP产物的克隆。     

香菇空间诱变突变体的分子生物学鉴定研究

香菇Cr04菌株经空间诱变后从中筛选出了农艺性状明显改善的突变菌株Cr04DZ。本研究用RAPD和AFLP技术对Cr04DZ与其地面对照菌株Cr04进行了DNA指纹分析,找出了它们之间的DNA多态性,从而从分子水平上证实了香菇经空间诱变后遗传物质发生了变化。此外,还比较了AFLP与RAPD检测香菇DNA多态性的效率,优化了对食用菌进行RAPD与AFLP分析的反应条件。通过对Cr04DZ及其对照株Cr04的RAPD及AFLP分析,筛选出了突变体的3个RAPD及29个AFLP多态性产物,已完成了3个AFLP产物的克隆。     

Identification and mapping of polymorphic RAPD markers of pea (Pisum sativum L.) genome

Various pea cultivars, lines, and mutants were studied by the RAPD method. Polymorphic fragments characteristic of certain pea genotypes and which can be used for identifying genotypes were detected. Inheritance of some polymorphic RAPD fragments was studied. Mendelian inheritance of these fragments was shown. By analyzing the data obtained in studies of RAPD polymorphism, genetic distances between different pea cultivars, lines, and mutants were calculated and a genealogic dendogram showing a varying extent of differences between RAPD patterns was constructed. Ten new RAPD markers linked to various pea genes were detected. Genetic distances between RAPD markers and genes to which they are linked were calculated, and the respective disposition of RAPD markers on chromosomes was established.     

Production of transgenetic sugarbeet (Beta vulgaris L.) plants resistant to phosphinothricin

A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) with vacuum infiltration has been developed. Aseptic 3-weeks old etiolated seedlings of two diploid O-type sugarbeet lines (KS3 and KS7) have been used for genetic transformation. Transgenic sugarbeet plants carrying the reporter beta-glucuronidase gene have been selected for their resistance to glufosinate ammonium herbicide. Integration of transgenes into sugarbeet genome was confirmed with GUS assay and PCR using primers for bar and gusA genes.     

Spread of herbicide‐resistant weedy rice (red rice,Oryza sativa L.) after 5 years of Clearfield rice cultivation in Italy

The weedy relative of cultivated rice, red rice, can invade and severely infest rice fields, as reported by rice farmers throughout the world. Because of its close genetic relationship to commercial rice, red rice has proven difficult to control. Clearfield (Cl) varieties, which are resistant to the inhibiting herbicides in the chemical group AHAS (acetohydroxyacid synthase), provide a highly efficient opportunity to control red rice infestations. In order to reduce the risk of herbicide resistance spreading from cultivated rice to red rice, stewardship guidelines are regularly released. In Italy, the cultivation of Cl cultivars started in 2006. In 2010, surveillance of the possible escape of herbicide resistance was carried out; 168 red rice plants were sampled in 16 fields from six locations containing Cl and traditional cultivars. A first subsample of 119 plants was analysed after herbicide treatment and the resistance was found in 62 plants. Of these 119 plants, 78 plants were randomly selected and analysed at the level of the AHAS gene to search for the Cl mutation determining the resistant genotype: the Cl mutation was present in all the resistant plants. Nuclear and chloroplast microsatellite markers revealed a high correlation between genetic similarity and herbicide resistance. The results clearly show that Cl herbicide‐resistant red rice plants are present in the field, having genetic relationships with the Cl variety. Finding plants homozygous for the mutation suggests that the crossing event occurred relatively recently and that these plants are in the F₂ or later generations. These observations raise the possibility that Cl red rice is already within the cultivated rice seed supply.     

Genetic diversity of inbred rye lines evaluated by RAPD analysis

This study presents an attempt to supply breeders of hybrid rye with more genetic information on inbred lines, using molecular markers. Eighteen polymorphic loci detected by means of the RAPD (Random Amplified Polymorphic DNA) technique and mapped on 2R-7R rye chromosomes, were applied to study genetic similarities among forty inbred lines of rye. The lines were grouped in four main clusters revealed on dendrogram, which was generally consistent with the pedigree data. Mapped RAPD markers were shown to be a useful tool for phenetic studies in rye. Additionally, a system of 20 polymorphic fragments, detected by three primers, was developed for fingerprinting of rye lines. The system of RAPD markers, which was developed in this study, should be helpful in characterisation of rye genetic stocks used for breeding.     

Two recessive gene inheritance for triallate resistance in Avena fatua L

Extensive use of the preemergence herbicide triallate over the last three decades has selected for resistant (R) Avena fatua L. populations in several areas of the United States and Canada. R plants are also cross-resistant to the unrelated pyrazolium herbicide difenzoquat. We made reciprocal crosses between inbred R and susceptible (S) lines to determine the genetic basis of triallate resistance. Seeds from parental lines and F(2) populations were treated with soil applications of 0.275, 0.55, or 1.1 kg/ha triallate in the greenhouse and plant heights recorded after 37 days. Surviving F(2) plants were selfed and the resulting F(3) families were screened with 1.1 kg/ha triallate. In the F(2) populations, assortment of S and R phenotypes fit a 15:1 segregation ratio, suggesting that resistance was controlled by the two independently segregating recessive genes TRR1 and TRR2. None of the 912 F(3) progeny from 51 R F(2) individuals was susceptible to triallate treatment, further supporting a two-gene mode of inheritance. There was a possible maternal effect on susceptibility at the highest triallate rate tested.     

Assessment of genetic fidelity of micropropagated plants of <Emphasis Type="Italic">Simmondsia chinensis</Emphasis> (Link) Schneider using RAPD and ISSR markers

RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeat) markers were screened to test the genetic integrity of jojoba (Simmondsia chinensis) plants multiplied through axillary bud multiplication from nodal segments. The in vitro raised plantlets were maintained for up to 12 in vitro subcultures. During the study a total of 48 (32 RAPD and 16 ISSR) primers were screened, out of which 24 RAPD and 13 ISSR primers produced a total of 191 (126 RAPD and 65 ISSR) clear, distinct and reproducible amplicons. The amplified products were monomorphic across all the selected micropropagated plants and were similar to the mother plant. The micropropagation protocol developed by our group for rapid in vitro multiplication is appropriate for clonal propagation of jojoba. The outcome supports the fact that axillary bud multiplication can also be used as one of the safest modes for the production of true-to-type plants.     

An examination of fitness costs of glyphosate resistance in the common morning glory,Ipomoea purpurea

Fitness costs are frequently invoked to explain the presence of genetic variation underlying plant defense across many types of damaging agents. Despite the expectation that costs of resistance are prevalent, however, they have been difficult to detect in nature. To examine the potential that resistance confers a fitness cost, we examined the survival and fitness of genetic lines of the common morning glory, Ipomoea purpurea, that diverged in the level of resistance to the herbicide glyphosate. We planted a large field experiment and assessed survival following herbicide application as well as fitness of the divergent selection lines in the absence of the herbicide. We found that genetic lines selected for increased resistance exhibited lower death compared to control and susceptible lines in the presence of the herbicide, but no evidence that resistant lines produced fewer seeds in the absence of herbicide. However, susceptible lines produced more viable seeds than resistant or control lines, providing some evidence of a fitness cost in terms of seed germination, and thus potential empirical support for the expectation of trait trade‐offs as a consequence of adaptation to novel environments.     

DNA polymorphism in the living fossil Ginkgo biloba from the eastern United States.

Random amplified polymorphic DNA (RAPD) analysis is a valuable tool in studying inter- and intra-specific genetic variations, patterns of gene expression, and for the identification of specific genes using nearly isogenic variants. Here we used RAPD analysis to study the genetic variation in Ginkgo biloba grown in the eastern United States. Our results support the evidence that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern than dye-stained gels or Southern blot hybridization of RAPD blots using probes made from purified PCR products. Using these techniques, we observed a high degree of relatedness among plants grown in certain localities although significant genetic variation may exist in the species, and could be a possible explanation for the observed variations in the efficacy of medications derived from G. biloba extract.