Use of molecular markers to accelerate the breeding of common bean lines resistant to rust and anthracnose

The main goal of this work was to introduce resistance genes for rust, caused by Uromyces appendiculatus, and anthracnose, caused by Colletotrichum lindemuthianum, in an adapted common bean cultivar through marker-assisted backcrossing. DNA fingerprinting was used to select plants genetically closer to the recurrent parent which were also resistant to rust and to race 89 of C. lindemuthianum. DNA samples extracted from the resistant parent (cv. Ouro Negro), the recurrent parent (cv. Rudá), and from BC₁, BC₂ and BC₃ resistant plants were amplified by the RAPD technique. The relative genetic distances in relation to the recurrent parent varied between 9 and 59% for BC₁, 7 and 33% for BC₂, and 0 and 7% for BC₃ resistant plants. After only three backcrosses, five lines resistant to rust and anthracnose with, approximately, 0% genetic distance in relation to the recurrent parent were obtained. These lines underwent field yield tests in two consecutive growing seasons and three of them presented a good yield performance, surpassing in that sense their parents and most of the reference cultivars tested.     

Evidence of gene introgression in apple using RAPD markers

Summary A genomic remnant of Malus floribunda clone 821 introgressed into the cultivated apple M. x domestica Borkh. was identified using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). Using a set of 59 oligonucleotide decamer primers, polymorphic DNA markers were identified among three pooled DNA samples. Based on the presence or absence of bands among bulked apple scab-resistant selections and cultivars, bulked scab-susceptible cultivars, and a M. floribunda clone 821 sample, one primer, A 15, identified amplified fragments in the scab-resistant bulked sample that was also unique to the M. floribunda clone 821. The unique band from M. floribunda clone 821 was amplified in four out of 17 scab-resistant selections/cultivars. This RAPD, designated OA15₉₀₀, identifies an introgressed fragment that has as yet no known function.     

Segregation distortion of Brassica carinata derived black rot resistance in Brassica oleracea

Three segregating F₂ populations were developed by self-pollinating 3 black rot resistant F₁ plants, derived from across between black rot resistant parent line 11B-1-12 and the susceptible cauliflower cultivar ‘Snow Ball’. Plants were wound inoculated using 4 isolates ofXanthomonas campestris pv. campestris (Xcc) race 4, and disease severity ratings of F₂ plants from the three populations were scored. A total of 860 arbitrary oligonucleotide primers were used to amplify DNA from black rot resistant and susceptible F₂ plants and bulks. Eight RAPD markers amplified fragments associated with completely disease free plants following black rot inoculation,which segregated in frequencies far lower than expected. Segregation of markers with black rot resistance indicates that a single, dominant major gene controls black rot resistance in these plants. Stability of this black rot resistance gene in populations derived from 11B-1-12 may complicate introgression into B. oleracea genotypes for hybrid production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variation within and between two cultivars of red clover (Trifolium pratense L.): Comparisons of morphological,isozyme, and RAPD markers

Summary Morphological, isozyme and random amplified polymorphic DNA (RAPD) markers were used to estimate genetic variation within and between cultivars of red clover (Trifolium pratense L.), an important temperate forage legume. Two cultivars of red clover, Essi from Europe and Ottawa from Canada, were evaluated. Six monogenic morphological characters were observed for 80 plants from each of these two cultivars. All six morphological loci were polymorphic in the cultivar Essi whereas only four loci were polymorphic in the cultivar Ottawa. Forty plants from each cultivar were assayed for isozyme markers. A total of 21 enzyme-coding loci with 43 alleles was detected using twelve enzyme systems. Thirteen and nine of these loci were polymorphic in Essi and Ottawa, respectively. The mean number of alleles per locus was 1.81 in Essi and 1.67 in Ottawa. Seventeen random 10-mer primers were screened for RAPD markers. Nine primers which gave clear and consistent amplified products were used to assay 20 individuals from each cultivar. Each primer gave from 7 to 20 amplified bands with an average of 14.8 bands per primer. One hundred and eight of 116 putative loci were polymorphic in Essi and 90 of 98 loci were polymorphic in Ottawa. High within-cultivar variation was observed in both cultivars using both isozyme and RAPD markers. This high polymorphism makes these markers useful for germplasm characterization and genetic studies in red clover.     

Screening fruit tree genetic resources in Belgium for disease resistance and other desirable characters

Summary The wide diversity of old fruit-tree cultivars originating or introduced into Belgium during the 18 ^th and 19 ^th centuries was collected as far as feasible over the last fifteen years at the State Plant Pathology Station in Gembloux. Out of the 2400 accessions now collected, one quarter was recovered from old public collections, and three quarters came from farms or gardens. The initial intention was to screen the material for disease resistance and other characters of agronomic interest with a view to using the best cultivars as breeding parents. However, as the collection developed, genetic resources conservation also became an objectiveper se. The collection presently contains 1150 apple, 850 pear and 300 plum accessions, and smaller numbers of other fruit species. Each accession is evaluated in an experimental orchard for at least ten years. In view of the growing public interest in old fruit-tree cultivars, the Plant Pathology Station has for several years been releasing to the nursery trade the better cultivars emerging from the evaluation, namely nine apple and four plum cultivars, and one peach cultivar. The principal features of the apple cultivars are presented in this paper. Since 1988, old apple and plum cultivars have been being used at the Station as parents in a breeding programme, with both controlled and open pollination. In some instances, old apple cultivars have also been crossed with a modern parent carrying the Vf gene for scab resistance. The preliminary observations on some of these seedlings are presented.     

Genetic diversity among elite Bulgarian barley varieties evaluated by RFLP and RAPD markers

Genetic variation among five elite winter barley cultivars (H. vulgare L.) currently grown in Bulgaria was assessed at the molecular level using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) markers. The present study sampled RFLPs in four well characterized multigene families in barley: the seed storage protein loci; the 18S, 5.8S and 26S ribosomal DNA loci; the loci coding for 5S ribosomal RNA and the loci coding subunit α of ATP-A complex in the mitochondrial genome. RFLPs were detected in three out of five investigated chromosomal loci in the barley cultivars studied. RAPD assay using arbitrary 10-base primers was applied to generate amplified length polymorphic markers in barley. Overall a total of 15 polymorphic phenotypes were found among the studied barley cultivars by using 11 out of 25 tested primers. All RAPDs were considered as dominant genetic markers except for two, where PCR and Southern blot analysis indicated the presence of codominant amplification products. Five RAPD polymorphisms in F₁ and F₂ progenies of the cross between Alpha and Obzor were inherited in Mendelian fashion. The determined values for the genetic variation proved a high genetic similarity among the tested cultivars. Genetic similarity (GS) calculated from RFLP and RAPD data ranged from 0.888 to 0.997 with a mean GS – 0.933. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purity control of F1-hybrid tomato cultivars by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were applied in purity control of hybrid seed production of tomato (Lycopersicon esculentum Mill.). DNA from three commercial F₁-hybrid cultivars and their parental lines was subjected to RAPD screening with 50 primers. Two of four primers which detected polymorphism between the parents tested, generated paternal-specific RAPDs, enabling a clear distinction to be made between hybrids and their maternal parents. In addition, combination of the polymorphic DNA products generated by these primers exhibited hybrid-specific patterns, enabling each cultivar to be identified. This result indicates the practical usefulness of RAPD markers in hybrid-tomato-seed purity-control tests and cultivar identification. The approach is advantageous in its rapidity and simplicity, particularly as an alternative for those cultivars for which lengthy and costly phenotypic tests are currently used.     

DNA polymorphisms in Oryza sativa L. and Lactuca sativa L. amplified by arbitrary primed PCR

Summary Thirty-five rice (Oryza sativa L.) varieties, including 18 japonica, 5 javanica and 12 indica subspecies and 12 lettuce (Lactuca sativa L.) varieties were identified taxonomically, using PCR with originally designed 21 RAPD (Random Amplified Polymorphic DNA) primers and 8 sequence-specific primers, used for amplifying four specific DNA fragments. Use of these primers revealed polymorphisms among varieties in rice and lettuce and facilitates DNA fingerprinting. Dendrograms of both species based on polymorphisms were constructed and genetical relationships were established. In rice, half the number of amplified bands were polymorphic and almost all varieties differentiated. However, differentiation of minor genetic alterations among somaclonal variants or mutants and their mother varieties was not feasible. In L. sativa, 47% of the amplified fragments were polymorphic and all 12 varieties were differentiated. Some of the PCR fragments were variety or type specific, which could be used for indicators for type-selection. The dendrogram obtained showed differentiated clusters of crisphead, leaf and butterhead type, findings in good accord with the classification based on the genetic background.     

Searching for DNA introgressed from wheat and for wheat-like grain proteins in a rice x wheat hybridization derivative

The DNA of a putative rice x wheat hybridization derivative (X Oryticum oryzoides) from China, the DNA of its parental rice cultivar and the DNA of a wheat line were digested with ten different restriction endonucleases, resolved by agarose electrophoresis, Southern blotted and hybridized using genomic wheat DNA as a probe. Phenol extracted, ethanol and cetyltrimethylammonium bromide precipitated DNA of the putative hybrid showed a restriction fragment length polymorphism (RFLP) different from that of the parental rice. When the DNA was further purified by Qiagen chromatography, the RFLP differences were not detected. Hence the apparent RFLP differences were probably due partial digestion of the less pure DNA preparations by the restriction endonucleases. No real introgressed fragments from wheat genome could be shown. The HpaII/MspI sites were more frequently digested with MspI than with HpaII in rice and hybridization derivative DNA, but the sites were evidently more frequently methylated in wheat DNA. Thus, in terms of methylation of the DNA, the hybridization derivative was much more like the rice parent than the wheat parent. The hybridization derivative showed a single endospermal protein (mass 19 kg mol^-1) not detected in the parental rice cultivar. This minor protein was soluble in buffered 50% isopropanol and precipitable with methanol. The results indicate that there are no or only short introgressed sequences from wheat in the rice/wheat derivative, a result which might be considered in breeding efforts with the hybrid derivative.     

Genetic variation among cultivars of red clover (Trifolium pratense L.) detected by RAPD markers amplified from bulk genomic DNA

Summary The use of random amplified polymorphic DNA (RAPD) markers obtained from bulked samples was investigated for cultivar identification in red clover. Pooled samples were examined in order to minimize variation within cultivars. To determine the appropriate number of individuals to include in the bulked samples representing each cultivar, DNA samples from two, three, four, five, ten and twenty individuals were pooled. Twenty was found to be an appropriate number of red clover individuals per bulk in order to amplify only the DNA sequences shared among most individuals in each cultivar. Fourteen 10-mer primers were used to amplify genomic DNA from combined leaf samples of 15 red clover cultivars from European, Japanese and North American origins. A total of 79 amplified products, of which 55 were polymorphic, was obtained. Cultivar-specific bands were observed with 13 primers. The amplification patterns obtained from two primers could distinguish all 15 red clover cultivars. Rogers' genetic distances for all 105 pairwise comparisons were calculated to evaluate relationships among these cultivars. Cluster analysis based on these genetic distances separated these 15 cultivars into three groups, with two of the groups consisting of a single Japanese cultivar each, while the third group included cultivars from European, North American, and Japanese origins.     

Radiomutants of chrysanthemum (Dendranthema grandiflora Tzvelev) of the Lady group: RAPD analysis of the genetic diversity

Random amplified polymorphic DNA (RAPD) markers were used to study the molecular characterization of 10 new radiomutants of chrysanthemum. The original cultivar ‘Richmond’ differed in genetic distance from its Lady group mutants. The analysis of genetic similarity indices revealed low diversity within the radiomutants. The dendrogram obtained after cluster analysis separated the new cultivars as a group that differed from the original cultivar ‘Richmond’. The Lady group cultivars, derived from one original cultivar by radiomutation, could be distinguished from each other by using RAPD markers of only a single primer or sets of two or three primers. Polymerase chain reaction analysis proved the efficiency of the RAPD method for DNA fingerprinting of the original cultivar ‘Richmond’ and its new radiomutants.     

Genetic diversity in tef [Eragrostis tef (Zucc) Trotter] and its relatives as revealed by Random Amplified Polymorphic DNAs

Tef is one of the staple cereal crops in Ethiopia. To evaluate genetic diversity of tef and its relatives, 47 accessions of tef, three accessions of E. pilosa, and six accessions of E. curvulawere analyzed using random amplified polymorphic DNA (RAPD) markers. The level of polymorphism among the wild species was extremely high, while low polymorphism was detected among tef accessions. All cultivars and wild species under study could be distinguished with the help of different primers, thereby indicating the potential of RAPD in the genetic fingerprinting of tef. Accessions from E. curvula and E. pilosa can be differentiated by a single selected primer. In spite of low polymorphism within tef, accessions under study could be distinguished by a combination of selected primers. Cluster analysis indicated that tef is a very closely related species to E. pilosa with 45%similarity, supporting the hypothesis that tef originated from E. pilosa based on morphological data. Given that RAPD are relatively quick, simple to use, and are not subjected to environmental influences, they provide a valuable new approach for the genetic fingerprinting and study of genetic diversity in tef. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparative assessment of DNA fingerprinting techniques (RAPD, ISSR, AFLP and SSR) in tetraploid potato (Solanum tuberosum L.) germplasm

Several DNA marker systems and associated techniques are available today for fingerprinting plant germplasm but information on their relative usefulness in particular crops is limited. The study investigated PCR based DNA fingerprinting in a set of 39 potato cultivars using RAPDs (20 primers), ISSRs (6 primers), AFLPs (2 primers) and SSRs (5 primer pairs). Results show that each of the four techniques can on their own, individually identify each cultivar, but that techniques differ in the mean number of profiles generated per primer (or primer pair) per cultivar, referred to as Genotype Index (GI). The order of merit based on this criterium and in this material was AFLPs (GI = 1.0), a multi-locus SSR (GI = 0.77),RAPDs (GI = 0.53), ISSRs (GI = 0.47) and single locus SSRs (GI = 0.36). Problems in relating banding patterns to individual loci and alleles for polyploid genomes, using these techniques as they are currently employed, are also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA标记在植物品种鉴定上的应用现状

为了全面地了解DNA指纹在品种鉴定上的利用现状,更重要的是促进DNA指纹技术切实有效的应用于植物品种鉴定,本研究归纳了目前开发出的可用于植物品种鉴定的DNA指纹技术,指出已有的DNA指纹品种鉴定技术的优势与不足,总结了DNA指纹应用于品种鉴定的分析措施,并认为受限于缺乏可以将DNA指纹转化为植物鉴定信息的理想措施,所以DNA标记还未真正有效地应用于生产实践中,因此提出一种基于DNA指纹的人工绘制品种鉴定简图(MCID)法是很重要的,本研究也重点阐述了MCID法的实施过程。     

Genetic variation and cultivar identification of Jamaican yam germplasm by random amplified polymorphic DNA analysis

Summary We have used random amplified polymorphic DNA (RAPD) analysis to characterize eleven cultivars of the five economically most important yam species grown in Jamaica (Dioscorea alata, D. cayenensis, D. rotundata, D. trifida and D. esculenta). Amplification of genomic DNA samples with nine different arbitrary 10mer primers revealed a total of 338 different band positions, ranging in size from 0.3 to 2.5 kb. RAPD patterns proved to be highly reproducible and somatically stable. While no variation was observed among plants belonging to the same cultivar, a large number of intervarietal and interspecific polymorphisms enabled us to reliably discriminate between all Jamaican cultivars investigated.     

Varietal identification in Cichorium intybus L. and determination of genetic purity of F1 hybrid seed samples, based on RAPD markers

Random amplified polymorphic DNA (RAPD) markers were used to distinguish between several Cichorium intybus genotypes, comprising four white witloof inbred lines, three red witloof experimental inbred lines and a number of F1 hybrids derived from two white parents. Amplification conditions and reproducibility of RAPD patterns were examined. Comparison of polymerase chain reaction (PCR) products obtained by using 100 10-mer arbitrary primers allowed identification of all the lines analysed. With several primers, we defined line-specific RAPD markers, while with others polymorphism was more extensive, revealing several RAPD markers for several lines. All the differences were confirmed both on individual heads and young seedlings for each genotype. Because of the Mendelian segregation of these molecular markers, this method was applied to evaluate the genetic purity of F1 hybrid seed samples.     

Fingerprinting temperate <Emphasis Type="Italic">japonica</Emphasis> and tropical <Emphasis Type="Italic">indica</Emphasis> rice genotypes by comparative analysis of DNA markers

This paper describes the relative efficiency of three marker systems, RAPD, ISSR, and AFLP, in terms of fingerprinting 14 rice genotypes consisting of seven temperatejaponica rice cultivars, three indica near-isogenic lines, three indica introgression lines, and one breeding line of japonica type adapted to high-altitude areas of the tropics with cold tolerance genes. Fourteen RAPD, 21 ISSR, and 8 AFLP primers could produce 970 loci, with the highest average number of loci (92.5) generated by AFLP. Although polymorphic bands in the genotypes were detected by all marker assays, the AFLP assay discriminated the genotypes effectively with a robust discriminating power (0.99), followed by ISSR (0.76) and RAPD (0.61). While significant polymorphism was detected among the genotypes of japonica and indica through analysis of molecular variance (AMOVA), relatively low polymorphism was detected within the genotypes of japonica rice cultivars. The correlation coefficients of similarity were significant for the three marker systems used, but only the AFLP assay effectively differentiated all tested rice lines. Fingerprinting of backcross-derived resistant progenies using ISSR and AFLP markers easily detected progenies having a maximum rate of recovery for the recurrent parent genome and suggested that our fingerprinting approach adopting the ‘undefined-element-amplifying’ DNA marker system is suitable for incorporating useful alleles from the indica donor genome into the genome of temperate japonica rice cultivars with the least impact of deleterious linkage drag.     

A genetic map of macadamia based on randomly amplified DNA fingerprinting (RAF) markers

The first genetic linkage map of macadamia (Macadamia integrifolia and M. tetraphylla) is presented. The map is based on 56 F₁ progeny of cultivars ‘Keauhou’ and ‘A16’. Eighty-four percent of the 382 markers analysed segregated as Mendelian loci. The two-way pseudo-testcross mapping strategy allowed construction of separate parental cultivar maps. Ninety bridging loci enabled merging of these maps to produce a detailed genetic map of macadamia, 1100 cm in length and spanning 70–80% of the genome. The combined map comprised 24 linkage groups with 265 framework markers: 259 markers from randomly amplified DNA fingerprinting (RAF), five random amplified polymorphic DNA (RAPD), and one sequence-tagged microsatellite site (STMS). The RAF marker system unexpectedly revealed 16 codominant markers, one of them a putative microsatellite locus and exhibiting four distinct alleles in the cross. This molecular study is the most comprehensive examination to date of genetic loci of macadamia, and is a major step towards developing marker-assisted selection for this crop. This revised version was published online in July 2006 with corrections to the Cover Date.     

用AFLP技术构建棉花雄性不育三系及其杂种F1的DNA指纹图谱

实验应用AFLP同位素标记法,获得了棉花细胞质雄性不育系、保持系、恢复系及其杂种F1的DNA指纹图谱.较为详尽地介绍了有关AFLP的技术核心及注意点,并探讨了其适用于棉花研究的方法.结果表明,AFLP是一种十分有效的DNA指纹技术,它具有多态性丰富、稳定性高、重复性好等优点.     

Random amplified polymorphic DNA variation among German and Dutch asparagus cultivars

DNA polymorphism among nine cultivars of Asparagus officinalis L. was measured using random amplified polymorphic DNA (RAPD). Of 69 reproducible amplification products from 12 arbitrary decamer primers, 49 RAPD markers were polymorphic and could be used to distinguish six German and three Dutch asparagus cultivars. Even with very small sample sizes, genetic similarity measurements based on the RAPD data allowed accurate grouping of the nine cultivars into distinct clusters, with the exception of two individuals which clustered to closely related varieties. Two German cultivars showed high genetic similarity and were distinct from the remaining German varieties. The German and Dutch cultivars were clearly separated by a relatively large genetic distance.