Cytoplasmic Molecular Markers Research Articles

Hybrid breeding boosted molecular genetics in rye

The history of rye (Secale cereale L.) breeding began from the first targeted selections made in Germany by the Probsteier Seed Cooperative around 1850, and over 150 years of breeding yielded a tremendous amount of results. Rye has also long been used as cytological subject due to its low number of chromosomes and their size. However, genetic findings in rye up to the early 1980s were rather scant. About 120 genes could be assigned to seven linkage groups. However, since then development of new methods such as C-banding procedures, in situ DNA hybridization, enzymology and molecular marker techniques achieved an enormous progress. Particularly, the latter of these was driven by the agronomic success of hybrid breeding in rye. The basic genetic knowledge resulting from intra- and interspecies genetic diversity assay and phylogenetic studies in the Secale genus used nuclear and cytoplasmic molecular markers to facilitate successful selection of parents for hybrid breeding and hence contribute to improvement of heterosis effect. The main achievements in rye hybrid breeding and genetic mechanisms exploited for different hybrid breeding strategies are discussed. The exploitation of landraces or wild rye species gene pools via advanced backcross QTL analysis or introgression libraries to increase the heterosis effect is a prospective strategy of marker-assisted hybrid breeding in rye. Prediction of hybrid performance can also be improved significantly by marker-assisted selection and genomic selection based on genome-wide marker data. The findings in rye genetics (including phylogenetic, mapping and population studies) are reviewed in their relation with the hybrid breeding purposes and demands. Overall, about 450 morphological and biochemical traits are mapped throughout the the genome plus about 5,000 DNA markers. They are not only associated with individual chromosome or segments but also efficiently used for comparative mapping (evolutionary studies), introgression monitoring, QTL tagging, and marker-assisted selection.

Hybrid breeding boosted molecular genetics in rye

History of rye (Secale cereale L.) breeding began from the first targeted selections made in Germany by the Probsteier Seed Cooperative around 1850, and over 150 years breeding yielded a tremendous amount of results. Rye has also long been used as cytological subject due to its low number of chromosomes and their size. However, genetic findings in rye up to the early 1980s were rather scant. About 120 genes could be assigned to seven linkage groups. Only through the development of new methods such as C-banding, in situ DNA hybridization, enzymology and molecular marker techniques achieved an enormous progress. Particularly, the latter was driven by agronomic success of hybrid breeding in rye. The basic genetic knowledge resulted from intra- and interspecies genetic diversity assay and phylogenetic studies in the genus Secale using nuclear and cytoplasmic molecular markers facilitated successful selection of parents for hybrid breeding and hence contributed to improvement of heterosis effects. Main achievements in rye hybrid breeding and genetic mechanisms exploited for different hybrid breeding strategies are discussed. The utilsation of landraces and wild relatives via advanced backcross procedures, combined by QTL analysis and introgression libraries, contributed to increase of heterotic effects. Those marker-assisted approaches became the basis of recent hybrid breeding of rye. Prediction of hybrid performance can also be improved significantly by marker-assisted selection and genomic selection based on genomewide marker data. The findings in rye genetics (including phylogenetic, mapping and population studies) are reviewed in their relation with the hybrid breeding purposes and demands. Overall, about 450 morphological and biochemical traits are mapped throughout the genome plus about 5,000 DNA markers. They are not only associated with individual chromosome or segments but also efficiently used for comparative mapping (evolutionary studies) introgression monitoring, QTL tagging, and marker-assisted selection.

Characterization of Gypsophila species and commercial hybrids with nuclear whole-genome and cytoplasmic molecular markers

The genus Gypsophila contains about 150 annual and perennial flowering plant species native to the temperate regions of Europe and Asia. Nowadays Gypsophila species are present worldwide as garden ornamental plants. Although Gypsophila is one of the most economically important ornamental crops, little is known about its genetic variability and the relationships among the different wild species, cultivars, and commercial hybrids. The aim of our work was to analyze genetic distances among 5 wild species and 13 commercial hybrids of Gypsophila with similar phenotypes but unknown origin. For this purpose, we have used amplified fragment length polymorphism, target region amplification polymorphism, and inter simple sequence repeat whole-genome markers and chloroplast simple sequence repeat (cpSSR), targeting chloroplast DNA. Nuclear markers were found to distinguish all the analyzed samples while cpSSR markers were found to discriminate the different wild species, but could not sufficiently separate the commercial hybrids. This notwithstanding, the data obtained allowed us to cluster the commercial hybrids into different sub-groups and to determine the relationships with the putative species of origin.

Genetic diversity in Vietnamese melon landraces revealed by the analyses of morphological traits and nuclear and cytoplasmic molecular markers

Genetic diversity among 59 melon landraces from Vietnam was studied by analyzing morphological traits and molecular markers. The morphological characters of the melon landrace fruits were highly diversified. Among the five types of cultivated melon, Dua le and Dua yang were classified as Conomon var. makuwa, whereas Dua gang was classified as Conomon var. conomon, and Dua bo was classified as Momordica. However, Dua thom could not be classified into a proper group or variety. The gene diversity based on random amplified polymorphic DNA (RAPD) and single sequence repeat analyses was small and equivalent to that of Chinese Conomon. A cluster analysis revealed that Dua bo, Dua le, Dua yang, and Dua gang were grouped in cluster II. Clusters III and IV consisted mainly of Conomon accessions from China and Japan. Dua thom was classified into cluster V with landraces from Yunnan Province, China. The comparison of a RAPD profile with 291 melon accessions from Africa and Asia clearly showed that Dua thom and Yunnanese landraces were closely related with the small-seed type melons from Myanmar, Bangladesh, and northeastern India. The other four types were related closely with Conomon and Agrestis accessions from China, Korea, and Japan, indicating their involvement in the differentiation and establishment of the Conomon group in East Asia.

Construction of cytoplasmic molecular markers distinguishing Danio rerio from Gobiocypris rarus at high identity domains based on MP-PCR strategy and Sybr Green I detection

To distinguish the cytoplasm of Danio rerio from that of Gobiocypris rarus, we cloned G. rarus COXI and constructed cytoplasmic molecular markers at the high identity domains of COXI by mutated primer PCR (MP-PCR for short). Then Sybr Green I was used to detect the single amplicon. As a result, we succeeded in getting the cytoplasmic molecular markers, G.M COXI and Z.M COXI, by MP-PCR strategy. They were used to detect the sperm-derived mtDNA in the sexual hybrid embryos (D. rerio female symbolxG. rarus male symbol) before the sphere stage. In the present study, all results demonstrate that MP-PCR approach and Sybr Green I detection are feasible to construct the molecular markers to identify genes that shared high identity.

Application of Cleaved Amplified Polymorphic Sequence Method for Analysis of Cytoplasmic Genome among Aurantioideae Intergeneric Somatic Hybrids

Somatic hybridization allows the creation of new patterns of nuclear, mitochondrial and chloroplastic association. It is therefore necessary to master cytoplasmic molecular markers to determine the genetic origin of both organelles of plantlets obtained from protoplasts fusion. In the case of Citrus and related genera, only southern blot hybridization and restriction fragment-length polymorphism (RFLP) techniques were used for this task until now. Here, we describe the use in the Aurantioideae subfamily, of a simple and non labeling cleaved amplified polymorphic sequence (CAPS) technique, to determine the cytoplasmic genome origin of intergeneric somatic hybrids. Mitochondrial and chloroplastic universal primers previously selected for population genetic studies in Quercus by Demesure et al. (1995) are used with some modifications. The variability of cytoplasmic genome among somatic fusion partners is detected by coupling amplification and restriction reactions. Digested DNA fragments are analyzed by agarose gel electrophoresis (PCR-RFLP). This technique has been applied for the analysis of the cytoplasmic constitution of somatic hybrids arising from intergeneric, intersubtribal and intertribal combinations. Systematic transmission of the mitochondria from protoplasts isolated from embryogenic callus parents was confirmed.

Associations among cytoplasmic molecular markers, gender, and components of fitness in Silene vulgaris, a gynodioecious plant.

It has been suggested that the dynamics of chloroplast DNA (cpDNA) or mitochondrial DNA (mtDNA) genetic markers used in studies of plant populations could be influenced by natural selection acting elsewhere in the genome. This could be particularly true in gynodioecious plants if cpDNA or mtDNA genetic markers are in gametic disequilibrium with genes responsible for sex expression. In order to investigate this possibility, a natural population of the gynodioecious plant Silene vulgaris was used to study associations among mtDNA haplotype, cpDNA haplotype, sex and some components of fitness through seed. Individuals were sampled for mtDNA and cpDNA haplotype as determined using restriction fragment length polymorphism (RFLP) methods, sex (female or hermaphrodite), fruit number, fruit set, seeds/fruit and seed germination. The sex of surviving germinating seeds was also noted. All individuals in the population fell into one of two cytoplasmic categories, designated haplotypes f and g by a unique electrophoretic signature in both the mtDNA and cpDNA. The subset of the population carrying haplotype g included a significantly higher proportion of females when compared with the sex ratio of the subset carrying the f haplotype. Haplotype g had a significantly higher fitness when measured by fruit number, fruit set and seeds/fruit, whereas haplotype f had significantly higher fitness when measured by seed germination. Offspring of individuals carrying haplotype g included a significantly greater proportion of females when compared with offspring of individuals carrying the f haplotype. Other studies of gynodioecious plants have shown that females generally have higher fitness through seed than hermaphrodites, but in this study not all fitness differences between haplotypes could be predicted from differences in haplotype-specific sex ratio alone. Rather, some differences in haplotype-specific fitness were due to differences in fitness between individuals of the same sex, but carrying different haplotypes. The results are discussed with regard to the potential for hitchhiking selection to influence the dynamics of the noncoding regions used to designate the cpDNA and mtDNA haplotypes.

Population genetics, molecular markers and the study of dispersal in plants

Summary 1 Long‐distance dispersal events are biologically very important for plants because they affect colonization probabilities, the probabilities of population persistence in a fragmented habitat, and metapopulation structure. They are, however, very difficult to investigate because of their low frequency. We reviewed the use of molecular markers in the population genetics approach to studying dispersal. With these methods the consequences of long‐distance dispersal are studied, rather than the frequency of the dispersal events themselves. 2 Molecular markers vary, displaying different amounts of variation and different modes of inheritance: they may be either dominant or codominant, and may or may not be subjected to genetic recombination. Use of markers has inspired the development of maximum likelihood techniques that take the evolutionary history of alleles into account while estimating gene flow. 3 Inferring seed dispersal rates from indirect measurements of gene flow involves three steps: (i) quantifying genetic differentiation among populations and using this to estimate the rate of gene flow; (ii) producing a genetic dispersal curve by regressing geographical distance among populations against the amount of gene flow; and (iii) separating seed‐mediated from pollen‐mediated gene flow, by comparing differentiation in nuclear vs. cytoplasmic molecular markers. In this way, potentially very low levels of gene flow can be detected. 4 The indirect approach is based on a number of assumptions. The validity of each assumption should be assessed by independent methods or the estimates of gene flow and dispersal should be mainly used in a comparative context. In metapopulations, with frequent extinction and colonization, the relationship between genetic differentiation and gene flow is not straightforward, and other methods should be used. 5 Highly variable molecular markers, especially microsatellites, have facilitated a direct genetic approach to measuring gene flow, based on parental analyses. 6 The population genetic approach provides different information about dispersal than ecological methods. Thus population genetic and ecological methods may supplement each other, and together lead to a better insight into the dispersal process than either of the methods on its own.

Cytoplasmic-nuclear male sterility in pearl millet: comparative RFLP and transcript analyses of isonuclear male-sterile lines

The identification of diagnostic cytoplasmic molecular markers is of prime interest to pearl millet breeders wishing to identify sources of cytoplasmic-nuclear male sterility (CMS) which can be used as an alternative to the single source currently used in the production of F1 hybrid seed. Here, we report the classification of five pearl millet CMS sources based on RFLP analysis of isonuclear lines carried out using mitochondrial gene-specific DNA probes in combination with eight restriction endonucleases. On the basis of RFLP data, the five CMS cytoplasms can be distinguished from each other and from the isonuclear fertile cytoplasm. In addition, based on cox1, cox3, atp6 and atp9 polymorphisms, these lines can be classified into two major groups: one corresponds to A5, Aegp, Av and A1 cytoplasms, and the other consists of the A4 cytoplasm. Our results suggest that a rearrangement involving the cox1 gene might be related to CMS in the first group (A5, Aegp, Av and A1), whereas a rearrangement within the atp6/cox3 cluster region might be related to CMS in the second group (A4).