Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato

Asr1, Asr2 andAsr3 are three homologous clones isolated from tomato whose expression is believed to be regulated by abscisic acid (ABA); the corresponding genes thus participate in physiological and developmental processes such as responses of leaf and root to water stress, and fruit ripening. In this report, results obtained with Near Isogenic Lines reveal thatAsr1, Asr2 andAsr3 represent three different loci. In addition, we map these genes on the restriction fragment length polymorphism (RFLP) map of the tomato genome by using an F₂ population derived from an interspecific hybrid crossL. esculentum × L. penelli. RFLP data allow us to map these genes on chromosome 4, suggesting that they belong to a gene family. The elucidation of the genomic organization of theAsr gene family may help in understanding the role of its members in the response to osmotic stress, as well as in fruit ripening, at the molecular level.     

Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small subunit multi-gene family

Of the eight nuclear genes in the plant multi-gene family which encodes the small subunit (rbcS) of Petunia (Mitchell) ribulose bisphosphate carboxylase, one rbcS gene accounts for 47% of the total rbcS gene expression in petunia leaf tissue. Expression of each of five other rbcS genes is detected at levels between 2 and 23% of the total rbcS expression in leaf tissue, while expression of the remaining two rbcS genes is not detected. There is considerable variation (500-fold) in the levels of total rbcS mRNA in six organs of petunia (leaves, sepals, petals, stems, roots and stigmas/anthers). One gene, SSU301, showed the highest levels of steady-state mRNA in each of the organs examined. We discuss the differences in the steady-state mRNA levels of the individual rbcS genes in relation to their gene structure, nucleotide sequence and genomic linkage.     

Characterisation of cDNAs for ribulose bisphosphate carboxylase small subunit genes in different species of Avena (Poaceae)

ABSTRACT

We report the cloning and sequencing of 14 rbcS cDNAs in six species of Avena (Poaceae) with different genome and ploidy levels. The nucleotide sequences 504 bp long were aligned with the published sequences of cultivated hexaploid oat, wheat, barley, rye, rice and corn and subjected to cladistic and phenetic analyses. The parsimonious analysis generated a tree with a topology very similar to the phenogram generated by the Neighbor-Joining analysis based on the Jukes and Cantor distances. Within the monophyletic assemblage of the tribe Aveneae, consistent clades composed of rbcS clones belonging to different species are recognized. It is suggested that they correspond to orthologous genes belonging to different subfamilies, and that the “within-species?d homogenisation may have occurred at a slow rate with respect to species evolution. In the monophyletic group of Pooideae, the topologies place barley rbcS sequences closer to wheat and rye than to oat sequences. This grouping agrees with most taxonomic and phylogenetic views.     

Development of an RFLP linkage map in diploid peanut species

An RFLP linkage map of peanut has been developed for use in genetic studies and breeding programs aimed at improving the cultivated species (Arachis hypogaea L.). An F₂ population derived from the interspecific hybridization of two related diploid species in the sectionArachis (A. stenosperma ×A. cardenasii) was used to construct the map. Both random genomic and cDNA clones were used to develop the framework of the map. In addition, three cDNA clones representing genes coding for enzymes involved in the lipid biosynthesis pathway have been mapped in peanut. Of the 100 genomic and 300 cDNA clones evaluated, 15 and 190, respectively, revealed polymorphisms among the parents of our mapping population. Unfortunately, a large number of these produced complex banding patterns that could not be mapped. Of the 132 markers analyzed for segregation, 117 are distributed among 11 linkage groups, while 15 have not yet been associated with any other marker. A total map distance of approximately 1063 cM has been covered to-date.     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.     

A New Retroposed Gene in <Emphasis Type="Italic">Drosophila</Emphasis> Heterochromatin Detected by Microarray-Based Comparative Genomic Hybridization

A genomic pattern of new gene origination is often dependent on a genomic method that can efficiently identify a statistically adequate number of recently originated genes. The heterochromatic regions have often been viewed as genomic deserts with low coding potential and thus a low flux of new genes. However, increasing reports revealed unexpected roles of heterochromatic regions in the evolution of genes and genomes. We identified recently retroposed genes that originated in heterochromatic regions in Drosophila, by developing microarray-based comparative genomic hybridization (CGH) with multiple species. This new gene family, named Ifc-2h, originated in the common ancestor of the clade of D. simulans, D. mauritiana, and D. sechellia. The sequence features and phylogenetic distribution indicated that Ifc-2h resulted from the retroposition from its parental gene, Infertile crescent (Ifc), and integrated into heterochromatic region of common ancester of the three sibling species 2 million years ago. Expression analysis revealed that Ifc-2h had developed a new expression pattern by recruiting a putative regulatory element from its target sequence. The distribution of indel variation in Ifc-2h of D. simulans and D. mauritiana revealed a significant sequence constraint, suggesting that the Ifc-2h gene may be functional. These analyses cast fresh insight into the evolution of heterochromatin and the origin of its coding regions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

Linkage mapping of starch branching enzyme III in rice (Oryza sativa L.) and prediction of location of orthologous genes in other grasses

 The chromosomal position of Starch Branching Enzyme III (SBEIII) was determined via linkage to RFLP markers on an existing molecular map of rice (Oryza sativa L.). A cDNA of 890 bp was generated using specific PCR primers designed from available SBEIII sequence data and used as a probe in Southern analysis. The SBEIII cDNA hybridized to multiple restriction fragments, but these fragments mapped to a single locus on rice chromosome 2, flanked by CDO718 and RG157. The detection of a multiple-copy hybridization pattern suggested the possibility of a tandemly duplicated gene at this locus. The map location of orthologous SBE genes in maize, wheat, and oat were predicted based on previously published genetic studies and comparative maps of the grass family. Received : 5 August 1996 / Accepted : 13 September 1996     

Overcoming the species hybridization barrier by ploidy manipulation in the genus Oryza

In most eudicot and monocot species, interspecific and interploidy crosses generally display abnormalities in the endosperm that are the major cause of a post‐zygotic hybridization barrier. In some eudicot species, however, this type of hybridization barrier can be overcome by the manipulation of ploidy levels of one parental species, suggesting that the molecular mechanisms underlying the species hybridization barrier can be circumvented by genome dosage. We previously demonstrated that endosperm barriers in interspecific and interploidy crosses in the genus Oryza involve overlapping but different mechanisms. This result contrasts with those in the genus Arabidopsis, which shows similar outcomes in both interploidy and interspecific crosses. Therefore, we postulated that an exploration of pathways for overcoming the species hybridization barrier in Oryza endosperm, by manipulating the ploidy levels in one parental species, might provide novel insights into molecular mechanisms. We showed that fertile hybrid seeds could be produced by an interspecific cross of female tetraploid Oryza sativa and male diploid Oryza longistaminata. Although the rate of nuclear divisions did not return to normal levels in the hybrid endosperm, the timing of cellularization, nucellus degeneration and the accumulation of storage products were close to normal levels. In addition, the expression patterns of the imprinted gene MADS87 and YUCCA11 were changed when the species barrier was overcome. These results suggest that the regulatory machinery for developmental transitions and imprinted gene expression are likely to play a central role in overcoming species hybridization barriers by genome dosage in the genus Oryza.     

Chloroplast DNA inheritance and variation in <Emphasis Type="Italic">Leucadendron</Emphasis> species (Proteaceae) as revealed by PCR-RFLP

The inheritance of chloroplast DNA (cpDNA) in Leucadendron species was studied by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. A total of 100 progeny from five interspecific crosses involving seven parental species were tested, and all progeny exhibited the cpDNA restriction fragment pattern of the female parent, indicating that cpDNA in Leucadendron is maternally inherited. PCR-RFLP was also employed to study cpDNA variation among 21 Leucadendron species. Parsimony analysis using a heuristic search resulted in a phylogenetic tree that showed limited agreement to the taxonomic classification of Leucadendron species, based on morphological characteristics. The incongruence between cpDNA phylogenetic and taxonomic groupings in Leucadendron may be due to reticulate evolution involving a combination of hybridization and introgression, convergent evolution and/or lineage sorting at the interspecific, intersubsectional and intersectional levels.     

Polyploidy, hybridization and reticulate evolution: lessons from the Brassicaceae

The family Brassicaceae is well known for its large variation in chromosome numbers, common occurrence of polyploids and many reports of interspecific gene flow. The present review summarizes studies from the past decades on polyploidization and hybridization events, recognizing them as important evolutionary forces in the family. Attention is drawn to the issue of the reconstruction of reticulated pattern of evolution resulting from allopolyploid and homoploid hybrid speciation. The research of various authors on several Brassicaceae genera is presented and discussed in the context of our current understanding of polyploid and hybrid evolution. Model species, Arabidopsis thaliana and Brassica taxa, are referred to only marginally, major focus is on a comprehensive survey of studies on about a dozen best explored non-model genera (e.g. Cardamine, Draba, Rorippa, Thlaspi). The increasing amount of genetic and genomic resources available for Brassicaceae model species provides excellent opportunities for comparative genetic and genomic studies. Future research directions and challenges are thus outlined, in order to obtain more detailed insights into the evolution of polyploid and hybrid genomes.     

Comparative mapping in grasses. Oat relationships

The development of RFLP linkage maps in hexaploid and diploid oat allows us to study genetic relationships of these species at the DNA level. In this report, we present the extension of a previously developed diploid oat map (Avena atlantica x A. hirtula) and its molecular-genetic relationships with wheat, rice and maize. Examination of 92–99% of the length of the oat genome map with probes common to Triticeae species, rice or maize showed that 84, 79 and 71%, respectively, was conserved between these species and oat. Generally, the orders of loci among chromosomes homoeologous to oat chromosomes A and D were the most conserved and those of chromosomes homoeologous to oat chromosome G were the least conserved. Conservation was observed for blocks ranging from whole chromosomes 101 cM long to small segments 2.5 cM long containing two loci. Comparison of the homoeologous segments of Triticeae, rice and maize relative to oat indicated that certain regions have been maintained in all four species. The relative positions of major genes governing traits such as seed storage proteins and resistance to leaf rusts have been conserved between cultivated oat and Triticeae species. Also, the locations of three vernalization/or photo-period response genes identified in hexaploid oat correspond to the locations of similar genes in homoeologous chromosomes of wheat, rice or maize. The locations of the centromeres for six of the seven oat chromosomes were estimated based on the homoeologous segments between oat and Triticeae chromosomes.     

Asr genes belong to a gene family comprising at least three closely linked loci on chromosome 4 in tomato

Asr1, Asr2 andAsr3 are three homologous clones isolated from tomato whose expression is believed to be regulated by abscisic acid (ABA); the corresponding genes thus participate in physiological and developmental processes such as responses of leaf and root to water stress, and fruit ripening. In this report, results obtained with Near Isogenic Lines reveal thatAsr1, Asr2 andAsr3 represent three different loci. In addition, we map these genes on the restriction fragment length polymorphism (RFLP) map of the tomato genome by using an F₂ population derived from an interspecific hybrid crossL. esculentum × L. penelli. RFLP data allow us to map these genes on chromosome 4, suggesting that they belong to a gene family. The elucidation of the genomic organization of theAsr gene family may help in understanding the role of its members in the response to osmotic stress, as well as in fruit ripening, at the molecular level.     

Inheritance of resistance derived from the B-genome of Brassica against Phoma lingam in rapeseed and the development of molecular markers

 Genes of the B genome of Brassica conditioning Phoma resistance at the epicotyle were transferred into Brassica napus by interspecific hybridization. The recombinant lines expressed high resistance similar to that of the donor parents. Unlike the oligo- or poly-genically inherited resistance of B. napus known so far, the B-genome resistance genes of the recombinant lines behaved monogenically dominant. No significant differences in the level of resistance or in the phenotype of the resistance mechanisms were observed among homozygous resistant plants when the different B-genome origins investigated, i.e. B. nigra, B. juncea and B. carinata, were compared. Therefore it was assumed that the resistance genes of each B-genome species and the resistance mechanisms of the species are identical. Temperature increased the expression of internal lesions caused by Phoma lingam. High summer temperatures in the greenhouse led to faster development of tissue damage at the epicotyle of plants, resulting in significant deviations in segregation ratios, when fixed scores were used for disease classification. Independent of origin, the three B-genome resistance genes were introgressed at the same location of the rapeseed genome. The arrangement and distances of closely linked RFLP markers on linkage-groups were similar to those of the same markers on linkage group six of the rapeseed map. It is concluded that the B-genome resistance genes were introgressed by homoeologous recombination after allosyndetical pairing of B-genome chromosomes with the A- or C-genome chromosomes. Received: 3 April 1998/Accepted: 22 April 1998     

Mitochondrial DNA Polymorphism in Natural Populations of the Drosophila virilis Species Group

Restriction fragment length polymorphism (RFLP) analysis has been used to evaluate mitochondrial DNA (mtDNA) variation in 12 sibling species forming the Drosophila virilis species group. The variation thresholds corresponding to the interspecific and interstrain levels have been determined. The results indicate that interspecific hybridization has significantly contributed to the evolutionary history of the virilis species group.     

Foot differentiation and genomic plasticity in Hydra: lessons from the PPOD gene family

In Hydra, developmental processes are permanently active to maintain a simple body plan consisting of a two-layered, radially symmetrical tube with two differentiated structures, head and foot. Foot formation is a dynamic process and includes terminal differentiation of gastric epithelial cells into mucous secreting basal disc cells. A well-established marker for this highly specialized cell type is a locally expressed peroxidase (Hoffmeister et al. 1985). Based on the foot-specific peroxidase activity, the gene PPOD1 has been identified (Hoffmeister-Ullerich et al. 2002). Unexpectedly, this approach led to the identification of a second gene, PPOD2, with high sequence similarity to PPOD1 but a strikingly different expression pattern. Here, we characterize PPOD2 in more detail and show that both genes, PPOD1 and PPOD2, are members of a gene family with differential complexity and expression patterns in different Hydra species. At the genomic level, differences in gene number and structure within the PPOD gene family, even among closely related species, support a recently proposed phylogeny of the genus Hydra and point to unexpected genomic plasticity within closely related species of this ancient metazoan taxon. Electronic supplementary material Supplementary material is available in the online version of this article at     

Species‐specific coordinated gene expression and trans‐regulation of larval color pattern in three swallowtail butterflies

SUMMARY The diversity of butterfly larval color pattern has been attracted to people since Darwin's time; however, its molecular mechanisms still remain largely unknown. Larval body markings often differ completely between closely related species under natural selection. The final instar larvae of the swallowtail butterflies Papilio xuthus and Papilio polytes show a green camouflage pattern, whereas those of Papilio machaon show a warning color pattern, although P. xuthus and P. machaon are closely related species. To identify the genes that contribute to species divergence, we compared the expression pattern of eight pigment‐associated genes between three Papilio species. The spatial expression pattern of melanin‐related genes coincided with the species‐specific cuticular markings. We newly found that the combination of bilin‐binding protein and yellow‐related gene (YRG) correlated perfectly with larval blue, yellow, and green coloration. To distinguish whether the interspecific differences in pigment‐associated genes are caused by cis‐regulatory changes or distribution differences in trans‐regulatory proteins, we compared species‐specific mRNA expression in an F1 hybrid specimen. Px‐YRG and Pp‐YRG showed a similar expression pattern, suggesting that the change in expression of YRG is caused mainly by changes in the distribution of trans‐regulatory proteins. Our findings shed light on the gene regulatory networks for butterfly larval color pattern.     

Perturbation of Parentally Biased Gene Expression during Interspecific Hybridization

Interspecific hybridization often induces epigenetic remodeling that leads to transposon activation, gene expression changes, and loss of imprinting. These genomic changes can be deleterious and contribute to postzygotic hybrid incompatibility. In Arabidopsis, loss of genomic imprinting of PHERES1 and presumed failure of Polycomb Repressive Complex contributes to seed inviability observed in A. thaliana X A. arenosa interspecific hybrids. We used this species pair to further analyze the relationship between parentally biased gene expression and postzygotic hybrid incompatibility using two A. thaliana accessions, Col-0 and C24, with differential seed survival. We found that parentally biased expression was perturbed to a similar degree in both A. thaliana hybrids for PHERES1, HDG3, and six other normally paternally expressed genes. We propose that early genome remodeling and loss of imprinting of seed development genes induces lethality in both compatible and incompatible hybrids.     

Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, andexpression analysis

Background  

In recent years, the relaxin family of signaling molecules has been shown to play diverse roles in mammalian physiology, but little is known about its diversity or physiology in teleosts, an infraclass of the bony fishes comprising ~ 50% of all extant vertebrates. In this paper, 32 relaxin family sequences were obtained by searching genomic and cDNA databases from eight teleost species; phylogenetic, molecular evolutionary, and syntenic data analyses were conducted to understand the relationship and differential patterns of evolution of relaxin family genes in teleosts compared with mammals. Additionally, real-time quantitative PCR was used to confirm and assess the tissues of expression of five relaxin family genes in Danio rerio and in situ hybridization used to assess the site-specific expression of the insulin 3-like gene in D. rerio testis.