Assessment of genetic diversity of native species in Izu Islands for a discriminate choice of source populations: Implications for revegetation of volcanically devastated sites

In using native species for revegetation, it is necessary choose source populations carefully to reduce the risk of planting suboptimal germplasm. To make preliminary recommendations for native species to use in the revegetation of a volcanically devastated area on Miyake Is., Japan, we investigated the genetic variation of Alnus sieboldiana, Miscanthus sinensis ssp. condensatus, and Polygonum cuspidatum var. terminalis in the Izu Islands and on the Izu Peninsula based on chloroplast DNA (cpDNA) sequence variations and amplified fragment length polymorphisms (AFLPs). The amount and pattern of differentiation differ between organelle and nuclear markers, suggesting the necessity of evaluation based on both types of markers. Within-population diversity did not vary among populations, suggesting that it does not need to be considered in the choice of a source population. The pattern and degree of differentiation varied among species, and geographical proximity did not necessarily accord with genetic similarity, suggesting that the site of an appropriate source population varies among species and should be determined empirically rather than by assuming that close proximity predicts genetic similarity. The Izu Peninsula populations deviated from the island populations in all species. Comparison of cpDNA sequences with those of related species indicates the possibility of hybridization with related species on the Izu Peninsula, suggesting that seeds collected from populations where related species live sympatrically should not be used for revegetation. These findings indicate the need to assess the genetic diversity empirically by using organelle and nuclear markers to avoid unintended consequences of genetic mixing associated with revegetation.     

Molecular characterization of reciprocal crosses of Aerides vandarum and Vanda stangeana (Orchidaceae) at the protocorm stage

Aerides vandarum and Vanda stangeana are two rare and endangered vandaceous orchids with immense floricultural traits. The intergeneric hybrids were synthesized by performing reciprocal crosses between them. In vitro germination response of the immature hybrid embryos was found to be best on half-strength Murashige and Skoog medium supplemented with 20% (v/v) coconut water/liquid endosperm from tender coconut. Determination of hybridity was made as early as the immature seeds or embryos germinated in vitro, using randomly amplified polymorphic DNA (RAPD) markers. Out of 15 arbitrarily chosen decamer RAPD primers, two were found to be useful in amplification of polymorphic bands specific to the parental species and their presence in the reciprocal crosses. However, a decisive profile that can identify the reciprocal crosses could not be provided by RAPD. Amplification of the trnL-F non-coding regions of chloroplast DNA of the parent species and hybrids aided easy identification of the reciprocal crosses from the fact that maternal inheritance of chloroplast DNA held true for these intergeneric hybrids. Subsequent restriction digestion of the polymerase chain reaction (PCR) amplified trnL-F non-coding regions of chloroplast DNA also consolidated the finding. Such PCR-based molecular markers could be used for early determination of hybridity and easy identification of the reciprocal crosses.     

Attempted pollen-mediated plant transformation employing genomic donor DNA

Summary Experiments were conducted to test the validity of previous reports of pollen-mediated plant transformation utilizing genomic donor DNA. Multiple Mendelian markers were employed in Zea mays L. and Lycopersicon esculentum Mill, to detect transformation events. Pollen from multiple recessive (recipient) lines was incubated with genomic DNA from multiple dominant (donor) lines, under various conditions. Treated pollen was subsequently used for pollinations on multiple recessive females, and resulting seeds were screened for transformation events. Over 200 crosses were made in tomato, and over 80 crosses were made in corn. Over 600 resulting seedlings were tested in tomato and over 800 seeds were screened in corn. Because multiple markers were used, 4,937 potential transformation events were screened. No clear-cut transformation events were observed. Therefore, using well-defined multiple markers, we have been unable to confirm the earlier claims of high efficiency pollen-mediated transformation employing genomic donor DNA.     

Discrimination of species and strains of basidiomycete genusCoprinus by random amplified polymorphic DNA (RAPD) analysis

All five examined strains ofCoprinus cinereus could be clearly discriminated from the strains of five otherCoprinus species by RAPD patterns with 12 of 13 primers. Also one specimen of unknownCoprinus strain was identified to beC. cinereus by this method. The RAPD patterns were similar among the strains in the same species; many common DNA fragments were recognized as well as some strain-specific DNA fragments. Thus all seven strains ofC. cinereus and all four strains ofC. angulatus examined could be distinguished individually. Diakryotic strains showed the combined RAPD patterns of the two monokaryotic strains constituting the dikaryon. The combined RAPD markers observed in the dikaryons were segregated in their basidiospore progeny. All 18 randomly picked progeny showed different combinations of RAPD markers from the parental strains.     

Molecular Marker Analysis of Differentially Aged Seeds of Soybean and Safflower

Storage of seeds for extended periods causes a number of degradative changes related to the aging process such as decreased seedling vigor and reduced germination. In this study, molecular markers were used to study the aging process in seeds of two different plants species. Seeds of three differentially aged seed groups, including control (un-aged), naturally aged, and accelerated aging, from soybean (Glycine max) and safflower (Carthamus tinctorius) were evaluated for genetic variability using random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) markers. For both plant species, naturally aged and accelerated aged groups clustered together with RAPD markers, whereas control and naturally aged seeds showed similarity in both AFLP and SSR profiles. Based on these findings, it can be concluded that observed changes in DNA profiles of seeds from different aged groups did not contribute to accumulation of genetic variations of the same magnitude. Therefore, seed of similar viability must be selected for molecular marker analysis for plant variety protection, among other comparative studies.     

RAPD characterisation of two neotropical hybrid legumes

An investigation of randomly amplified polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker distribution was made for two well-characterised hybrids and their parents,Leucaena leucocephala andL. esculenta andParkinsonia aculeata andCercidium praecox. Three chloroplast DNA (cpDNA) markers identified the maternal parent of eachL. leucocephala ×L. esculenta hybrid. Fifteen species-diagnostic RAPD markers (invariant in one taxon and absent from the other) were always present in theLeucaena hybrid and assumed to be of nuclear origin, whilst three RAPD markers showed expression patterns identical to the cpDNA markers and were assumed to be of organellar origin. No RAPD or PCR-RFLP taxon-diagnostic markers were discovered for eitherP. aculeata orC. praecox. However, 21 RAPD markers were species-specific (polymorphic within one taxon but absent from the other) and Southern analysis indicated that none of the markers were of organellar origin. Only 67% additivity of markers specific toP. aculeata andC. praecox was demonstrated in the hybrids between these two species, whilst inLeucaena 97% additivity was demonstrated. Differences between the two hybridising situations were related to the behaviour of the molecular markers and the biology of the species.     

Gene flow among different taxonomic units: evidence from nuclear and cytoplasmic markers in Cedrus plantation forests

Hybridization and introgression are important natural evolutionary processes that can be successfully investigated using molecular markers and open- and controlled-pollinated progeny. In this study, we collected open-pollinated seeds from Cedrus atlantica, Cedrus libani and C. libani × C. atlantica hybrids from three French-plantation forests. We also used pollen from C. libani and Cedrus brevifolia to pollinate C. atlantica trees. The progeny were analyzed using three different types of molecular markers: RAPDs, AFLPs and cpSSRs. Chloroplast DNA was found to be paternally inherited in Cedrus from the progeny of controlled-crosses. Heteroplasmy, although possible, could not be undoubtedly detected. There was no indication of strong reproductive isolating barriers among the three Mediterranean Cedrus taxa. Gene flow between C. atlantica and C. libani accounted for 67 to 81% of viable open-pollinated seedlings in two plantation forests. We propose that Mediterranean Cedrus taxa should be considered as units of a single collective species comprising two regional groups, North Africa and the Middle East. We recommend the use of cpSSRs for monitoring gene flow between taxa in plantation forests, especially in areas where garden specimens of one species are planted in the vicinity of selected seed-stands and gene-conservation reserves of another species.Communicated by D.B. Neale     

Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule

Summary Species-specific highly repeated DNA sequences can be used to screen the progeny of protoplast fusions combining different species. Such probes are easy to clone and can be detected by fast methods, e.g., hybridization to total genomic DNA. Furthermore, due to their high copy number, hybridization signals are strong and represent more than one locus, unlike isozymes or resistance markers. After cloning and screening for species-specific DNA sequences we characterized the highly repeated DNA sequences of the solanaceous species Solanum acaule and Lycopersicon esculentum var. gilva. DNA sequencing and hy ridization revealed a prominent, tandemly arranged satellite DNA repeat of 162 bp in Lycopersicon esculentum and a different satellite repeat of 183 bp, also tandemly organized, in Solanum acaule. Each repeat is absent in the respective other species. Therefore, we have used these DNA repeats as markers to distinguish regenerated interspecific somatic hybrids from the respective fusion partners. These hybrids were clearly identified by Southern hybridization and dot-blot assays to the respective ³²P-labelled satellite DNA.     

A PCR-based marker tightly linked to the nematode resistance gene,Mi, in tomato

A PCR-based codominant marker has been developed which is tightly linked to Mi, a dominant genetic locus in tomato that confers resistance to several species of root-knot nematode. DNA from tomato lines differing in nematode resistance was screened for random amplified polymorphic DNA markers linked to Mi using decamer primers. Several markers were identified. One amplified product, REX-1, obtained using a pair of decamer primers, was present as a dominant marker in all nematode-resistant tomato lines tested. REX-1 was cloned and the DNA sequences of its ends were determined and used to develop 20-mer primers. PCR amplification with the 20-mer primers produced a single amplified band in both susceptible and resistant tomato lines. The amplified bands from susceptible and resistant lines were distinguishable after cleavage with the restriction enzyme Taq I. The linkage of REX-1 to Mi was verified in an F₂ population. This marker is more tightly linked to Mi than is Aps-1, the currently-used isozyme marker, and allows screening of germplasm where the linkage between Mi and Aps-1 has been lost. Homozygous and heterozygous individuals can be distinguished and the procedure can be used for rapid, routine screening. The strategy used to obtain REX-1 is applicable to obtaining tightly-linked markers to other genetic loci. Such markers would allow rapid, concurrent screening for the segregation of several loci of interest.     

Mating system and seed variation of <Emphasis Type="Italic">Acacia</Emphasis> hybrid (<Emphasis Type="Italic">A. mangium</Emphasis> × <Emphasis Type="Italic">A. auriculiformis</Emphasis>)

The mating system and seed variation of Acacia hybrid (A. mangium × A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean±s.e. t _m = 0.86±0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%–70%), followed by among pods within inflorescence (29%–37%), and the least variation among inflorescences within tree (<1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.     

A highly reliable sex diagnostic PCR assay for mass screening of papaya seedlings

Female plants of several dioecious angiosperms are commercially valued for production of fruits or seeds, viz. papaya, nutmeg, pistachio, kiwi fruit and jojoba. To make the cultivation profitable it is necessary to grow more female than male plants. To discriminate between male and female plants, sex-specific molecular markers have been identified in a few dioecious species such as Silene and pistachio. However, accurate and convenient sex diagnostic methods for early sexing of seedlings are not available to date. For the first time, we report here a PCR-based Seedling Sex Diagnostic Assay (SSDA) specially designed for early sexing of papaya seedlings. We have developed a male-specific SCAR marker in papaya by cloning a male-specific RAPD (831 bp) fragment and designing longer primers. The potential of this SCAR marker is further exploited to develop a simplified and highly accurate sex diagnostic assay by (1) including an internal PCR control, (2) following a single-step DNA extraction procedure and (3) optimising the PCR conditions to simultaneously amplify male-specific and control bands from the crude leaf extract. This diagnostic approach would be of great commercial significance to papaya growers as well as to seed companies and plant nurseries for early identification of female seedlings of dioecious species. In principle, this experimental design could be easily applied to molecular analysis of any agriculturally important trait for which specific DNA probes could be identified and hence opens new avenues of research in the field of genetic diagnostics of plants.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

Hybridisation processes in sympatric populations of pines Pinus sylvestris L., P. mugo Turra and P. uliginosa Neumann

Natural hybridisation was postulated between the closely related pine species Pinus sylvestris and the P. mugo complex, however no clear evidence on propagation of mature hybrids in nature has been documented so far. To test the hybridisation hypothesis we applied chloroplast DNA (cpDNA) markers and isozymes in the analyses of 300 individuals representing the variety of morphological forms in the sympatric populations of P. sylvestris, P. mugo and P. uliginosa at the peat bog complex in the Sudety Mts., Poland. Additionally, the haplotypes of paternally inherited cpDNA of 149 open pollinated progeny derived from seeds were compared to the haplotypes of parental trees to access the intensity and direction of contemporary hybridisation. The morphologically highly variable polycormic (multi-stemmed) hybrids between P. mugo and P. uliginosa were identified. The second group of hybrids was found among the monocormic (single-stemmed) P. sylvestris-like individuals carrying the cpDNA from P. mugo complex. Hybrids of P. sylvestris as a pollen donor and P. mugo or P. uliginosa as a mother were not found, either in the group of examined trees, or among the open pollinated progeny. The results indicate that numerous hybrids can exist in the sympatric population of the species studied and that gene flow can successfully proceed from P. mugo complex to P. sylvestris. Hybridisation and ecological selection seems to play a significant role in diversification and evolution of the investigated species.     

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.)

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/–) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F₂ progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F₂ progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.     

Interspecific hybridization in natural populations ofCyrtandra (Gesneriaceae) on the Hawaiian Islands: Evidence from RAPD markers

Interspecific hybridization among Hawaiian species ofCyrtandra (Gesneriaceae) was investigated using randomly amplified polymorphic DNA (RAPD) markers. Thirty-three different primers were used to investigate interspecific hybridization for 17 different putative hybrids based on morphological intermediacy and sympatry with putative parental species. RAPD data provided evidence for the hybrid origin of all putative hybrid taxa examined in this analysis. However, the patterns in the hybrid taxa were not found to be completely additive of the patterns found in the parental species. Markers missing in the hybrid taxa can be attributed to polymorphism in the populations of the parental species and the dominant nature of inheritance for RAPD markers. Unique markers found within hybrid taxa require further explanation but do not necessarily indicate that the taxa are not of hybrid origin. The implications suggest that these interspecific hybridization events had, and continue to have, an effect on the adaptive radiation and conservation biology ofCyrtandra.     

Distribution of parental DNA markers inEncelia virginensis (Asteraceae: Heliantheae), a diploid species of putative hybrid origin

Morphological, geographical and ecological evidence suggests thatEncelia virginensis is a true-breeding diploid species derived from hybrids ofE. actoni andE. frutescens. To test this hypothesis, we examined the chloroplast and nuclear DNA of severalEncelia species. PCR amplification targeted three separate regions of chloroplast DNA:trnK-2621/trnK-11,rbcL/ORF106, andpsbA3/TrnI-51, which amplify 2600bp, 3300bp and 3200bp fragments respectively. Restriction fragment analysis of chloroplast DNA revealed no variation that could be used to discriminate between the parent species. A RAPD analysis using 109 dekamer primers was used to analyze the nuclear genome.Encelia actoni andE. frutescens were distinguished by several high-frequency RAPD markers. In populations ofE. virginensis, these markers were detected in varying proportions, and no unique markers were found. Evidence from the nuclear genome supports the hypothesis thatE. virginensis is of hybrid origin. ThatE. virginensis may have arisen by normal divergent speciation followed by later introgression remains a possibility, however, and is not formally ruled out here. Diploid hybrid speciation inEncelia differs from other documented cases in that there are no discernible chromosome differences between the species, and all interspecific hybrids are fully fertile. In addition, apparent ecological selection against backcross progeny provides an external barrier to reproduction between F₁ progeny and the parental species. These characteristics suggest that hybrid speciation inEncelia may represent an alternative model for homoploid hybrid speciation involving external reproductive barriers. In particular, this may be the case for other proposed diploid hybrid taxa that also exhibit little chromosomal differentiation and have fertile F₁s.     

Gene-assisted selection for high molecular weight glutenin subunits in wheat doubled haploid breeding programs

Molecular markers based on DNA sequence variations of the coding and/or promoter regions of the wheat (Triticum aestivum L.) HMW glutenin genes located at the Glu-1 loci were developed. Markers characteristic of alleles Glu-A1-1a (encoding Ax1 subunit) and Glu-A1-1c (encoding Ax2* subunit) at the Glu-A1 locus, alleles Glu-B1ak (encoding Bx7* subunit) and Glu-B1al for overexpressed Bx7 subunit at the Glu-B1 locus and alleles Glu-D1-1a (encoding Dx2 subunit) and Glu-D1-1d (encoding Dx5 subunit) at the Glu-D1 locus were tested using genomic DNA of haploid leaf tissue. A method for simultaneously extracting DNA from 96 haploid leaf tissue pieces is described. Two of the developed markers were dominant and two were co-dominant. A F₁-derived population segregating for all HMW glutenin genes was used to test the validity of the markers and their usefulness in doubled haploid breeding programs. SDS-PAGE analysis of seed storage protein was performed on seeds from the doubled haploid lines. A total of 299 lines were tested with the DNA markers on the haploid tissue and validated by protein analysis of the corresponding DH seeds. PCR markers and SDS-PAGE analysis showed between 2 and 8.5% discrepancies depending on the marker. Applications of DNA markers for gene-assisted-selection of haploid tissue and use in breeding programs are discussed. Advantages and disadvantages of dominant and co-dominant markers are outlined.     

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression

Summary Nearly isogenic lines (NILs) of rice (Oryza sativa) differing at a locus conferring resistance to the pathogen Xanthomonas oryzae pv. oryzae were surveyed with 123 DNA markers and 985 random primers using restriction fragment length plymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. One chromosome 11 marker (RG103) detected polymorphism between the NILs that cosegregated with Xa21. All other chromosome 11 DNA markers tested were monomorphic between the NILs, localizing the Xa21 introgressed region to an 8.3 cM interval on chromosome 11. Furthermore, we identified two polymerase chain reaction (PCR) products (RAPD2148 and RAPD818) that detected polymorphisms between the NILs. Genomic sequences hybridizing with RAPD818, RAPD248 and RG103 were duplicated specifically in the Xa21 NIL. All three markers cosegregated with the resistance locus, Xa21, in a F₂ population of 386 progeny. Based on the frequency with which we recovered polymorphic Xa21-linked markers, we estimated the physical size of the introgressed region to be approximately 800 kb. This estimation was supported by physical mapping (using pulsed field gel electrophoresis) of the sequences hybridizing with the three Xa21-linked DNA markers. The results showed that the three Xa21-linked markers are physically close to each other, with one copy of the RAPD818 sequences located within 60 kb of RAPD248 and the other copy within 270 kb of RG103. None of the enzymes tested generated a DNA fragment that hybridized with all three of the markers indicating that the introgressed region containing the resistance locus Xa21 is probably larger than 270 kb.     

A set of simple PCR markers converted from sequence specific RFLP markers on tomato chromosomes 9 to 12

A set of 24 simple PCR markers was generated for tomato chromosomes 9, 10, 11 and 12. Polymorphism was sought for between Lycopersicon esculentum and one of six other Lycopersicon species (L. parviflorum, L. cheesmanii, L. hirsutum, L. pennellii, L. peruvianum, and L. chilense). PCR primers, which were designed from mapped RFLP sequences, were used to amplify genomic DNA of the different species and the PCR amplification products were screened for polymorphism by testing restriction enzymes. With this approach, 24 (71%) of the 34 selected RFLPs were converted into simple PCR markers. By using a reference population, the map positions of these markers relative to the original RFLP markers were verified. These markers are locus specific and can be efficiently used for alignment of linkage maps, mapping target genes and marker assisted selection.