Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee

Summary The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 11 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.     

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea)

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.     

DNA methylation in stingless bees with low and high heterochromatin contents as assessed by restriction enzyme digestion and image analysis.

BACKGROUND: The stingless bee genus Melipona has been divided into two groups, based on their heterochromatin content. Melipona quadrifasciata and Melipona rufiventris have low and high levels of heterochromatin, respectively. Since condensed chromatin may be rich in methylated DNA sequences, M. quadrifasciata and M. rufiventris nuclei may contain different amounts of methylated CpG. These differences could be assessed by comparing Feulgen-DNA values obtained by image analysis of cells treated with the restriction enzymes Msp I and Hpa II that distinguish between methylated and unmethylated DNA. Msp I and Hpa II cleave the sequence -CCGG-, but there is no cleavage by Hpa II if the cytosine of the central CG dinucleotide is methylated. METHODS: Malpighian tubules of M. quadrifasciata and M. rufiventris were treated with Msp I and Hpa II prior to the Feulgen reaction, and analyzed by automatic scanning microspectrophotometry. RESULTS: The Feulgen-DNA values for the heterochromatin of M. rufiventris and for the small heterochromatin and some euchromatin domains of M. quadrifasciata mostly decreased after treatment with Msp I, but were unchanged after treatment with Hpa II. CONCLUSION: CpG methylation, although detected in diverse chromatin compartments in different bee species, may induce silencing effects required for the same cell physiology.     

Direct amplification of length polymorphisms (DALP), or how to get and characterize new genetic markers in many species.

Direct amplification of length polymorphisms (DALP) uses an arbitrarily primed PCR (AP-PCR) to produce genomic fingerprints and to enable sequencing of DNA polymorphisms in virtually any species. Oligonucleotide pairs were designed to each produce a specific multi-banded pattern and all the fragments thus generated can be directly sequenced with the same two universal M13 sequencing primers. This strategy combines the advantages of a high resolution fingerprint technique and the possibility of characterizing the polymorphisms. The use of family members as templates in the multi-locus detection step allows a direct test of allele transmission, as well as early mapping of the markers or selection of loci associated with some traits or diseases. We used this method to detect micro-deletions/insertions and microsatellite DNA loci useful in population genetics studies, but it could be applied in many other fields of biology, such as genome mapping for definition of polymorphic sequence tagged sites, directly localized on a genetic map.     

Transferability of microsatellite primers developed for stingless bees to four other species of the genus Melipona

Microsatellite markers are a useful tool for ecological monitoring of natural and managed populations. A technical limitation is the necessity for investment in the development of primers. Heterologous primers can provide an alternative to searching for new loci. In bees, these markers have been used in populational and intracolonial genetic analyses. The genus Melipona has the largest number of species among bee genera, about 70, occurring throughout the Neotropical region. However, only five species of the genus Melipona have specific microsatellite markers. Given the great diversity of this genus, this number is not representative. We analyzed the transferability of 49 microsatellite loci to four other species of the genus Melipona (M. scutellaris, M. mondury, M. mandacaia, and M. quadrifasciata). Four individuals of each species, from different localities, were used in amplification tests. Primer pairs described for five Melipona species and for Trigona carbonaria were tested. Among the 49 loci, 22 gave amplification products for all four species, while three gave nonspecific bands and five showed no amplification products. The remaining loci varied in the pattern of amplification, according to the species examined. The number of alleles ranged from 1 to 6. The results demonstrate the possibility of using these heterologous markers in other Melipona species, increasing the number of loci that can be analyzed and contributing to further genetic analyses of intra- and intercolonial structure, which is required for conservation measure planning, genetic improvement and resolution of taxonomic problems.     

Development of molecular markers using MFLP linked to a gene conferring resistance to Diaporthe toxica in narrow-leafed lupin ( Lupinus angustifolius L.)

Phomopsis stem blight (PSB) caused by Diaporthe toxica is a major disease in narrow-leafed lupin ( Lupinus angustifolius L.). The F(2) progeny and the parental plants from a cross between a breeding line 75A:258 (containing a single dominant resistance gene Phr1 against the disease) and a commercial cultivar Unicrop (susceptible to the disease) were used for development of molecular markers linked to the disease resistance gene. Two pairs of co-dominant DNA polymorphisms were detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. Both pairs of polymorphisms were isolated from the MFLP gels, re-amplified by PCR, sequenced, and converted into co-dominant, sequence-specific and PCR-based markers. Linkage analysis by MAPMAKER suggested that one marker (Ph258M2) was 5.7 centiMorgans (cM) from Phr1, and the other marker (Ph258M1) was 2.1 cM from Ph258M2 but further away from Phr1. These markers are suitable for marker-assisted selection (MAS) in lupin breeding.     

Using AFLP to resolve phylogenetic relationships in a morphologically diversified plant species complex when nuclear and chloroplast sequences fail to reveal variability

Inferring phylogenetic relationships among closely related plant species is often difficult due to the lack of molecular markers exhibiting enough nucleotide variability at this taxonomic level. Moreover, gene tree does not necessary represent the true species tree because of random sorting of polymorphic alleles in different lineages. A solution to these problems is to use many amplified fragment length polymorphisms (AFLP) distributed throughout the whole genome, to infer cladistic and phenetic among-species relationships. Phylogenetic relationships among interfertile species of Trollius L. (Ranunculaceae) were investigated using nuclear DNA (ITS1+5.8S rRNA+ITS2) and chloroplast DNA (trnL intron and trnL-trnF intergene spacer) sequences, and AFLP markers. ITS sequences were not informative at the intrageneric level, but confirmed the sister relationship between Trollius and Adonis genera, and provided new information on the phylogenetic relationships among five Ranunculaceae genera. Chloroplast DNA was more informative among Trollius species, but not consistent with the sections previously described. AFLP proved to be a powerful tool to resolve the complex genetic relationships between the morphological entities constituting the genus Trollius. Although as much as 76.1% of the total AFLP variability was found within a priori defined morphological groups, the remaining 23.9% variability differentiating groups was sufficient to generate congruent and robust cladistic and phenetic trees. Several morphological traits, independent from those used to define groups, were mapped onto the molecular phylogeny, and their evolution discussed in relation to the absence/presence of pollinator-seed parasite Chiastocheta flies.     

Isolation of Y chromosome-specific microsatellites in the horse and cross-species amplification in the genus Equus

Y chromosome polymorphisms such as microsatellites or single nucleotide polymorphisms represent a paternal counterpart to mitochondrial DNA (mtDNA) for evolutionary and phylogeographic studies. The use of Y chromosome haplotyping in natural populations of species other than humans is still hindered by the lack of sequence information necessary for polymorphism screening. Here we used representational difference analysis (RDA) followed by a screen of a bacterial artificial chromosome (BAC) library for repetitive sequences to obtain polymorphic Y-chromosomal markers. The procedure was performed for the domestic horse (Equus caballus) and we report the first six Y-chromosomal microsatellite markers for this species. Three markers were also useful for haplotyping taxa of the zebra/ass lineage. Y-chromosomal microsatellite markers show a single haplotype in the domestic horse, whereas notable variation has been observed in the other members of the genus Equus.     

Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.     

Identification of apple cultivars using RAPD markers

Summary Eleven apple cultivars were differentiated using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). The variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in their presence or absence was chosen to create a differentiating band pattern. A key is proposed by which one can differentiate apple cultivars using commercially available prime.     

Screening apples for OPD20/600 using sequence-specific primers

Apple scab, caused by Venturia inaequalis (Cke.) Wint., is the most serious disease of apple trees in many areas of the world. Resistance to V. inaequalis, derived from the small-fruited species Malus floribunda 821, is determined by a major dominant gene, Vf. Using random decamer primers, we identified a RAPD marker, OPD20/600, which is linked to the Vf gene. OPD20/600 was then cloned and sequenced. Sequence-specific primers based on the marker were used to further screen M. floribunda 821, 7 scab-susceptible apple cultivars, 10 scab-resistant apple cultivars, and 28 scab-resistant Coop selections. The sequence-specific primers allowed identification of polymorphisms of OPD20/600 based on the presence or absence of a single band. The advantages of sequence-specific primers over decamer primers for developing genetic markers are discussed.     

Temperature gradient capillary electrophoresis (TGCE)–a tool for the high-throughput discovery and mapping of SNPs and IDPs

Temperature gradient capillary electrophoresis (TGCE) can be used to distinguish heteroduplex from homoduplex DNA molecules and can thus be applied to the detection of various types of DNA polymorphisms. Unlike most single nucleotide polymorphism (SNP) detection technologies, TGCE can be used even in the absence of prior knowledge of the sequences of the underlying polymorphisms. TGCE is both sensitive and reliable in detecting SNPs, small InDel (insertion/deletion) polymorphisms (IDPs) and simple sequence repeats, and using this technique it is possible to detect a single SNP in amplicons of over 800 bp and 1-bp IDPs in amplicons of approximately 500 bp. Genotyping data obtained via TGCE are consistent with data obtained via gel-based detection technologies. For genetic mapping experiments, TGCE has a number of advantages over alternative heteroduplex-detection technologies such as celery endonuclease (CELI) and denaturing high-performance liquid chromatography (dHPLC). Multiplexing can increase TGCEs throughput to 12 markers on 94 recombinant inbreds per day. Given its ability to efficiently and reliably detect a variety of subtle DNA polymorphisms that occur at high frequency in genes, TGCE shows great promise for discovering polymorphisms and conducting genetic mapping and genotyping experiments.Electronic Supplementary Material Supplementary material is available for this article at     

The identification of random amplified polymorphic DNA markers for daylength insensitivity in oat.

Daylength insensitive accessions of Avena sativa L. are being used to develop cultivars that will flower normally when grown under short or long photoperiods. Field data indicate that the insensitivity trait is under the control of a single dominant gene, designated Di1. The random amplified polymorphic DNA (RAPD) technique and bulk segregant analysis of daylength sensitive and insensitive plants were used to find markers for this gene. Five of 200 random decamer primers tested produced polymorphic bands, which were shown to be linked to the trait using 30 homozygous insensitive and 30 homozygous sensitive F3 individuals. Three of the markers produced a band in the presence of the dominant allele, and two in its absence. Segregation analysis showed that markers 221 and 136 could be mapped to within 9.8 +/- 4.6 and 13.9 +/- 5.4 cM of the trait, respectively; that is, close enough to be useful in a breeding program. A study of different cultivars suggested that the band produced by primer 136 is actually the more closely linked marker and the only one present in the original Di1 gene donor CAV2700. The possibility of using both markers in populations derived from different cultivars is discussed.     

Exon-primed intron-crossing (EPIC) PCR markers of Helicoverpa armigera (Lepidoptera: Noctuidae)

Applying microsatellite DNA markers in population genetic studies of the pest moth Helicoverpa armigera is subject to numerous technical problems, such as the high frequency of null alleles, occurrence of size homoplasy, presence of multiple copies of flanking sequence in the genome and the lack of PCR amplification robustness between populations. To overcome these difficulties, we developed exon-primed intron-crossing (EPIC) nuclear DNA markers for H. armigera based on ribosomal protein (Rp) and the Dopa Decarboxylase (DDC) genes and sequenced alleles showing length polymorphisms. Allele length polymorphisms were usually from random indels (insertions or deletions) within introns, although variation of short dinucleotide DNA repeat units was also detected. Mapping crosses demonstrated Mendelian inheritance patterns for these EPIC markers and the absence of both null alleles and allele 'dropouts'. Three examples of allele size homoplasies due to indels were detected in EPIC markers RpL3, RpS6 and DDC, while sequencing of multiple individuals across 11 randomly selected alleles did not detect indel size homoplasies. The robustness of the EPIC-PCR markers was demonstrated by PCR amplification in the related species, H. zea, H. assulta and H. punctigera.     

Evaluation of the neotropical stingless bee Melipona quadrifasciata (Hymenoptera: Apidae) as pollinator of greenhouse tomatoes

The Neotropical stingless bee Melipona quadrifasciata Lepeletier was evaluated for pollinating tomatoes (variety Rodas; long-life hybrid) in greenhouses under plastic and with a hydroponic system and "organic concepts" in Minas Gerais State, Brazil. Flowers not pollinated did not set any fruit. Pollination by bees plus manual pollination did not differ from either bee or manual pollination. Maximum fruit diameter, fruit height, and roundness (quotient between maximum fruit diameter and fruit height) were not significantly different between treatments, but fruit visited by M. quadrifasciata had 10.8% less seeds (dry mass) than manual pollination. This apparently low efficiency of M. quadrifasciata pollination was attributed to the overlap of only 30 min between highest bee foraging activity and highest flower stigma receptivity. Thus, it was concluded that M. quadrifasciata is a feasible pollinator of greenhouse tomatoes because of 1) the observed increase in fruit quality with lower mechanical injury than traditional manual pollination, 2) no significant decrease in fruit size, and 3) high price of such product in the market. Some considerations for sustainable use of M. quadrifasciata as greenhouse pollinator are presented. Although techniques for keeping captive colonies of M. quadrifasciata are currently available, the sole current method for acquiring new colonies is removing them from the forest, and if demand was created for large numbers of colonies for commercial use, techniques for captive rearing must be developed to prevent serious declines in wild populations.     

Molecular determination of species boundaries in corals: genetic analysis of the Montastraea annularis complex using amplified fragment length polymorphisms and a microsatellite marker

Analyses of DNA have not been widely used to distinguish coral sibling species. The three members of the Montastraea annularis complex represent an important test case: they are widely studied and dominate Caribbean reefs, yet their taxonomic status remains unclear. Analysis of amplified fragment length polymorphisms (AFLPs) and a microsatellite locus, using DNA from sperm, showed that Montastraea faveolata is genetically distinct. One AFLP primer yielded a diagnostic product (880 bp in M. faveolata 920 bp in M. franksi and M. annularis) whose homology was established by DNA sequencing. A second primer revealed a 630 bp band that was fixed in M. faveolata, and rare in M. franksi and M. annularis; in this case homologies were confirmed by Southern hybridizations. A tetranucleotide microsatellite locus with several alleles exhibited strong frequency differences between M. faveolata and the other two taxa. We did not detect comparable differences between M. annularis and M. franksi with either AFLPs (12 primers screened) or the microsatellite locus. Comparisons of AFLP patterns obtained from DNA from sperm, somatic tissues, and zooxanthellae suggest that the technique routinely amplifies coral (animal) DNA. Thus analyses based on somatic tissues may be feasible, particularly after diagnostic differences have been established using sperm DNA.     

Identification of Capsicum species using SNP markers based on high resolution melting analysis

Single nucleotide polymorphisms (SNPs) derived from both nuclear and cytoplasmic DNA sequences were developed to identify distinct species of Capsicum. Species identification was achieved by detecting allelic variations of these type of markers via high resolution melting analysis (HRM). We used the HRM polymorphisms of COSII markers and the Waxy gene from the nuclear sequence, in addition to the intergenic spacer between trnL and trnF from cytoplasmic DNA as our SNP markers. A total of 31 accessions of Capsicum, representing six species, were analyzed using this method. As single markers were insufficient for identifying Capsicum species, combinations of all markers unambiguously identified all six. A phylogeny based on the SNP markers was consistent with the current taxonomy of Capsicum species. These observations demonstrate that the markers developed in this study are useful for rapid identification of new germplasm for management of Capsicum species.     

Intensive Linkage Mapping in a Wasp (Bracon Hebetor) and a Mosquito (Aedes Aegypti) with Single-Strand Conformation Polymorphism Analysis of Random Amplified Polymorphic DNA Markers

The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito, Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A. aegypti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.