Discordance in the distribution of markers of different inheritance systems (nDNA, mtDNA, and chromosomes) in superspecies complex Mus musculus as a result of extensive hybridization in Primor'e

The genetic structure of eight Mus musculus L. populations in Primorskii krai was studied with the use of taxon-specific markers of different inheritance systems: nDNA (RAPD), mtDNA (D-loop), and chromosomes. The results obtained demonstrate that although the compared nuclear marker characteristics (nDNA and chromosomes) have the same basis they are not linke with each other and, moreover, are often mutually inconsistent. Discordance in the inheritance of the marker characteristics in most of the animals studied is a result of extensive hybridization involving two to four house mouse subspecies. To identify taxon-specific nuclear markers revealed by RAPD, they were cloned and sequenced, and their localization on chromosomes was determined. It was found that some fragments similar in size consist of two different comigrating sequences that are localized on different chromosomes and belong to different subspecies. All sequenced anonymous markers are localized in protein-coding genes. The functions of genes containing the marker sequences have been established. Differences in the taxon-specific RAPD fragments are associated with changes in the structure of important functional genes, and this can be considered as a significant genetic marker.     

Identification and mapping of polymorphic RAPD markers of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) genome

Various pea cultivars, lines, and mutants were studied by the RAPD method. Polymorphic fragments characteristic of certain pea genotypes and which can be used for identifying genotypes were detected. Inheritance of some polymorphic RAPD fragments was studied. Mendelian inheritance of these fragments was shown. By analyzing the data obtained in studies of RAPD polymorphism, genetic distances between different pea cultivars, lines, and mutants were calculated and a genealogic dendogram showing a varying extent of differences between RAPD patterns was constructed. Ten new RAPD markers linked to various pea genes were detected. Genetic distances between RAPD markers and genes to which they are linked were calculated, and the respective disposition of RAPD markers on chromosomes was established.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 341–348.Original Russian Text Copyright © 2005 by Koveza, Kokaeva, Konovalov, Gostimsky.     

Identification and mapping of polymorphic RAPD markers of pea (Pisum sativum L.) genome

Various pea cultivars, lines, and mutants were studied by the RAPD method. Polymorphic fragments characteristic of certain pea genotypes and which can be used for identifying genotypes were detected. Inheritance of some polymorphic RAPD fragments was studied. Mendelian inheritance of these fragments was shown. By analyzing the data obtained in studies of RAPD polymorphism, genetic distances between different pea cultivars, lines, and mutants were calculated and a genealogic dendogram showing a varying extent of differences between RAPD patterns was constructed. Ten new RAPD markers linked to various pea genes were detected. Genetic distances between RAPD markers and genes to which they are linked were calculated, and the respective disposition of RAPD markers on chromosomes was established.     

Mapping of avirulence genes in the rice blast fungus, Magnaporthe grisea, with RFLP and RAPD markers

Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoi and AVR1-Ku86 were closely linked to telomere RFLPs such as marker TelG (6 cM from AVR1-MedNoi) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.     

Isolation of sex-specific AFLP markers in Spotted Halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>)

Spotted Halibut (Verasper variegatus) is a commercially important marine fish species. In the present study, to isolate sex-specific markers in Spotted Halibut, we screened the genomes of Spotted Halibut by AFLP technique with 64 different primer combinations. Two primer combinations, MseI-CAG/EcoRI-ACC and MseI-CAT/EcoRI-AGG, produced a female-specific fragment in all females (n = 88) and in no males (n = 60, except 3 individuals), respectively. Both fragments were excised from the gel, cloned and characterized. The first fragment (named VevaF533) was 533 bp long, while the length of the second one (named VevaF218) was 218 bp. The two sequences showed no similarity to each other, and to the known sequences existing in the GenBank database using BLASTn. Cross-species amplification showed that the marker VevaF218 is a species-specific marker which is present in Spotted Halibut females but absent in Barfin Flounder (Verasper moseri). So this marker could be used for discriminating unambiguously between Spotted Halibut females and Barfin Flounder. Examination of the patterns of inheritance of VevaF218 in an interspecific hybrid family (V. variegatus ♂×V. moseri ♀) showed a female-specific pattern of inheritance from mother to daughter, implying that the marker VevaF218 is located on the female sex chromosome. This study provides a reliable AFLP-based genetic sexing of Spotted Halibut that could be useful for genetic mapping of the sex chromosomes and identification of sex-linked genes.     

RAPDs as molecular markers for the detection of Aegilops markgrafii chromatin in addition and euploid introgression lines of hexaploid wheat

 Aegilops markgrafii contains resistance genes to powdery mildew, leaf rust and stripe rust, and also has high crude protein and lysine contents, which can be useful for wheat improvement. These important traits are localized on different chromosomes. Disomic Triticum aestivum-Ae. markgrafii addition lines and euploid introgression lines showing leaf-rust and powdery mildew resistance were screened with RAPDs to detect chromosome-specific markers which can accelerate the breeding process. RAPD markers for all six available disomic addition lines were obtained. The additional chromosomes B, C, D, E, F and G were identified by three, three, three, two, one and seven primers, respectively. All three chromosome-B-specific RAPD markers demonstrated the presence of alien chromatin in the leaf-rust-resistant 42-chromosome introgression lines as well as in the segregating progeny. The three chromosome-C-identifying primers also demonstrated the presence of that chromosome in powdery mildew-resistant euploid introgression lines. The substitution lines (5A)5C and (5D)5C with different genetic backgrounds for both parents, in comparison to the lines mentioned above, showed the chromosome C-specific band with only two of the three primers. The chromosome F-specific primer and a primer evident on all the Ae. markgrafii chromosomes analysed did not generate the expected fragments on the chromosome F_del addition line, indicating that the markers are located on the deleted part of chromosome F. Received: 20 August 1996 / Accepted 17 January 1997     

Nuclear mitochondrial pseudogenes

As has been demonstrated recently, the transfer of genetic material from mitochondria to the nucleus and its integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) are incorporated in the nuclear genome as noncoding sequences, which are called nuclear mitochondrial pseudogenes (NUMT pseudogenes or NUMT inserts). In various eukaryotes, NUMT pseudogenes are distributed through different chromosomes to form a “library” of mtDNA fragments, providing important information on genome evolution. The escape of mtDNA from mitochondria is mostly associated with mitochondrial damage and mitophagy. Fragments of mtDNA may be integrated into nuclear DNA (nDNA) during repair of double-strand breaks (DSBs), which are caused by endogenous or exogenous agents. DSB repair of nDNA with a capture of mtDNA fragments may occur via nonhomologous end joining or a similar mechanism that involves microhomologous terminal sequences. An analysis of the available data makes it possible to suppose that the NUMT pseudogene formation rate depends on the DSB rate in nDNA, the activity of the repair systems, and the number of mtDNA fragments leaving organelles and migrating into the nucleus. Such situations are likely after exposure to damaging agents, first and foremost, ionizing radiation. Not only do new NUMT pseudogenes change the genome structure in the regions of their integration, but they may also have a significant impact on the actualization of genetic information. The de novo integration of NUMT pseudogenes in the nuclear genome may play a role in various pathologies and aging. NUMT pseudogenes may cause errors in PCR-based analyses of free mtDNA as a component of total cell DNA because of their coamplification.     

Development and Study of SCAR Markers in Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

In order to develop more specific markers that characterize particular regions of the pea genome, the data on nucleotide sequences of RAPD fragments were used for choosing more extended primers, which may be helpful in amplifying a fragment corresponding to the particular DNA region. Of the 14 STS markers obtained from 14 polymorphic RAPD fragments, 12 were polymorphic, i.e., they are SCAR markers that can be used in genetic analysis. The transition from complex RAPD spectra to amplification of a particular SCAR marker substantially facilitates analysis of large samples for the presence or absence of the examined fragment. Inheritance of the developed SCAR markers was studied in F₁ and F₂. SCAR markers were used to identify various pea lines, cultivars, and mutants. It was established that the study of amplification of STS markers in various pea genotypes at varying temperatures of annealing and the comparison with amplification of the original RAPD fragments in the same genotypes provide an approach for analysis of RAPD polymorphism origin.     

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.     

Genetic linkage map of ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers

 The potential of PCR-based markers for construction of a genetic linkage map in Einkorn wheat was investigated. From a comparison of polymorphisms between two Einkorn wheats, Triticum monococcum (Mn) and T. boeoticum (Bt), we obtained 49 polymorphic bands produced by 33 primers for inter-simple sequence repeat (ISSR) and 36 polymorphic bands shown by 25 combinations of random amplified polymorphic DNA (RAPD) primers for mapping in 66 individuals in the F₂ population. Although 44 ISSR fragments and 29 RAPD fragments statistically showed a 3 : 1 segregation ratio in the F₂ population, only 9 markers each of the ISSR and RAPD bands were able to be mapped on the RFLP linkage map of Einkorn wheat. ISSR markers were distributed throughout the chromosomes. The mapped positions of the ISSR markers seemed to be similar to those obtained by the RFLP markers. On the other hand, 4 of the 9 RAPD markers could map the RFLP marker-poor region on the short arm of 3A^m, suggesting a potential to map novel regions containing repetitive sequences. Comparisons of the genetic linkage map of Einkorn wheat to the linkage map and cytological map of common wheat revealed that the marker orders between the two maps of Einkorn wheat and common wheat coincided except for 4A, which harbors chromosome rearrangements specific for polyploid wheats, indicating a conservatism between the two genomes. Recombinations in Einkorn wheat chromosomes took place more frequently around the centromere and less at the distal part of chromosomes in comparison to those in common wheat. Nevertheless, recombinations even in Einkorn wheat chromosomes were strongly suppressed around the centromere. In fact, the markers located within 1 cM of the centromere were located almost in the central part of the chromosome arm. Received: 7 June 1997 / Accepted: 17 June 1997     

Genetic variation,population structure and identification of yellow catfish, <Emphasis Type="Italic">Mystus nemurus</Emphasis> (C&;V) in Thailand using RAPD,ISSR and SCAR marker

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four subpopulations of Mystus nemurus in Thailand. The 7 RAPD and 7 ISSR primers were selected. Of 83 total RAPD fragments, 80 (96.39%) were polymorphic loci, and of 81 total ISSR fragments, 75 (92.59%) were polymorphic loci. Genetic variation and genetic differentiation obtained from RAPD fragments or ISSR fragments showed similar results. Percentage of polymorphic loci (%P), observed number of alleles, effective number of alleles, Nei’s gene diversity (H) and Shannon’s information index revealed moderate to high level of genetic variations within each M. nemurus subpopulation and overall population. High levels of genetic differentiations were received from pairwise unbiased genetic distance (D) and coefficient of differentiation. Mantel test between D or gene flow and geographical distance showed a low to moderate correlation. Analysis of molecular variance indicated that variations among subpopulations were higher than those within subpopulations. The UPGMA dendrograms, based on RAPD and ISSR, showing the genetic relationship among subpopulations are grouped into three clusters; Songkhla (SK) subpopulation was separated from the other subpopulations. The candidate species-specific and subpopulation-specific RAPD fragments were sequenced and used to design sequence-characterized amplified region primers which distinguished M. nemurus from other species and divided SK subpopulation from the other subpopulations. The markers used in this study should be useful for breeding programs and future aquacultural development of this species in Thailand.     

Localization of BAC clones on mitotic chromosomes of <Emphasis Type="Italic">Musa acuminata</Emphasis> using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization

A bacterial artificial chromosome (BAC) library of banana (Musa acuminata) was used to select BAC clones that carry low amounts of repetitive DNA sequences and could be suitable as probes for fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes. Out of eighty randomly selected BAC clones, only one clone gave a single-locus signal on chromosomes of M. acuminata cv. Calcutta 4. The clone localized on a chromosome pair that carries a cluster of 5S rRNA genes. The remaining BAC clones gave dispersed FISH signals throughout the genome and/or failed to produce any signal. In order to avoid the excessive hybridization of repetitive DNA sequences, we subcloned nineteen BAC clones and selected their ‘low-copy’ subclones. Out of them, one subclone gave specific signal in secondary constriction on one chromosome pair; three subclones were localized into centromeric and peri-centromeric regions of all chromosomes. Other subclones were either localized throughout the banana genome or their use did not result in visible FISH signals. The nucleotide sequence analysis revealed that subclones, which localized on different regions of all chromosomes, contained short fragments of various repetitive DNA sequences. The chromosome-specific BAC clone identified in this work increases the number of useful cytogenetic markers for Musa.     

A contiguous sequence in spinach nuclear DNA is homologous to three separated sequences in chloroplast DNA

Summary A 3.4-kbp nuclear (n) DNA sequence has greater than 99% sequence homology to three segments of the chloroplast (cp) genes rps2, psbD/C, and psaA respectively. Each of these cpDNA segments is less than 3 kbp in length and appears to be integrated, at least in part, into several (>5) different sites flanked by unique sequences in the nuclear genome. Some of these sites contain longer homologies to the particular genes, while others are only homologous to smaller parts of the cp genes. Both the cpDNA fragments found in the nuclear genome and their flanking nDNA sequences are invested with short repeated A-T rich sequences but, apart from a hexanucleotide sequence and a palindromic sequence identified near each recombination point, there is no obvious structure that can suggest a mechanism of DNA transfer from the chloroplast to the nucleus in spinach.     

Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato

We have cloned and sequenced six RAPD fragments tightly linked to the Tm-1 gene which confers tomato mosaic virus (ToMV) resistance in tomato. The terminal ten bases in each of these clones exactly matched the sequence of the primer for amplifying the corresponding RAPD marker, except for one in which the 5-endmost two nucleotides were different from those of the primer. These RAPD clones did not cross-hybridize with each other, suggesting that they were derived from different loci. From Southern-hybridization experiments, five out of the six RAPD clones were estimated to be derived from middle- or high-repetitive sequences, but not from any parts of the ribosomal RNA genes (rDNA), which are known to be tightly linked with the Tm-1 locus. The remaining clone appeared to be derived from a DNA family consisting of a few copies. These six RAPD fragments were converted to sequence characterized amplified region (SCAR) markers, each of which was detectable using a pair of primers having the same sequence as that at either end of the corresponding RAPD clone. All pairs of SCAR primers amplified distinct single bands whose sizes were the same as those of the RAPD clones. In four cases, the SCAR markers were present in the line with Tm-1 but absent in the line without it, as were the corresponding RAPD markers. In the two other cases, the products of the same size were amplified in both lines. When these SCAR products were digested with different restriction endonucleases which recognize 4-bp sequences, however, polymorphisms in fragment length were found between the two lines. These co-dominant markers are useful for differentiating heterozygotes from both types of homozygote.     

RAPD markers of mitochondrial origin exhibit lower population diversity and higher differentiation than RAPDs of nuclear origin in Douglas fir

We developed a method of screening RAPD markers for the presence of organelle DNA products using enriched organelle DNA probes, then used these markers to compare the structure of nuclear and mitochondrial RAPD diversity in Douglas fir. Of 237 screened RAPD fragments from 25 primers, 16% were identified as originating in the mitochondrial genome and 3% in the chloroplast genome. The mitochondrial DNA probe correctly distinguished fragments with known maternal inheritance (which is exclusive for the mitochondrial genome in the Pinaceae), and neither of the organelle probes hybridized to biparentally inherited fragments. Mitochondrial RAPD markers exhibited low diversity within populations compared to nuclear RAPD diversity ( H _S = 0.03 and 0.22, respectively), but were much more highly differentiated than were fragments of nuclear origin at both the population ( G _ST = 0.18 and 0.05, respectively) and racial levels ( G _ST = 0.72 and 0.25, respectively). Both nuclear and mitochondrial DNA based phylogenetic analyses identified the varieties as monophyletic groups; the nuclear RAPD markers further separated the north and south interior races.     

Genetic diversity and species identification in the endangered white abalone (<Emphasis Type="Italic">Haliotis sorenseni</Emphasis>)

In 2001, the white abalone Haliotis sorenseni became the first marine invertebrate in United States waters to receive federal protection as an endangered species. Prior to the endangered species listing, 20 abalone were collected as potential broodstock for a captive rearing program. Using DNA from these animals, we have developed genetic markers, including five nuclear microsatellite loci and partial sequences of one nuclear (VERL) and two mitochondrial (COI and CytB) genes, to assess genetic variability in the species, aid in species identification, and potentially track the success of future outplanting of captive-reared animals. All five microsatellite loci were polymorphic and followed expectations of simple Mendelian inheritance in laboratory crosses. Each of the wild-caught adult abalone exhibited a unique composite microsatellite genotype, suggesting that significant genetic variation remains in natural populations. A combination of nuclear and mitochondrial gene sequencing demonstrated that one of the original wild-caught animals was, in fact, not a white abalone, but H. kamtschatkana (possibly subspecies assimilis). Similarly, another animal of uncertain identity accidentally collected by dredging was also shown to be H. kamtschatkana. Inclusion of these two animals as broodstock could have resulted in unintentional hybridizations detrimental to the white abalone recovery program. Molecular genetic identifications will be useful both in preventing broodstock contamination and as markers for future restocking operations.     

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae)

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlantica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.     

PERCHED AT THE MITO‐NUCLEAR CROSSROADS: DIVERGENT MITOCHONDRIAL LINEAGES CORRELATE WITH ENVIRONMENT IN THE FACE OF ONGOING NUCLEAR GENE FLOW IN AN AUSTRALIAN BIRD

Relationships among multilocus genetic variation, geography, and environment can reveal how evolutionary processes affect genomes. We examined the evolution of an Australian bird, the eastern yellow robin Eopsaltria australis, using mitochondrial (mtDNA) and nuclear (nDNA) genetic markers, and bioclimatic variables. In southeastern Australia, two divergent mtDNA lineages occur east and west of the Great Dividing Range, perpendicular to latitudinal nDNA structure. We evaluated alternative scenarios to explain this striking discordance in landscape genetic patterning. Stochastic mtDNA lineage sorting can be rejected because the mtDNA lineages are essentially distinct geographically for > 1500 km. Vicariance is unlikely: the Great Dividing Range is neither a current barrier nor was it at the Last Glacial Maximum according to species distribution modeling; nuclear gene flow inferred from coalescent analysis affirms this. Female philopatry contradicts known female‐biased dispersal. Contrasting mtDNA and nDNA demographies indicate their evolutionary histories are decoupled. Distance‐based redundancy analysis, in which environmental temperatures explain mtDNA variance above that explained by geographic position and isolation‐by‐distance, favors a nonneutral explanation for mitochondrial phylogeographic patterning. Thus, observed mito‐nuclear discordance accords with environmental selection on a female‐linked trait, such as mtDNA, mtDNA–nDNA interactions or genes on W‐chromosome, driving mitochondrial divergence in the presence of nuclear gene flow.     

Mitotic chromosomal abnormalities and DNA polymorphism in the Nile tilapia (Oreochromis niloticus,Linnaeus, 1758) as a biomarker for water pollution by heavy metals

Mitotic chromosomal aberrations and DNA polymorphism (RAPD marker) were carried out on the Nile tilapia Oreochromis niloticus collected from five sites in Minia governorate, Egypt to test their applicability as biomonitors for heavy metal contaminants of water. The diploid chromosome number of O. niloticus population was 2 n = 44. Different types of chromosomal aberrations were recorded (e.g., deletion, ring, centromeric attenuation, end-to-end association, dicentric chromosome, stickiness chromosomes, endomitosis, fragments and chromatid gap). The chromosomal aberrations varied between O. niloticus population collected from five sites, and the most common type was ring (R) chromosomes. Samples obtained from Bahr Yousef and Irrigation drain exhibited the highest aberration frequency. The frequency of chromosomal aberration was positively correlated with the concentration of heavy metals where their concentration in the surface water of Irrigation drain and Bahr Yousef exceeded the limits defined by WHO as well as the concentration of Pb in muscles. The RAPD marker was also used to identify genetic variation among Nile tilapia samples collected from five different water sources. It created polymorphic and unique bands that can be used as genetic markers to track DNA variations. The dendrogram also revealed that exposure to heavy metal pollution causes gradual accumulation of variance, whereas areas subjected to environmental stress showed higher genetic variation and clustered together.     

两个不同地区东方田鼠杂交子代RAPD标记分析

目的研究不同地区东方田鼠杂交后产下的子代的遗传特性.方法筛选4条10bp随机引物对宁夏和洞庭湖地区东方田鼠杂交子代的基因组进行随机扩增多态DNA(RAPD)分析,并对不同地区亲代以及子代相互之间的基因组DNA进行相似性分析.结果①所有东方田鼠均有相同的扩增片段出现;②两个不同地区的亲代分别有特异性片般;③亲代的DNA带型均能在子代中找到;④同一胎次东方田鼠之间基因共享度大约在72%～96%之间.结论RAPD分析能在一定程度上反映出东方田鼠种的特性以及亚种的特异性,而且同一胎次之间基因共享度较高.