Genetic linkage map in sour cherry using RFLP markers

 Restriction fragment length polymorphism (RFLP) linkage maps of two tetraploid sour cherry (Prunus cerasus L., 2n=4x=32) cultivars, Rheinische Schattenmorelle (RS) and Erdi Botermo (EB), were constructed from 86 progeny from the cross RS×EB. The RS linkage map consists of 126 single-dose restriction fragment (SDRF, Wu et al. 1992) markers assigned to 19 linkage groups covering 461.6 cM. The EB linkage map has 95 SDRF markers assigned to 16 linkage groups covering 279.2 cM. Fifty three markers mapped in both parents were used as bridges between both maps and 13 sets of homologous linkage groups were identified. Homoeologous relationships among the sour cherry linkage groups could not be determined because only 15 probes identified duplicate loci. Fifty nine of the markers on the linkage maps were detected with probes used in other Prunus genetic linkage maps. Four of the sour cherry linkage groups may be homologous with four of the eight genetic linkage groups identified in peach and almond. Twenty one fragments expected to segregate in a 1 : 1 ratio segregated in a 2 : 1 ratio. Three of these fragments were used in the final map construction because they all mapped to the same linkage group. Six fragments exhibited segregation consistent with the expectations of intergenomic pairing and/or recombination. Received: 1 April 1998 / Accepted: 9 June 1998     

Molecular characterization of UV-treated sugar beet somaclones using RFLP markers

Sugar beet plants regenerated from UV-treated calluses were examined by restriction fragment length polymorphism (RFLP) analysis to determine the extent of somaclonal variation occurring at the DNA level. In total, 50 random sugar beet DNA sequences were used to screen 42 somaclones for genetic alterations. Three polymorphisms were detected among the 7 644 alleles analysed. From these data a mutation frequency of 0.03 ± 0.02% per allele was estimated. This frequency is in agreement with similar studies of somaclonal DNA variation using molecular markers and lies in the upper range of the spontaneous gene mutation frequencies found in plants. The two probegenotype combinations showing independent polymorphisms, were further analysed using the restriction enzymes Bam HI, Eco RI, Eco RV and Hind III. Both polymorphisms are likely to result from structural rearrangements rather than from point mutations. Differences in methylation among 10 of the investigated somaclones were tested for by comparing Hpa II and Msp I generated RFLP patterns. The somaclones showed extensive methylation, but no differences in their degree of methylation. Cytological analysis revealed 34 diploid, 8 tetraploid, but no aneuploid plants.     

Genetic linkage mapping in peach using morphological,RFLP and RAPD markers

We have constructed a genetic linkage map of peach [Prunus persica (L.) Batsch] consisting of RFLP, RAPD and morphological markers, based on 71 F₂ individuals derived from the self-fertilization of four F₁ individuals of a cross between New Jersey Pillar and KV 77119. This progeny, designated as the West Virginia (WV) family, segregates for genes controlling canopy shape, fruit flesh color, and flower petal color, size and number. The segregation of 65 markers, comprising 46 RFLP loci, 12 RAPD loci and seven morphological loci, was analyzed. Low-copy genomic and cDNA probes were used in the RFLP analysis. The current genetic map for the WV family contains 47 markers assigned to eight linkage groups covering 332 centi Morgans (cM) of the peach nuclear genome. The average distance between two adjacent markers is 8 cM. Linkage was detected between Pillar (Pi) and double flowers (Dl) RFLP markers linked to Pi and flesh color () loci were also found. Eighteen markers remain unassigned. The individuals analyzed for linkage were not a random sample of all F₂ trees, as an excess of pillar trees were chosen for analysis. Because of this, Pi and eight other markers that deviated significantly from the expected Mendelian ratios (e.g., 121 or 31) were not eliminated from the linkage analysis. Genomic clones that detect RFLPs in the WV family also detect significant levels of polymorphism among the 34 peach cultivars examined. Unique fingerprint patterns were created for all the cultivars using only six clones detecting nine RFLP fragments. This suggests that RFLP markers from the WV family have a high probability of being polymorphic in crosses generated with other peach cultivars, making them ideal for anchor loci. This possibility was examined by testing RFLP markers developed with the WV family in three other unrelated peach families. In each of these three peach families respectively 43%, 54% and 36% of RFLP loci detected in the WV family were also polymorphic. This finding supports the possibility that these RFLP markers may serve as anchor loci in many other peach crosses.     

Genetic mapping of jointless-2 to tomato chromosome 12 using RFLP and RAPD markers

Abscission zones are specialized regions in plants, usually located at the base of most plant parts, such as flowers, fruit and leaves, where organs are shed. Although a great deal of information is known about the physiological and biochemical events that lead to organ shedding, very little is known of the molecular events that lead to the formation of the abscission zone itself. In tomato, two recessive mutations have been discovered that completely suppress the formation of flower and fruit pedicel abscission zones, i.e., jointless (j) and jointless-2 (j-2), both tentatively localized to chromosome 11 about 30 cM apart. Because the study of the control of abscission zone development is important for both basic and applied research we are using a map-based cloning approach to identify the jointless genes. The first step in any positional cloning experiment is to establish segregating mapping populations for the target gene and identify closely linked molecular markers that flank the locus. In this study, bulked segregant analysis was used to identify a RAPD marker associated with the j-2 locus, RPD140. To determine the chromosome location of RPD140, we converted it to an RFLP marker that was then mapped on the Cornell reference tomato map in a marker-dense region of chromosome 12. To verify that the j-2 locus was located on tomato chromosome 12, we used nine chromosome 12 RFLP markers linked with RPD140 to map the j-2 gene in an interspecific F₂ mapping population of 151 plants segregating for j-2. The j-2 gene was localized to a 3.0-cM interval between RPD140 and TG618 on tomato chromosome 12. Received: 29 March 1999 / Accepted: 13 October 1999     

Analysis of mosquito bloodmeals using RFLP markers

An important variable in the amplification of arthropod vector-borne diseases is the degree of contact between human hosts and mosquito vectors. To analyze this interaction, a DNA based method was developed to differentiate human bloodmeals from other sources in the mosquito Anopheles stephensi (Diptera: Culicidae) Liston. A portion of the host mitochondrial DNA cytochrome B genes were PCR amplified and classified to the species level based on their restriction fragment length polymorphism (RFLP). The cytochrome B sequences showed sufficient interspecific polymorphism to distinguish between human, cow, sheep, chicken, and guinea pig hosts. XhoI could distinguish human from other vertebrates whereas TaqI alone could separate the others. The importance of these results in epidemiological studies of malaria and other vector borne diseases is discussed.     

Genetic characterization of Latin-American Creole cattle using microsatellite markers

Genetic diversity in and relationships among 26 Creole cattle breeds from 10 American countries were assessed using 19 microsatellites. Heterozygosities, F-statistics estimates, genetic distances, multivariate analyses and assignment tests were performed. The levels of within-breed diversity detected in Creole cattle were considerable and higher than those previously reported for European breeds, but similar to those found in other Latin American breeds. Differences among breeds accounted for 8.4% of the total genetic variability. Most breeds clustered separately when the number of pre-defined populations was 21 (the most probable K value), with the exception of some closely related breeds that shared the same cluster and others that were admixed. Despite the high genetic diversity detected, significant inbreeding was also observed within some breeds, and heterozygote excess was detected in others. These results indicate that Creoles represent important reservoirs of cattle genetic diversity and that appropriate conservation measures should be implemented for these native breeds in order to minimize inbreeding and uncontrolled crossbreeding.     

Genetic characterization of Colombian Brahman cattle using microsatellites markers

Genetic structure and diversity of 3789 animals of the Brahman breed from 23 Colombian regions were assessed. Considering the Brahman Zebu cattle as a single population, the multilocus test based on the HW equilibrium, shows significant differences (P < 0.001). Genetic characterization made on the cattle population allowed to examine the genetic variability, calculating a H _o = 0.6621. Brahman population in Colombia was a small subdivision wthin populations (F _it = 0.045), a geographic subdivision almost non-existent or low differentiation (F _st = 0.003) and the F _is calculated (0.042) indicates no detriment to the variability in the population, despite the narrow mating takes place or there is a force that causes the variability is sustained without inbreeding actually affect the cattle population. The outcomes of multivariate analyses, Bayesian inferences and interindividual genetic distances suggested that there is no genetic sub-structure in the population, because of the high rate of animal migration among regions.     

Genetic characterization of local Sudanese sheep breeds using DNA markers

Genetic diversity was investigated among 11 local Sudanese sheep populations. These populations were: Desert, with 6 sub-populations; Nilotics; Arid upland; West African; and Nilodesert, with 2 sub-populations. 15 microsatellites were used, a total number of 263 alleles being found, with expected heterozygosity ranging from 0.726 to 0.811. Principal component analysis revealed a distinct demarcation between the West African population and all other populations in Sudan and a moderate distance of Desert Dongla sheep from all other Desert populations. Structure modelling clustered West African, Arid upland and Nilotics populations in one group, and divided the Desert and Nilodesert populations into 2 mixed clusters with incomplete demarcation, reflecting cross breeding between these populations. Mitochondrial analysis indicated the dominance of the B haplotype in all populations, with about 10% of sheep carrying the A haplotype. The presence of the A and B haplotypes suggest that Sudan may be a contact zone between Asian and African sheep ancestors.     

Genetic characterization of Brazilian strains of Aspergillus flavus using DNA markers

The Aspergillus genus belongs to a filamentous fungal group characterized by wide dispersion in the environment. Some species are associated with diseases, especially in immunocompromised patients, while others are of economical importance due to aflatoxin production or biotechnological applications. Its species identification is nowadays performed by traditional techniques combined with molecular markers, resulting in a higher efficiency of isolate characterization. In the present study, internal transcribed spacer, inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of Aspergillus flavus and strains of other species of the A. flavus group. High genetic diversity was revealed by RAPD and by ISSR, in which the use of the (GACA)4 primer yielded a higher diversity than with the (GTG)5 primer, although the latter showed a characteristic banding profile for each species. These data were used to create a similarity matrix for the construction of dendrograms by means of the UPGMA method. The ISSR and RAPD profiles showed that among the strains previously identificated as A. flavus, one should be A. oryzae, one A. parasiticus and two A. tamarii. On the other hand, a strain previously identified as A. parasiticus should be A. flavus. All these strains were retested by traditional methods and their new species identification was confirmed. These results strongly support the need for using molecular markers as an auxiliary tool in differentiating fungal species and strains.     

Genetic characterization of four wild species of Chinese marmots using microsatellite markers

Marmots are large ground squirrels, and 14 species have been reported in the world, including four species of marmots (Himalayan marmot, Tarbagan marmot, gray marmot and long-tailed marmot) living in China. Although these biological resources are abundant in China, information regarding their genetic features is lacking, hampering further study regarding them. The aims of this research were to evaluate genetic variations of four species of Chinese wild marmots, and analyzed kinship of these marmot populations. In the current study, we collected samples of four species of Chinese wild marmot and analyzed the effective allele number, gene diversity, the Shannon index, and polymorphism information to evaluate genetic variations using 13 microsatellite loci. Based on Nei’s genetic distance using the unweighted pair group method, we constructed a dendrogram to analyze the population kinship. We determined that all four Chinese marmot species had high genetic polymorphisms and departure from Hardy-Weinberg equilibrium. The Chinese marmots to be divided into two large groups: Himalayan marmot was independent group. Tarbagan marmot, gray marmot and long-tailed marmot were others; Tarbagan marmot and gray marmot showed a close kinship with each other, but long-tailed marmot did not have a close relationship with the other species. The high polymorphisms and the kinship of Chinese marmot populations were correlated with geographical terrain of their habitat. Himalayan marmot was characterized as living in unique alpine meadows in Qinghai-Tibet plateau and was affected by terrain; however, Tarbagan marmot, gray marmot and long-tailed marmot were characterized as living in grassland or alpine grassland and were not affected by terrain. Genetic features of Chinese wild marmots were investigated in this study. This may give using information regarding protection of Chinese wild marmot resource and further application of biomedical research.     

Prediction of hybrid performance in grain sorghum using RFLP markers

Heterosis is an important component of hybrid yield performance. Identifying high yielding hybrids is expensive and involves testing large numbers of hybrid combinations in multi-environment trials. Molecular marker diversity has been proposed as a more efficient method of selecting superior combinations. The aim of this study was to investigate the value of molecular marker-based distance information to identify high yielding grain sorghum hybrids in Australia. Data from 48 trials were used to produce hybrid performance-estimates for four traits (yield, height, maturity and stay green) for 162 hybrid combinations derived from 70 inbred parent lines. Each line was screened with 113 mapped RFLP markers. The Rogers distances between the parents of each hybrid were calculated from the marker information on a genome basis and individually for each of the ten linkage groups of sorghum. Some of the inbred parents were related so the hybrids were classified into 75 groups with each group containing individual hybrids that showed similar patterns of Rogers distances across linkage groups. Correlations between hybrid-group performance and hybrid-group Rogers distances were calculated. A significant correlation was observed between whole genome-based Rogers distance and yield ( r = 0.42). This association is too weak to be of value for identifying superior hybrid combinations. One reason for the generally poor association between parental genetic diversity and yield may be that important QTLs influencing heterosis are located in particular chromosome regions and not distributed evenly over the genome. Variation in the sign and magnitude of correlations between Rogers distance and hybrid-group performance for particular linkage groups observed in this study support this hypothesis. The concept of using diversity on individual linkage groups to predict performance was explored. Using data from just two linkage groups 38% of the variation in hybrid performance for grain yield could be explained. A model combining phenotypic trait data and parental diversity on particular linkage groups explained 71% of the variation in grain yield and has potential for use in the selection of heterotic hybrids.     

Towards a saturated sorghum map using RFLP and AFLP markers

 A near-saturated sorghum genetic linkage map was produced using RFLP, AFLP and morphological markers. First a composite, essentially RFLP-based genetic linkage map was obtained from analyses of two recombinant inbred populations. This map includes 343 loci for 11 linkage groups spanning 1352 cM. Since this map was constructed with many previously mapped heterologous probes, it offers a good basis for synteny studies. Separately, an AFLP map was obtained from the analysis of 168 bands revealed from 12 primer pair combinations. It includes 137 loci for 11 linkage groups spanning 849 cM. Taking into account the different data sets, we constructed a combined genetic linkage map including 443 loci spanning 1899 cM. Two main features are to be noted: (1) the distribution of AFLPs along the genome is not uniform; (2) an important stretching of the former core map is induced after adding the AFLPs. Received: 10 May 1998 / Accepted: 13 July 1998     

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     

Isolation, characterization, and physical localization of 33 human X-chromosome RFLP markers

In a search for highly polymorphic X-specific loci, the X-chromosome DOE Ch35 phage library (LAOXNL01) was screened with three oligonucleotides representative of minisatellite consensus sequences. A total of 170 clones containing human inserts were isolated by hybridization to the oligonucleotide sequences; each was tested for polymorphism on five random female DNAs with six restriction enzymes. Among the 53 clones demonstrating a polymorphic pattern, 47 were of distinct origin. Twelve of the polymorphisms (23%) were determined to be autosomal. Polymorphisms for the remaining 35 clones were characterized, These polymorphisms represent 33 new X-chromosome RFLP loci, since two pairs of clones detected partially overlapping patterns. A pattern of similar length variation with multiple enzymes ("VNTR-type") was demonstrated in 6 (50%) of the 12 non-X-polymorphic clones. However, only 3 (9%) of the 33 X polymorphic loci showed VNTR-like patterns, suggesting a decreased amount of VNTR polymorphism on the X chromosome. The 33 polymorphic X loci were physically localized with a set of rodent x human somatic cell hybrid DNAs representing nine different X-chromosome breakpoints.     

Genetic characterization and breed assignment in five Italian sheep breeds using microsatellite markers

The knowledge of the genetic relationship and admixture among neighbouring populations is crucial for conservation efforts. The aim of this study was to analyse the genetic diversity of five Italian sheep breeds (Appenninica, Garfagnina Bianca, Massese, Pomarancina and Zerasca) using a panel of 24 microsatellite markers. Blood samples from 226 individuals belonging to the aforementioned populations were obtained and genotyped. All the investigated breeds showed a significant heterozygote deficiency caused by the high level of inbreeding indicated also by the high level of F_IS (0.146). Genetic differentiation between breeds was moderate (F_ST = 0.05) but significant and the individuals could be assigned to their breeds with an high success rate even if the inter-individual distances showed that few animals clustered separately from the other individuals of the same breed, especially for Pomarancina breed. The genetic distances reflect the historical knowledge of these breeds and some patterns of ancestral and recent gene flow between neighbour populations arise. The clustering analysis detects the presence of six clusters. Massese and Zerasca breeds were grouped together as well as Appenninica and Pomarancina with the latter forming two distinct clusters equally represented. The formation of this last breed is occurred with the absorption of individuals of the Appenninica breed and the gene flow probably continued in these recent years allowing the presence of a population substructure for Pomarancina breed. Such substructure supports the high level of heterozygote deficiency found for this breed despite the relatively high population size. The five populations analysed presented some genetic similarities but a clear uniqueness of the populations has been showed for almost all of them. Special attention to monitor genetic variability and to organize mating plans should be given especially for the three endangered breeds.     

Further characterization of nuclear DNA RFLP markers that distinguish African and European honeybees

Two nuclear honeybee DNA probes, 12R1C1 and 2A2, were reported previously to detect restriction fragment patterns specific to African and neotropical African honeybee populations. Individual drones and workers from several additional Old and New World populations, African and European, were tested further with these probes. With probe 12R1C1, only two of several Hhal fragment patterns were seen among haploid drone progeny of each queen bee, indicating that the patterns represented alleles at a single locus. Four alleles detected by probe 12R1C1 were described previously, three of which had been found only in populations of African descent. In this study, one of the three was found at a low frequency among samples from western Europe, northern Mexico, and the United States. However, ten additional alleles were discovered in South African drones, six of which were seen also in neotropical African colonies. With probe 2A2, only one or the other of two Alul restriction fragments was detected in drones indicating that the fragments represented alles at a single locus. One of the two alleles, seen previously only in populations of African descent, was found at a very low frequency in bees from western Europe and northern Mexico.     

Genetic characterization of Iranian native Bombyx mori strains using amplified fragment length polymorphism markers

Genetic relationships within and among seven Iranian native silkworm strains was determined by DNA fingerprinting by using amplified fragment length polymorphism (AFLP) markers. In total, 189 informative AFLP markers were generated and analyzed. Estimates of Nei's gene diversity for all loci in individual strains showed a higher degree of genetic similarity within each studied strain. The highest and the least degrees of gene diversity were related to Khorasan Pink (h = 0.1804) and Baghdadi (h = 0.1412) strains, respectively. The unweighted pair-group method with arithmetic average dendrogram revealed seven strains of silkworm, Bombyx mori (L.), resolving into two major clusters. The highest degree of genetic similarity was related to Baghdadi and Harati White, and the least degree was related to Guilan Orange and Harati Yellow. The genetic similarity estimated within and among silkworms could be explained by the pedigrees, historical and geographical distribution of the strains, effective population size, inbreeding rate, selection intensity, and gene flow. This study revealed that the variability of DNA fingerprints within and among silkworm strains could provide an essential basis for breeders in planning crossbreeding strategies to produce potentially hetrotic hybrids in addition to contributing in conservation programs.     

Genetic variability of the wild diploid wheat Triticum urartu revealed by RFLP and RAPD markers

 Genetic variability among 49 accessions of Triticum urartu was estimated by RFLP and RAPD marker analyses, and the two data sets were compared. One T. timopheevii accession and two accessions of T. durum and T. aestivum, respectively, were included to identify T. urartu accessions closely related to these polyploid wheats. Twenty eight RFLP clones and 29 RAPD primers generated 451 and 155 polymorphic bands, respectively. The three accessions from Armenia clustered together and were well separated from all other accessions, which showed less pronounced geographical patterns. Genetic similarity and co-phenetic values calculated with RAPD markers were very similar to those calculated with RFLP markers for the intraspecific comparisons, but not for the interspecific comparisons. The identification of individual T. urartu accessions which are more related to polyploid wheats than others was not possible. Received: 14 May 1996 / Accepted: 13 September 1996