Physical mapping of four RAPDs in the B chromosome of maize

 Four DNA fragments were amplified specifically from the B chromosome by PCR using random 10-base oligonucleotides as primers. The location of the fragments in the B chromosome was determined based on whether or not they were amplified from the hypo- ploid DNA generated by four B-A translocations, three of which break in the proximal euchromatic region and the fourth in the distal one-third of the heterochromatic region on the B long arm. Since the hypoploid DNA carries the portion of the B chromosome distal to the breakpoint of a translocation, the presence of a fragment in the hypoploid DNA, but not in the control (which is devoid of any B chromatin), indicates that the fragments is located in the B region distal to the breakpoint in the B long arm. Two fragments were mapped to the euchromatic region and two others to either the distal portion of the euchromatic region or the proximal two-thirds of the heterochromatic region. These fragments in turn mapped three B-A translocations whose breakpoints were located in the euchromatic region. Received: 22 May 1996 / Accepted: 30 August 1996     

Analysis of specific RAPD- and ISSR-fragments in somaclonal maize (Zea mays L.) and development of SCAR markers based on them

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter Simple Sequence Repeats) markers were used to analyse the genetic divergence between the regenerated plants derived from callus cultures and the original maize line A188. Analysis of polymorphism by using 38 RAPD- and 10 ISSR-oligonucleotide primers showed that the differences between eight examined somaclones and the original line ranged from 6.5 to 23%. As confirmed using new primers, the regenerants derived from callus cultures grouped into two clusters according to their origin. The regenerants isolated from calluses grown for eight months differed from one another and the original line to a larger extent than the regenerants obtained from two-month callus cultures. In some somaclones, molecular marking of the regenerants revealed specific RAPD and ISSR fragments that were absent in other somaclones or the original maize line. On the basis of six specific fragments (five RAPD and one ISSR), SCAR (Sequence Characterized Amplified Region) markers were developed. Specific polymorphism revealed with random primers was completely confirmed using five SCAR markers. Polymorphism of one SCAR marker differed from that revealed with random primers. Five SCAR fragments were inherited as simple dominant traits. One SCAR fragment displayed codominant inheritance.     

The development of species-specific AFLP-derived SCAR and SSCP markers to identify mantis shrimp species

Molecular Biology Reports - Mantis shrimp has become commercially valuable in many countries, while the commercially aquaculture still unsuccessful. The stable supply of the species-specific...     

Physical mapping of RFLP markers on four chromosome arms in maize using terminal deficiencies

Terminal deficiencies (TDs) generated by the r-XI deletion system in maize were used to physically map RFLP markers on the short arm of chromosome 2 (2S) and the long arm of chromosome 6 (6L), chromosome 8 (8L), and chromosome 10 (10L). Five TDs on 2S, 8 on 6L, 10 on 8L, and 20 on 10L were isolated using the recessive morphological markers lg1, py1, j1(gl18), and sr2, respectively, for selection. Two exceptional TDs on 2S and 8L also have a second breakpoint on the long arm of chromosome 2 (2L) and 8L, respectively. The physical mapping of RFLP probes in relation to TD breakpoints was done by Southern hybridization. The five TDs on 2S divide chromosome 2 into four regions, all of which are distinguishable by RFLP markers. Likewise, three remaining chromosome arms are divided by TDs into RFLP-marked regions: 8 TDs divide 6L into five regions, 10 TDs divided 8L into seven regions, and 20 TDs divide 10L into three regions. The linear order of the physical map of 6L and 8L is consistent with that of the genetic maps, but that of 2L and 10L is not. Four groups of markers on 2S as well as 2L, and two on 10L are in reverse order in the physical map compared with the genetic maps. Other intriguing results are that breakpoints of TDs on 6L and 8L are distributed throughout the selected region, but most of those on 2L and 10L cluster in a region near the centromere; a single TD arose after fertilization. Received: 17 March 1997 / Accepted: 26 June 1997     

Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species.

RAPD markers were used to characterize the genetic diversity and relationships of root-knot nematodes (RKN) (Meloidogyne spp.) in Brazil. A high level of infraspecific polymorphism was detected in Meloidogyne arenaria, Meloidogyne exigua, and Meloidogyne hapla compared with the other species tested. Phylogenetic analyses showed that M. hapla and M. exigua are more closely related to one another than they are to the other species, and illustrated the early divergence of these meiotically reproducing species from the mitotic ones. To develop a PCR-based assay to specifically identify RKN associated with coffee, three RAPD markers were further transformed into sequence-characterized amplified region (SCAR) markers specific for M. exigua, Meloigogyne incognita and Meloidogyne paranaensis, respectively. After PCR using the SCAR primers, the initial polymorphism was retained as the presence or absence of amplification. Moreover, multiplex PCR using the three pairs of SCAR primers in a single reaction enabled the unambiguous identification of each species, even in mixtures. Therefore, it is concluded that the method developed here has potential for application in routine diagnostic procedures.     

Genetic analysis of Pinus strobus and Pinus monticola populations from Canada using ISSR and RAPD markers: development of genome-specific SCAR markers

Pinus is the largest genus of conifers, containing over 100 species and is also the most widespread genus in the Northern Hemisphere. Pinus monticola and P. strobus are two closely related and economically important species in Canada. Morphological and allometric characteristics have been used to assess genetic variation within these two species but these markers are not reliable due to ecological variations. The purpose of the present study was to determine the level of genetic diversity within and among Canadian populations from the two species using molecular markers and to identify and characterize genome-specific inter-simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers. The level of genetic variation among populations was much lower for P. monticola than P. strobus. For both species, the among population variation values were smaller than within population variation. The populations from P. monticola were more closely genetically related than populations from P. strobus based on ISSR and RAPD analyses. Six ISSR and four RAPD markers specific to either P. monticola or P. strobus were cloned and sequenced. Primer pairs flanking these specific sequences were designed and genome specific SCAR markers for P. monticola and P. strobus were developed and characterized.     

Specific genetic markers for wheat, spelt, and four wild relatives: comparison of isozymes, RAPDs, and wheat microsatellites.

Three types of markers-isozymes, RAPDs (random amplified polymorphic DNAs), and wheat microsatellites- were tested on wheat, spelt, and four wild wheat relatives (Aegilops cylindrica, Elymus caninus, Hordeum marinum, and Agropyron junceum). The aim was to evaluate their capability to provide specific markers for differentiation of the cultivated and wild species. The markers were set up for subsequent detection of hybrids and introgression of wheat DNA into wild relatives. All markers allowed differentiation of the cultivated from the wild species. Wheat microsatellites were not amplified in all the wild relatives, whereas RAPDs and isozymes exhibited polymorphism for all species. The dendrograms obtained with RAPD and isozyme data separated Swiss wheat cultivars from those collected in Austria and England, while no difference was found between Swiss spelt and wheat. RAPD data provided a weak discrimination between English and Austrian E. caninus. The microsatellite-based dendrogram discriminated populations of Ae. cylindrica, but no clear separation of H. marinum from E. caninus was revealed. The similarity matrices based on the three different sets of data were strongly correlated. The highest value was recorded between the matrices based on RAPDs and isozymes (Mantel's test, r = 0.93). Correlations between the similarity matrix based on microsatellites and matrices based on RAPDs and isozymes were lower: 0.74 and 0.68, respectively. While microsatellites are very useful for comparisons of closely related accessions, they are less suitable for studies involving less-related taxa. Isozymes provide interesting markers for species differentiation, but their use seems less appropriate for studies of within-species genetic variation. RAPDs can produce a large set of markers, which can be used for the evaluation of both between- and within-species genetic variation, more rapidly and easily than isozymes and microsatellites.     

Determining the plasmotypic structure of rye populations by SCAR markers

In rye (Secale cereale L.), 2 types of cytoplasmic male sterility are known: Pampa type (CMS-P) and Vavilovii type (CMS-V). As an alternative method to the conventional plasmotype-genotype interaction test, for identification of the cytoplasm type, the use of sequence-characterised amplified region (SCAR) markers was validated in this study. In over 2600 individual rye plants, representing 26 populations originating from Poland (18 cultivars), Iran (5 populations of primitive rye), and South America (3 populations), the cytoplasm type was determined by using a set of 3 SCAR markers. For about 10% of these individuals, the plasmotype-genotype interaction test was performed in parallel. The results of both tests were fully consistent. In the majority of the Polish populations, CMS-V was present, and only 4 populations contained CMS-P. Primitive Iranian populations contained predominantly normal cytoplasm, and only occasionally CMS-P was identified in them. South American populations displayed a mixture of normal cytoplasm, CMS-P and CMS-V. This work validates the use of SCAR markers as a reliable and quick method to determine the plasmotypic diversity of rye populations on a large scale.     

Characterization of Mimban maize landrace from North-Eastern Himalayan region using microsatellite markers

The North-Eastern Himalayan (NEH) region of India is endowed with rich maize genetic resources which is important from both genetics and evolutionary viewpoints. Mimban landrace of maize is a popular choice in Mizoram as food among the locals due to its stickiness caused by recessive wx1 gene resulting in high amylopectin in the grains. In the present study, a set of 24 Mimban accessions possessing high amylopectin (mean 89.72%, range 80.2–93.7%) content were analyzed. 93 SSRs markers generated a total of 334 alleles with a range of 2–9 and mean of 3.59 alleles per locus. Polymorphism information content varied from 0.117 to 0.829 with an average of 0.528. A total of 20 unique and 24 rare alleles were detected. Twenty-seven major alleles with individual frequencies exceeding 0.70 were also identified across the accessions. Cluster analyses classified 24 genotypes into three major clusters each having 2, 14 and 9 accessions. The clustering pattern was largely congruent with the geographical information. Diverse origin of the accessions was also depicted by the SSR based principal coordinate analysis. These accessions with high amylopectin content from diverse clusters may be crossed to derive heterotic hybrid and also might be used for novel gene identification. Thus information generated here possesses great potential in their utilization in the waxy corn genomics and breeding and emphasizes the need for further exploration of unique trait specific genepool from unexplored areas. This is the first report of molecular characterization of Mimban landrace accessions from NEH region.

     

The heterogeneity of B-chromosome DNA: no evidence for a B-chromosome specific repetitive DNA correlated with B-chromosome effects on meiotic pairing in the triticinae

The compositional heterogeneity of DNAs of A (normal) and B (supernumerary) chromosomes of Aegilops speltoides, Ae. mutica and Triticum aestivum has been compared in order to elucidate the mechanism of B-chromosome disruption of meiotic pairing in interspecific hybrids. Comparisons of % heterologous association after DNA/DNA hybridation at C₀t 10^?2 (highly repetitious DNA) and C₀t 100 (moderately repetitious DNA), and comparisons of nucleotide base divergence (ΔT_ms) and thermal elution profiles of homologous and heterologous duplexes, show that genotypes of Aegilops spp., having large numbers of Bs, do not carry additional families of repetitious DNA exclusive to B-chromosomes. Neither the presence of Bs nor the direction of DNA/DNA hybridisation affect the above parameters. No cryptic DNA satellites were revealed in A- and B-chromosome DNA after sedimentation in actinomycin D-CsCl gradients; and there were no significant differences in buoyant densities of main-band DNA. Mean melting temperatures (T_m); transition temperatures (ΔT) and numbers and positions of peaks of dissociating DNA fractions in profiles of differentiated melting curves of native DNAs were similar in strictly comparable denaturation conditions. One small AT-rich (< 5%) DNA fraction correlated with speltoides Bs was revealed; however, no corresponding fraction is associated with mutica Bs. The overall similarity in numbers and base composition of families of DNA (repetitious and unique) of As and Bs is discussed in relation to the origin of Bs and the origin of the meiotic diploidising system in haploid T. aestivum.     

Genetic relationships among Native American maize accessions of the Great Plains assessed by RAPDs

Genetic variation among 15 accessions of Native American maize from the Great Plains was investigated using random amplified polymorphic DNA (RAPD). RAPDs revealed very high levels of polymorphism among accessions. Banding patterns ranged in percentage polymorphism from 46.7% to 86.2% with an overall mean of 70.7% for the primers analyzed. The construction of genetic relationships using cluster analysis and principal coordinates analysis revealed that RAPDs are successful in confirming hypothesized relationships and in identifying misclassified specimens. Furthermore, the phenogram fails to reveal a strong correspondence between genetic relationships and the geographical position of Native Americans prior to contact. This provides support for the hypothesis that multiple introductions of maize into the Great Plains via trade may have resulted in the great morphological variation found among accessions in the region. Based on these data, it is unlikely that a separate Great Plains race of maize can be distinguished. In general, we conclude that RAPDs are potentially very useful in organizing seed collections and understanding intraspecific genetic differentiation. Received: 10 March 1999 / Accepted: 25 March 1999     

Identification of Prunus armeniaca cultivars by RAPD and SCAR markers

Nineteen cultivars of apricot (Prunus armeniaca) were distinguished using random amplified polymorphic DNA (RAPD) markers. One decamer out of 44 used was useful to differentiate cultivars of the Campania Region from those of Northern Italy, North America and Greece. A sequence characterized amplified region (SCAR) marker was obtained. The results provide a protocol to fingerprint DNA of apricots as an efficient way to quality control and fraud prevention.     

Use of inter-simple sequence repeat markers to develop strain-specific SCAR markers forFlammulina velutipes

In this paper, we report for the first time on authentication of Flammulina velutipes cultivars by using strain-specific sequence-characterized amplified region (SCAR) markers developed from inter-simple sequence repeat (ISSR) markers. The genomic DNA polymorphism was analyzed by the ISSR technique in 7 strains of F. velutipes presently cultivated in China on a commercial scale. Eight primers selected from 20 ISSR primers amplified 104 clear and stable bands, of which 81 bands were polymorphic. Among the selected primers, primer ISSR9 can distinguish strain No. 12 from the other 6 strains by amplifying a unique and reproducible band of approximately 750 bp. According to the sequence of the strain-specific fragment, a pair of SCAR primers was designed to diagnose strain No. 12 on the molecular level. The validity of the SCAR marker was confirmed by using DNA samples from another 12 strains of F. velutipes obtained from different parts of China. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for F. Velutipes.     

RAPD and ISSR-assisted identification and development of three new SCAR markers specific for the Thinopyrum elongatum E (Poaceae) genome

Diploid Thinopyrum elongatum, a wild relative of wheat, contains many agronomically desirable traits and has potential for increasing genetic variability and introducing desirable characters in this crop. Few molecular markers are available for rapid screening of T. elongatum genome segments in the wheat genetic background. We used 36 RAPD primers and 33 ISSR primers to screen for polymorphisms in the common wheat variety Chinese Spring and in T. elongatum. Two RAPD markers and one ISSR marker, designated OPF03(1407), LW10(1487) and UBC841(701), were identified and were specific for the T. elongatum E genome. Three pairs of primers flanking these specific sequences were designed to produce SCAR markers. All three SCAR markers were T. elongatum E genome-specific. Two of these SCAR markers, SCAR(807) and SCAR(577), were present in all seven T. elongatum chromosomes, while SCAR(839) was specific for T. elongatum chromosomes 2E and 3E. These newly developed SCAR markers should be useful for detecting alien genome chromatin or chromosome segments in the genetic background of common wheat.     

Studies of chloroplast development in four maize mutants defective in chlorophyll biosynthesis

Four mutants of maize (Zea mays L.) defective in chlorophyll biosynthesis have been analyzed with regard to the sites of their lesions and their effects on chloroplast development. Two yellow mutants, which accumulate no detectable porphyrin precursors when grown in darkness, are defective in the conversion of protoporphyrin IX to magnesium protoporphyrin. Etioplasts of these mutants may develop elaborate lamellar membrane systems, but prolamellar bodies are never observed. Two mutants, which are necrotic when grown under illumination, develop normal (non-necrotic) leaf tissue in the dark and accumulate a small amount of magnesium protoporphyrin monomethyl ester, corresponding approximately to the amount of protochlorophyllide accumulated by normal plants. The etioplasts of these mutants contain noncrystalline bodies. The implications of these observations with respect to chloroplast development are discussed.Journal Paper No. J-9136 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 2035     

The development of two flanking SCAR markers linked to a sex determination locus in Salix viminalis L

Most studies of sex determination systems in plants involve dioecious annuals that have known sex chromosomes. Despite the absence of such structures in the majority of dioecious plants, gender seems to be under relatively strict genetic control in some species. Genetic markers linked to a female sex-determination locus in Salix viminalis L. have been discovered through bulked segregant analysis of three full-sib families using approximately 1,000 arbitrary primers. Two RAPD markers that were present in the common female parent as well as in predominantly female progeny of these families were subsequently sequenced and converted to sequence characterized amplified region (SCAR) markers. The two SCAR markers are correlated with gender in the three full-sib families and are present in 96.4% of the female progeny and 2.2% of the males, providing evidence of linkage to a putative female-specific locus associated with gender determination in S. viminalis. Estimates of recombination suggest that the two markers are flanking a putative sex determination locus, SDL-II, in certain families of S. viminalis.     

Development of strain-specific SCAR markers for authentication of Ganoderma lucidum

In this study we report the application of sequence-characterized amplified region (SCAR) markers in Ganoderma lucidum for strain identification, the first such study in this medicinal mushroom. One fragment unique to strain No. 9 was identified by inter-simple sequence repeats (ISSR), and then sequenced. Based on the specific fragment, one SCAR primer pair designated as GL₆₁₂F and GL₆₁₂R was designed to amplify a 612-bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer pair. The results showed that this SCAR marker can clearly distinguish strain No. 9 from other related Ganoderma lucidum strains. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for Ganoderma lucidum.     

Assessment of SCAR markers to design real‐time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

Aims: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real‐time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP‐154 and CFN‐535 in the rhizosphere. Methods and Results: Primers were designed based on strain‐specific SCAR markers and were screened for successful amplification of target strain and absence of cross‐reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real‐time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 × 10³ (for UAP‐154) and 4 × 10⁴ CFU g^?1 (for CFN‐535) in the maize rhizosphere. Inoculant quantification was effective from 10⁴ to 10⁸ CFU g^?1 soil. Conclusion: BOX‐based SCAR markers were useful to find primers for strain‐specific real‐time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Significance and Impact of the Study: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation‐independent monitoring methods were lacking. The real‐time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP‐154 and CFN‐535.     

Use of intersimple sequence repeats markers to develop strain-specific SCAR markers for Lentinula edodes

Although Lentinula edodes is the second most important cultivated mushroom worldwide, most industrially cultivated strains have been identified only through traditional phenotypic analysis. Here, we report for the first time the use of sequence characterized amplified region (SCAR) markers for strain differentiation. SCAR markers were created by first generating and sequencing single intersimple sequence repeats fragments, and then designing primers based on these sequences to amplify strain-specific fragments of a certain size. One SCAR primer pair, ISL450F/R7 (amplifying a band of c. 450 bp), was designed to identify one strain of L. edodes (strain No. 7). The SCAR primer pair was then used to correctly amplify the single unique fragment from DNA samples taken from a total of 85 strains representing three separate species. Our data provide the foundation for a precise and rapid PCR-based strain-diagnostic system for L. edodes.