The present study utilized dominant markers such as Random Amplified Polymorphic DNA (RAPD) for the evaluation of relatedness among 6 Piper species were included in this study and to ensure such technique for claiming intellectual property right (IPR) by the plant breeder. Further, the study also aimed at estimation of the genetic relatedness of these 6 morpho-agronomically contrasting species. Six Piper species were screened using RAPD with decamer primer of arbitrary sequence. Out of 100 primers screened 12 were selected which gave clear and bright fragments. DNA banding patterns generated by RAPD were recorded as ‘1’ for presence of the RAPD marker and ‘0’ for absence. Genetic distance between these 6 species was calculated based on the RAPD data set as per Squared Euclidean distances. Based on the number of bands all the species were grouped into 3 clusters and the dendrogram revealed maximum similarity between P. betel and P. longum and also in between P. nigrum and P. mullesua species, altogether forming one cluster. The standardized method can identify any of the single hybrid or species tested.
Genetic variations in two accessions of S. fruticosa (41117 and 41117A) was carried out employing random amplified polymorphic DNA (RAPD) and sequence-tagged-sites (STS) markers to establish the accession closely resembles to the mapping population developed using one of these as a anthracnose susceptible parent along with S. scabra cv Seca as resistant source. Sixty-four RAPD and 15 STS primers generated 469 and 59 fragments respectively. Of these, 90 (19.2%) RAPD and 6 (10.2%) STS fragments showed polymorphism between two accessions of S. fruticosa. When same primers sets were used with F2 progeny, the segregated RAPD fragments showed patterns belonging to S. scabra cv Seca×S. fruticosa 41117A rather S. scabra cv Seca×S. fruticosa 41117 cross. Of 15 STS primers pairs, SHPALF2/R2, SHST2F3/T2R16 and SHST1F3/T1R3 clearly demonstrated that 41117A line was closer to population as SHPALF2/R2 showed absence of a 480-bp fragment in S. fruticosa 41117 and present in other two parents i.e., S. scabra cv Seca and S. fruticosa 41117A and 78 F2 plants tested whereas rest two primer pairs amplified a fragment of smaller size in 41117 only. Though most of the STS primer sequences used were derived from S. scabra cv Fitzroy and S. hamata cv Verano, the level of reaction in S. fruticosa accessions indicated low genome specificity of STS primers and hence low level of genetic divergence in different species of Stylosanthes. Results also provided the list of potential STS and RAPD primers could be used in studying the genetic relationships among S. fruticosa accessions, which is the only species of the genus Stylosanthes native and endemic to the southern peninsular regions of India, for plant conservation and breeding programs.
Karyotype analyses of 3 Garra species, G. gotyla gotyla (Gray), G. kempi Hora and G. lissorhynchus (McClelland) from Arunachal Pradesh, India, are presented. All the 3 species have a diploid chromosome number of 2n=50, but different karyotype formulae. The species-specific karyotypes were observed in the 3 species. It was also tried to draw a possible phylogenetic relationship among the 3 species.
Four elite diploid populations of sugarbeet viz. IISR Comp-1, LKC-11, LS-6 and Ramonskaya-06 were examined using isozymes and molecular markers. Seventy-one bands consisting of 28 isomorphs of 6 isozyme systems viz. superoxide dismutase, guaiacol peroxidase, malate dehydrogenase, amylase, esterase and aspartate amino transferase were resolved. Molecular analysis was performed using 28 RAPD (random amplification of polymorphic DNA) and 4 inter simple sequence repeat (ISSR) primers. Highly polymorphic band profiles with 327 RAPD markers (11.67/primer) of 162–2862 bp and 39 ISSR markers (9.75/primer) of 160–1884 bp were obtained. Mean value of Polymorphic Information Content (PIC) for RAPD and ISSR primers was 0.41 and 0.44 respectively. Based on the binary matrix of presence/absence of bands, Dice coefficient of genetic similarity (GS) matrix was computed for each marker system, which ranged from 0.62–0.76 (mean value 0.726), 0.10–0.74 (mean value 0.27) and 0.12–0.86 (mean value 0.35) for isozyme, RAPD and ISSR markers. There was significant correlation (r=0.908) between the RAPD-GS and ISSR-GS matrices, however, the correlation of isozyme matrix vs. RAPD and ISSR pattern was low. GS coefficients of isozyme, RAPD and ISSR were used to cluster the genotypes based on the UPGMA method and the dendrograms obtained were compared to develop consensus phylogenetic trees using NTSYSpc. The X-axis contained IISR Comp-1 and R-06, and the Y-axis contained LS-6 and LKC-11. The much lower values of average genetic similarity based on RAPD (0.27) and ISSR (0.35) indicated the ability of DNA based markers to detect high degree of polymorphism among these populations suggesting thereby, the possibility of screening a higher number of anonymous loci in sugarbeet using these molecular markers as compared to isozymes to enable the selection of the best parents in order to obtain new genetic combinations.
Four species of Japanese Scleria were used for karyomorphological studies. Chromosome numbers of S. mikawana (2n=20) and S. levis (2n=48) were determined for the first time. Our finding of 2n=30 for S. parvula and 2n=48 for S. terrestris differs from those of previous studies. Two major groups were identified in the genus Scleria by karyomorphological data: (1) low chromosome numbers and x=5, (2) high chromosome numbers (2n=48). These karyomorphological data support the taxonomical treatment of 2 sections by Ohwi (1944). The high chromosome numbers may arise by polyploidy of low chromosome numbers because chromosome sizes were almost equal in both. Therefore, chromosomal evolution in the genus Scleria may be caused by polyploidy, rather than aneuploidy.
The karyotype of 4 species namely D. agumbensis, D. anomelani, D. truncata and D. cauverii have been analysed. The heterochromatin content has been characterized by using C- and Q-profiles. The Y- and 4th chromosome have shown a great degree of divergence in their form and size. The heterochromatin has played a very important role in bringing about this high degree of differentiation within the limit of 2n=8 among the members of the montium subgroup of Drosophila.
Genomic DNA was isolated from leaves of five species of stylo (Stylosanthes scabra, S. hamata, S. seabrana, S. humilis and S. viscosa), a range fodder legume using CTAB and SDS based extraction buffer ‘S’ method. Ethanol was used as leaf fixing solution instead of grinding in liquid nitrogen, which yielded high molecular weight DNA (>30 kb). DNA quality and quantity were comparable to those isolated with liquid nitrogen. Isolated DNA was suitable for RAPD, sequence tagged site (STS) and restriction enzyme digestion. This method does not require liquid nitrogen for fixation, grinding, or storage at −80°C, making it advantageous over other common protocols.
Adventitious root tips developing from base of cuttings and leaf samples of Herreria salsaparilha were used to determine the karyotype and to estimate DNA amount, respectively. Flow cytometry methodology was used with DAPI (4′,6-diamidino-2-phenylindole) and PI (propidium iodide) as specific DNA fluorochromes, and leaf samples of Pisum sativum (9.09 pg) as an internal standard. Cytogenetic techniques determined that H. salsaparilha has 29 pairs of homologous chromosomes (2n=58), 12 acrocentric pairs (length varying from 1.75 to 10.51 μm), 16 submetacentric pairs (1.30 to 3.45 μm), and 1 metacentric pair (2.8 μm). The DAPI- and PI-based flow cytometry analysis revealed that H. salsaparilha has nuclear DNA content 2C=5.5 pg, equivalent to approximately 5.31×109 base pairs. This karyotype asymmetric type may suggest an evolutionary process with involvement of polyploidization, being justified by the relative increased chromosome number (2n=58) and fusions.
A rare hybrid of Sorghum×Sugarcane was produced at Sugarcane Breeding Institute during 1996 which had a chromosome number 2n=66. This hybrid lacked vigor and was poor with respect to agronomic traits. Attempts were made to create genetic variation in this hybrid through tissue culture and colchicine treatment. Calli induction and their differentiation to plantlets were obtained on modified MS medium. Different concentrations of colchicine (25 mg/l, 50 mg/l, 75 mg/l and 100 mg/l) were added to the differentiation medium. There was no differentiation in the medium containing 75 mg/l and 100 mg/l colchicine, while calli treated with 25 mg/l and 50 mg/l colchicine produced 230 plants. The regenerated plants obtained were rooted, hardened and established in pots as well as in the field and cytomorphologically characterized. Though the somaclones were morphologically similar to the original hybrid in almost all the characters, the cytological analysis revealed the occurrence of polyploidy in these calli clones. Chimeric plants were also obtained during in vitro polyploidization. The results indicate that colchicine could be efficiently utilized for induction of polyploidy in Saccharum hybrids.
Diploid lowland Taraxacum classified as T. elatum, T. hondoense, T. longeappendiculatum, T. japonicum, T, maruyamanum and T. platycarpum according to Kitamura (1957) were studied cytologically. Karyotypic formulae of the 6 forms were as follows: T. platycarpum, 2n=16=2M+10m+4mcs; T. elatum, 2n=16=12m+4mcs: T. hondoense, 2n=16=2M+10m+4mcs; T. longeappendiculatum, 2n=16=12m+4mcs; T. japonicum, 2n=16=12m+4mcs; T. maruyamanum, 2n=16=14m+2mcs. The karyotypes from T. elatum, T. hondoense, T. longeappendiculatum, T. japonicum and T. platycarpum had similar formulae described as 2n=16=12(M+m)+4mcs. In contrast, that of T. maruyamanum was 2n=16=14m+2mcs. The karyological data support the taxonomic study by Serizawa (1986, 1995), who treated the native lowland diploid Taraxacum in Japan as 2 species, T. platycarpum and T. maruyamanum.
Genus Delphinium L. with about 320 species, distributed in North temperate regions of the world is represented by about 24 species in India, mainly found in Himalayan regions. Delphinium malabaricum (Huth) Munz is the only rare endemic species of the genus Delphinium restricted to Northern Western Ghats of peninsular India. It has medium-sized violet–blue–metallic blue spurred flowers of great ornamental value. The present paper describes distribution, morphology, ornamental potential and karyotype of the species. The chromosome number 2n=16 and n=8 is reported for the first time in this species and the karyotype is found to be moderately symmetrical (2b). Its utilization as an ornamental plant after its domestication seems to be better strategy for conservation and utilization of this rare endemic of Northern Western Ghats.
Karyological data for the southern short-tailed shrew, Blarinella wardi, were obtained from 3 specimens collected on Mt. Laojun, Lijiang District, Yunnan Province, China. We examined 35 metaphases of B. wardi. The diploid number (2n) was 32 and the fundamental autosome number (NFa) was 58. The karyotype consisted of 10 M and 4 SM pairs, one small A pair, one medium-sized M X chromosome, and one small SM Y chromosome. The autosomal complement contained one pair of M chromosomes (no. 10) bearing a secondary constriction on the proximal long arm. The range of diploid number in the genus Blarinella indicates 32 to 44. In comparison with previously reported data on the karyotype of B. griselda, the secondary constriction on one pair of metacentric and biarmed sex chromosomes are considered affinal characters of the genus Blarinella.
Polytene complement of Parasarcophaga ruficornis (Sarcophagidae: Diptera) has been analysed to provide data on intercalary heterochromatin (IH). The location of weak points and ectopic contacts has been attributed to the presence of IH in the polytene complement. The spontaneously occurring weak points appear to be distributed on all the 5 polytene chromosomes. The telomeric end of right arm of chromosome II often turns back to form an ectopic contact with the region 5B/C of the same chromosome, thus, resulting in the formation of a loop.
Cytological studies were made in hybrids between S. spontaneum L clone Iritty-2 (2n=64) and Erianthus arundinaceus (Retz.) Jesweit clone IK 76-99 (2n=60). The chromosome number of 19 hybrids ranged from 2n=78 to 86. Predominantly bivalent formation was observed at diakinesis and metaphase I stages in pollen mother cells of all the hybrids. At later meiotic stages abnormalities such as lagging chromosomes, multipolar spindles, abnormal planes of cytokinesis and micronuclei were present. In one hybrid CYM 04-391 with chromosome number 2n=80, the pollen mother cells had cytomixis and syncyte formation in the prophase stage. Certain syncytes had very high number of chromosomes in the form of bivalents at diakinesis and metaphase I, with nearly 80 to more than 600 bivalents. Fertile pollen was observed in few anthers of CYM 04-391 and the pollen size variation was high in it compared to that in the other similar hybrids.
Three distinct variants differing for their phenotypic appearance and floral characters were identified and isolated in M1 generation of 200 Gy gamma irradiated set in Lathyrus sativus. They were subjected to meiotic analysis to understand their genotypic sensitivity to induced cytological changes. One interesting abnormality encountered during the present study was nucleolar dysfunctioning in the treated plants. Despite of chromosomal abnormalities like laggards, stickiness, micronuclei, etc. chiasma frequency was found to increase in the 3 plants isolated at 200 Gy dose of gamma rays. Pollen fertility was also moderately affected.
The medicinal plant Jacaranda decurrens Cham., known as “carobinha-do-campo”, is native to “Cerrado” (open pasture land with patches of stunted vegetation, woody pasture) and it belongs to the family Bignoniaceae. It is used in the popular medicine for the syphilis treatment, diaphoresis, inflammations etc., being prepared in the form of infusions (leaves and bark). We evaluated the mutagenic and antimutagenic activity of J. decurrens in somatic cells of Drosophila melanogaster using the SMART/eye test (somatic mutation and recombination test), which is based on the pigmentation of Drosophila ommatidia. Deletions, point mutations, non-disjunction and somatic recombinations were detected. Larvae in the third instar, resulting in crossing between virgin females from the Yellow lineage and males from the White one were treated with three different concentrations of J. decurrens ethanolic gross extract (EGE): 10 mg/ml (dosage-1); 6.25 mg/ml (dosage-2) and 4.16 mg/ml (dosage-3). They were isolated for mutagenic activity evaluation and associated to urethane (URE) for the antimutagenic analysis. URE was used (4 mg/ml) for the positive control and distilled water for the negative one. Three experiments were carried out for both mutagenesis and antimutagenesis. In the evaluation of the mutagenesis, the amount of clones/cells found in the dosage-3 (10.14) was statistically significant when compared to the negative dosage control (2.31), leading to the conclusion that J. decurrens EGE has a mutagenic effect depending on the concentration. On the other hand, in the antimutagenetic analysis, the EGE showed an antimutagenic effect in the three concentrations (dosage-1=4.18; dosage-2=4.58; and dosage-3=3.24 clones/cells) when compared to the positive control (urethane=6.29 clones/cells). Compared to dosage-3 (more diluted), the EGE presented a larger modulator effect of the genetic damages caused by URE. Thus, considering the established experimental conditions, we concluded that J. decurrens EGE has, depending on the concentration, either a mutagenic effect or a protecting effect of DNA against URE damage.
Cytogenetical data in 3 populations of characid fish assigned to the “complex” of Astyanax scabripinnis from São Francisco river basin and Grande river basin, Minas Gerais State, Brazil, are presented for the first time. The same diploid number, 2n=50, was detected in the 3 populations, which has conspicuous differences involving karyotype morphology: 8M, 20SM, 6ST and 16A (Cambeba stream), 6M, 28SM, 6ST and 10A (Machado headwater), 6M, 24SM, 8ST and 12A (Pedra Branca stream). Differences involving amount and/or locations of heterochromatin blocks, number and position of nucleolar organizer regions (NORs) and CMA3 positive signals were also observed. Some aspects related to the chromosome diversification of Astyanax scabripinnis are discussed.
In the present investigation a method is demonstrated using bulk samples of genomic DNA to estimate the genetic relatedness among heterogeneous lucerne cultivars through RAPD markers. Each of 7 random primers employed generated 5 to 10 DNA fragments from the individual plant of 5 cultivars as well as bulked samples made of several plants per cultivar. The genetic distance (D) calculated from RAPD patterns obtained with bulked DNA samples ranged from 0.03 to 0.11 which largely corresponds to their known relatedness. The RAPD profiles of the bulk samples containing DNA from different numbers of individuals of the cultivars indicated bulking of 10 individuals DNA are necessary to get the representation of the most of the bands of the cultivars. Thus, the method demonstrated could be useful for estimating genetic relatedness among the heterogeneous crop like lucerne.
The overall compaction rate of DNA molecules in mitotic chromosomes of dioecious Coccinia indica and Trichosanthes dioica has been measured in the present investigation. The data indicates that the average packing ratio differs significantly in relation to sex and the compaction rate is comparatively high in male plant of these dioecious species. Based on the estimates of this packing ratio, a theoretical proposition on the possible evolution of sex chromosomes in relation to dioecy has been explained.
Chromosomes of Tilapia rendalli from river Iguaçu, particularly its nucleolus organizing regions, was analyzed by Ag-staining NOR (Ag-NOR), Chromomicyn A3 (CMA3) and fluorescence in situ hybridization techniques (FISH). Karyotypic analyses showed diploid number 2n=44, with 10 submetacentric (SM), 8 subtelocentric (ST) and 26 acrocentric (A) chromosomes, and constitutive pericentromeric heterochromatin standard for most chromosomes. Whereas Ag-NOR and CMA3 detected 2 chromosome pairs, FISH method revealed 3 chromosome pairs with fluorescent signs and, thus, additional 18S cistrons. Results show that additional rDNA sites was inactive. Whilst active pairs were heterochromatic and CMA3 positive, the inactive one was heterochromatic and CMA3 negative.