An interspecific hybrid between G. arboreum×G. anomalum was obtained by pollinating previous day bagged flowers of G. arboreum genetic male sterile line MPKV GMS with pollen from wild G. anomalum.The chromosome constitution in all the plants of G. arboreum and G. anomalum were found to be normal while interspecific hybrid showed mean chromosome association 1.081I+10.479II+0.000III+0.990IV with normal sporads. Interspecific hybrid showed anomalous behavior of univalent and formation of single and double chromatin bridges both during fist and second meiotic division of meiosis. Meiotic chromosome association was observed in nine plants of F2 generation. The cytological studies showed that F1 and F2 generations were having irregular pairing and unequal separation of chromosomes. This led to pollen sterility in these generations. In F2 plants some good plants identified with higher pollen fertility and more number of bolls per plant. Selections from these plants can help to introgress desired characters from G. anomalum to G. arboreum. Random Amplified Polymorphic DNA (RAPD) analysis was employed to characterize the F1 interspecific hybrid of G. arboreum MPKV GMS×G. anomalum and its nine segregants. A total fifteen primers of 10 mer were screened of which eight were selected based on polymorphism of amplified fragments, amplification intensity and the presence of informative markers. Maximum 98 amplified fragments were resolved on the gel for parents, F1 and F2's with OPC-19 primers, whereas, minimum 25 fragments were seen with OPD-04 primer. Similarity coefficient based on DNA amplification using amplified RAPD primer was estimated. The values for genetic similarity ranged from 0.14 to 0.77.
This research was the first cytogenetic study of the Malayan porcupine (Hystrix brachyura) from Songkla Zoo, Thailand. Blood samples were taken from two males and two females and then subjected to standard whole blood T-lymphocyte culture. The samples were harvested by colchicine-hypotonic-fixation-air-drying technique and followed by conventional staining and G-banding with Giemsa's. The results showed that the diploid number was 2n=66, and the fundamental number (NF) was 127 and 128 in male and female, respectively. The autosomes consist of 6 large submetacentric, 20 large acrocentric, 6 medium submetacentric, 18 medium acrocentric, 2 medium telocentric, 4 small submetacentric, 6 small acrocentric and 2 small telocentric chromosomes. We found the nucleolar organizer regions (NORs), the representative chromosome marker, which are located on the long arms of the pair submetacentric autosomes 9 and 13. The X chromosome was a large metacentric chromosome, while the Y chromosome was the smallest telocentric chromosome. G-banding technique indicated that the number of bands was 236. All of these results can be use as the useful basic genetic information for H. brachyuran cytogenetic study. The karyotype formula can be shown as following: 2n(66)=L6sm+La20+M6sm+Ma18+Mt2+S4sm+S6a+St2+sex-chromosomes
Our knowledge is the first report on karyotypic study of four toad species (genus Bufo) in Thailand namely; large-eared toad (Bufo macrotis Boulenger 1887), Indochinese dwarf toad (Bufo parvus Boulenger 1887), common Indian toad (Bufo melanostictus Schneider 1799) and river giant toad (Bufo asper Gravenhorst 1829). Blood samples were taken from 5 males and 5 females of each four toad species. After the standard whole blood T-lymphocyte culture in the presence of colchicine, the metaphase spreads were performed on microscopic slides and air-dried. Conventional staining, G-banding and C-banding techniques were applied to stain the chromosomes. The results indicated 2n=22 and fundamental number (NF) 44 in both male and female of four toad species. The autosomes of B. macrotis and B. melanostictus is being as 18 metacentric and 4 submetacentric chromosomes while B. parvus and B. asper is as 16 metacentric and 6 submetacentric chromosomes. G-banding technique showed a B. melanostictus's constriction on short arm of Y chromosome (the largest chromosome) but did not show on X chromosome. C-banding technique demonstrated a dark band constriction on Y chromosome of B. melanostictus, the representative of constitutive heterochromatin. However, there is no dark band constriction on X chromosome. So, we conclude that the sex determination of B. melanostictus is XY system. Although we do not treat B. macrotis, B. parvus and B. asper with G-banding and C-banding technique, we also predict that those three species have the same sex determination as B. melanostictus. We extremely appreciate to public our present research, the first cytogenetic study of B. macrotis and B. asper.
The karyotypic and C-banding analysis of five species of Indian frogs belonging to the genus Fejervarya (F. cf. limnocharis, F. cf. brevipalmata, F. cf. keralensis, F. rufescens and F. sahyadris), which are distributed in the Western Ghats, Southwestern India, was carried out. All species had 2n=26 chromosomes with five large and eight small pairs. Most chromosomes were metacentric or submetacentric, and only no. 9 pair of F. cf. brevipalmata was subtelocentric chromosome. Nos. 1, 12 and 13 were metacentric and no. 3 was submetacentric in all the species analyzed. In F. cf. brevipalmata, an achromatic gap was present in the pericentric region of no. 6 chromosome. The chromosome pair no. 11 of F. cf. keralensis had a secondary constriction with a prominent satellite in the short arm. In three of the five species, C-positive regions were confined to the centromere of each chromosome, but F. rufescens and F. sahyadris showed non-centromeric C-bands on both ends of three pairs of large chromosomes, in addition to the centromeric C-bands. None of the species had identifiable sex chromosomes. The results are compared with karyotypes of the other Fejervarya species and the possible causes for the inter-specific karyological divergence are discussed.
Inter-specific hybridization is mainly used to introgress pest and disease resistant genes. Vigna radiata and Vigna mungo are widely cultivated and consumed in India. These crops are highly affected by Mungbean Yellow Mosaic Virus (MYMV) disease and bruchid (Callasobruchus spp.) pest, leading to heavy yield loss. Vigna umbellata, a divergent Vigna species is found to be resistant to both MYMV and bruchids. Utilization of this species to introgress the resistance gene is marginal because of their inherent low crossability with the cultivated Vigna species. In the present study, V. umbellata was used as pollen donors in hybridization with V. radiata and V. mungo. Low percentage of pod set observed in the cross V. radiata×V. umbellata (12.89%) and V. mungo×V. umbellata (5.56%) indicated the presence of reproductive barriers that render introgression difficult. Aim of this study was to observe pre-fertilization barriers operating in V. radiata×V. umbellata and V. mungo×V. umbellata crosses. In vivo pollen germination and growth of pollen tubes were studied in inter-specific crosses using fluorescence microscope. The pollen grain germination on stigmatic surface was normal. A very common pre-fertilization barrier of slow rate of pollen growth, in addition to structural abnormalities in stigmatic and stylar regions was observed in both the crosses. However, the level of incompatibility was high in V. mungo×V. umbellata cross than V. radiata×V. umbellata. Measures to overcome incompatibility barriers before fertilization in these crosses to produce inter-specific hybrids are discussed.
Mouse diploid and tetraploid H-1 (ES) cells cultured in L15F10 medium (2nH1 and 4nH1 cells, respectively) without leukemia inhibitory factor (LIF) (2nH1(−) and 4nH1(−) cells, respectively) were examined for their growth and cellular characteristics. Though the doubling times of 2nH1(−) and 4nH1(−) cells were longer than those of 2nH1 and 4nH1 cells, the phase fractions of 2nH1(−) and 4nH1(−) cells were the same as those of 2nH1 and 4nH1 cells, suggesting that occasional cell deaths occurred. DNA loss in long-term culturing was observed in 4nH1(−) cells, but not in 2nH1(−) cells, suggesting that DNA decay was independent of LIF. The cell volume, cellular morphology, and the pluripotent potential of 2nH1(−) and 4nH1(−) cells were retained during long-term culturing, suggesting that 2nH1(−) and 4nH1(−) cells survived in the medium without LIF had similar characteristics to those of 2nH1 and 4nH1 cells, respectively.
Cytogenetic characteristics of 11 olive cultivars were studied considering their chromosome pairing and segregation. The cultivars studied possessed 2n=4x=46 chromosome number and formed only bivalents in metaphase of meiosis-I. The cultivars differed significantly in their chiasma frequency and distribution as well as chromosome pairing indicating partly their genetic distinctness. Meiotic abnormalities including laggard chromosomes and chromosome stickiness occurred in the cultivars studied. B-chromosomes, cytomixis and unreduced pollen grains were observed in some of the cultivars. Clustering and ordination of the olive cultivars based on cytogenetic characteristics showed distinctness of these cultivars. B-chromosomes and unreduced pollen grain formation are new in olive cytogenetics.
Karyotypic studies of 9 tetraploid cotton cultivars showed that all the cultivars possessed 2n=4x=52 chromosome number. The chromosomes were mostly metacentric (M or m) and sub-metacentric (sm) but sub-telocentric chromosomes (st) occurred in the cultivars Sahel, Zeta2, Tashkand and Siokra. The size of chromosomes varied from 0.82 to 4.30 μm in the cultivars studied. The highest size difference among the chromosomes was observed in the cultivar Siokra while the highest value of mean haploid total chromatin length and size of the longest chromosome occurred in Tashkand cultivar. The cotton cultivars differed significantly in the size of chromosomes and the size of chromosomes arms indicating the occurrence of quantitative change in the chromatin material (DNA) during the cultivars diversification. They differed in karyotypic formulae and symmetry of karyotype indicating the occurrence of chromosomes structural changes. Meiotic analysis showed mainly bivalent formation in most of the cultivars, however quadrivalents were formed in three cultivars and hexavalent was formed only in the cultivar Bakhtegan. The genotypes studied also differed significantly in chiasma frequency and chromosome pairing due to genetic differences.
The tribe Paullinieae (Sapindaceae) is exclusively neotropically distributed, and is characterized by apomorphic characters and considered a monophyletic natural group. Recently explored cytogenetical aspects suggest that the disploid chromosomal reduction, the increase in the chromosomal size and the diversification of highly repetitive DNA sequences are associated with the karyotypic evolution of this tribe. This work compares patterns of chromosome banding and the distribution of ribosomal DNA 18S-5.8S-26S in Cardiospermum grandiflorum Sw., Pullinia elegans Cambess., Urvillea chacoensis Hunz. and U. ulmacea Kunth. The studied species share the presence of a pattern of terminal C-Giemsa bands, differentiated for characteristics of heterochromatic regions. Terminal AT-rich bands occurred in C. grandiflorum (2n=2x=20) and U. chacoensis (2n=2x=22). Differing from the others, U. chacoensis presented prominent bands in the majority of chromosomes. The polyploid cytotype of U. ulmacea (2n=8x=88) possessed terminal bands CMA+ and DAPI+, forming heterochromatic blocks constituted by GC- and AT-rich repetitive DNA. On the other hand, P. elegans (2n=2x=24) presented a pattern of neutral bands after staining with CMA3/DAPI. The presence of GC-rich regions associated with 45S rDNA sites was a common characteristic in the studied species, nevertheless, variations in the NOR number might be useful for the differentiation of some species. Our results on karyological differences and resemblances of the studied species are discussed in relation to the systematics of the Paullinieae tribe.
The black fly, Simulium aureohirtum was geographically widespread, and encompasses a wide range of ecological conditions. A total of 1303 larvae from 21 sampling sites throughout Thailand were cytologically examined. Fourteen chromosome rearrangements were found and all were paracentric inversions. Most inversions (12 of 14) were present at low frequency and geographically restricted to a particular population, suggesting they originated recently. Significant genetic differentiation between populations were detected (FST=0.092, p=0.0002). Detection of an isolation by distance (r=0.497, p=0.004) suggests that gene flow was limited by geographic distance. Inversion frequency of IIL-1 was significantly associated with latitude (r=0.832, p<0.01), altitude (r=0.471, p<0.05) and minimum annual air temperature (r=−0.748, p<0.01) indicating that it might be maintained in populations by selection. The frequency of IIL-2 was significantly associated with altitude (r=0.778, p<0.01) and minimum annual air temperature (r=−0.570, p<0.01) but because it was present at low frequency (<7%), selection probably was not involved. This inversion more likely represents a relict of ancestral polymorphisms. Comparison of the chromosome banding patterns revealed that S. aureohirtum is more closely related to S. ruficorne than to the S. ornatipes complex.
Cytogenetic traits were examined in Aloe saponaria (2S), A. vera (2V and 4V), and the experimental diploid (VS) and triploid (VVS) hybrids. Karyotype 2n=2x=14 (8L+6S) was confirmed in 2S and 2V, whereas 2n=4x=28 (16L+12S) was ascribed to 4V. VS showed the expected karyotype 2n=2x=14 (8L+6S), with slight differences between the long (L) homologues. VVS also showed the expected karyotype 2n=3x=21 (12L+9S); however, several chromosome deviations reflecting the absence or addition of small (S) chromosomes, terminal deletions of variable size which led to the formation of atypical chromosomes, or the loss of a L chromosome were also observed. The implication of these karyological variations is discussed.
Drosera spathulata complex, which consists of diploid, tetraploid and hexaploid populations, is widely but disjunctively distributed from eastern part of Australia throughout the South East Asian countries, to Japan. Among these populations, high morphological changing has been found in this species. To investigate intraspecific DNA polymorphism, and to infer the polyploid origin, some populations and cultivars of the Drosera spathulata complex and close related species were investigated using molecular cluster analysis with nucleotide sequences of the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL), and the internal transcribed spacer (ITS) of 18S-26S nuclear rDNA sequences (ITS). The rbcL analysis to estimate the species as maternal inheritance revealed that the highest similarity to the hexaploid sequence was found in that of the tetraploid. In contrast, the nuclear rDNA analysis clarified that the hexaploid had two types of ITS sequences: One type of the sequence showed the highest sequence similarity to ITS in D. rotundifolia L. genome, while another type of sequence showed the highest sequence similarity to ITS in the tetraploid D. spathulata Labill. genome. These results suggested that the hexaploid genome could be derived from amphiploidizaion between ancestor species of D. rotundifolia as paternal genome, and the tetraploid D. spathulata as maternal genome.
Karyotype and morphology of mitotic chromosomes in four populations of medicinal plant Silybum marianum collected from various geographical locations of Iran and two cultivars of this species were studied. Chromosome characteristics were measured from 10 complete metaphase cells using Micromeasure software. Results showed that S. marianum is a diploid species with 2n=2x=34 chromosomes. The karyotype consisted of six pairs of metacentric, ten pairs of submetacentric, and one pairs of acrocentric chromosomes. One of the chromosomes (chromosome 1) had a secondary constriction on centromeric region of its short arm. Karyological characteristics of all materials studied were similar to each other, however, among different genotypes some variations were observed on chromosome arm ratios and relative lengths. According to Stebbin's asymmetry index, S. marianum is placed at 2B category.
Morphological and cytogenetic analyses were performed in populations of Glycyrrhiza species growing in Iran. Clustering of the species based on morphological characters indicated distinctness of the Glycyrrhiza species studied. The present study revealed the presence of 2n=2x=16 chromosome number in the species studied, reporting the chromosome number of G. triphylla for the first time. Moreover, multipolar cells, abnormal tetrads and unreduced pollen grain formation are reported in Glycyrrhiza for the first time. Clustering of the Glycyrrhiza species and populations based on morphological and cytogenetic data showed close relationship between G. glabra and G. triphylla and between G. echinata and G. aspera supporting taxonomic treatment of the genus Glycyrrhiza in Flora Iranica.
We searched for candidate genes for producing salt tolerant plants from the red alga Cyanidioschyzon merolae, which lives in an extreme environment (hot springs). Arabidopsis thaliana plants die under 0.1 M salt culture, whereas the red algal cells survived under 0.3 M salt for 7 d. However, their chloroplasts changed from green to white and they soon died under 0.4 M, which is the concentration of seawater. Genes that were selectively expressed at 2 h and 24 h in 0.3 M salt concentrations were examined by microarray analysis. Under salt stress, the numbers of highly expressed genes at 2 h increased from 70 to 95 after culture for 24 h. The highly expressed genes included those encoding proteins similar to low molecular weight heat shock proteins, heat shock protein 70, and S-adenosylmethionine (SAM) synthetase. On the base of the present data and on the known metabolic functions of the proteins, we suggest that the SAM synthetase gene from C. merolae is a candidate gene for genetic engineering to produce salt tolerance plants.