Meiotic Configurations Research Articles

Meiotic behaviour and its implication on species inter-relationship in the genus Curcuma (Linnaeus, 1753) (Zingiberaceae).

In this paper, detailed meiotic analysis was investigated in seven species of Curcuma (Linnaeus, 1753) which can contribute significantly to our understanding about species inter-relationship, speciation and evolution. The species were divided into two groups viz., Group I having 2n = 42 (C. comosa Roxburgh, 1810, C. haritha Mangaly & M.Sabu, 1993, C. mangga Valeton & Zijp, 1917, and C. motana Roxburgh, 1800) and Group II with 2n = 63 (C. caesia Roxburgh, 1810, C. longa Linnaeus, 1753 and C. sylvatica Valeton, 1918). Both groups display varying degree of chromosome associations. Group I species showed the prevalence of bivalents, however occasional quadrivalents besides univalents were also encountered. About 48% of the PMCs analyzed in C. mangga showed 21 bivalents (II) meiotic configurations, 32% in C. comosa and 16% in C. haritha. Group II species as expected showed the presence of trivalents besides bivalents, univalents and quadrivalents. About 32% of the PMCs analyzed at MI in C. sylvatica showed 21 trivalents (III) meiotic configurations, 24% in C. longa and 8% in C. caesia. Overall, low frequency of multivalent associations as compared to bivalents indicates that Curcuma is an allopolyploid complex. Moreover, x = 21 is too high a basic number, therefore, we suggest that the genus Curcuma has evolved by hybridization of species with different chromosome numbers of 2n = 24 and 18, resulting in a dibasic amphidiploid species.

Cytological and Morphological Evaluation of Interspecific Hybrids between Trifolium repens and T. uniflorum

A significant constraint to the vegetative persistence of white clover (Trifolium repens L.) is summer moisture stress. A closely related species, T. uniflorum L., has a robust root system that could provide drought resistance. Hybridization of these two species leads to the generation of fertile hybrids. A study was conducted to generate F1 hybrids and first backcrosses (BC1) to white clover, and to evaluate the fertility, meiotic chromosome behavior, and plant morphology of these hybrids. Marker chromosome counts of the F1 and BC1 individuals confirmed hybridity. Meiotic configurations of five F1 and five BC1 plants indicated close homology between the two species and homoeologous pairing of white clover subgenomes. To evaluate phenotypes, clones of 56 individual genotypes (seven F1, 32 BC1, and 17 elite white clover plants) were grown in sand. After 13 mo, the leaves, stolons, and roots were measured and the dry weights of shoots and roots were determined by destructive harvest. Pattern analysis of the genotype‐by‐trait data identified four progeny groups. The F1 progeny were confined to the group with the lowest mean expression for most root and shoot traits. Most of the BC1 progeny grouped with the elite white clovers, but two BC1 formed a group with superior vigor and morphology, combining the best root and shoot traits of both parents. These BC1 individuals with similar shoot morphology to white clover and a robust root system, similar to T. uniflorum, are being integrated into a program to breed drought‐resistant hybrids to replace white clover in dry environments.

Does chromosomal change restrict gene flow between house mouse populations (Mus musculus domesticus)? Evidence from microsatellite polymorphisms

Chromosomal change is recognized as an important force in speciation-with-gene-flow models. Although Robertsonian (Rb) translocations contribute to hybrid unfitness and modify recombination patterns, they are generally not considered as efficient as inversions in reducing gene flow. The present study analyses two hybridizing chromosomal races of the house mouse (Mus musculus domesticus), one carrying nine Rb fusions (2n = 22) and the other carrying the ancestral karyotype (2n = 40). Chromosomal heterozygosity involves simple meiotic configurations, the underdominance of which is not as efficient in promoting speciation as more complex chromosomal differences. The pattern of divergence between these races was investigated using 32 microsatellite loci distributed over all autosomes. The results highlighted a relatively large differentiation between the two chromosomal races. The contribution of variation in recombination patterns to this differentiation was supported by the contrasting levels of divergence between proximal vs. non-proximal microsatellite loci. Clinal analyses of the Rb fusions through the hybrid zone uncovered differences in steepness between metacentrics, suggesting associated differences in selection which did not involve linked genic incompatibilities. This study illustrates the significant role of Rb-associated changes in recombination in promoting or maintaining divergence between chromosomal races of the house mouse.

Possible origin of Triticum petropavlovskyi based on cytological analyses of crosses between T. petropavlovskyi and tetraploid, hexaploid, and synthetic hexaploid (SHW-DPW) wheat accessions

Intraspecific hybridization between Triticum petropavlovskyi Udacz. et Migusch., synthetic hexaploid wheat (SHW-DPW), and tetraploid and hexaploid wheat, was performed to collect data on seed set, fertility of F1 hybrid, and meiotic pairing configuration, aiming to evaluate the possible origin of T. petropavlovskyi. Our data showed that (1) seed set of crosses T. petropavlovskyi × T. polonicum and T. petropavlovskyi × T. aestivum cv. Chinese Spring was significantly high; (2) fertility of hybrids T. petropavlovskyi × T. polonicum and T. petropavlovskyi × T. aestivum ssp. yunnanense was higher than that of the other hybrids; (3) fertility of F1 hybrids SHW-DPW × T. dicoccoides and SHW-DPW×T. aestivum ssp. tibetanum was significantly high; and (4) c-value of T. petropavlovskyi × T. polonicum and T. petropavlovskyi × T. aestivum cv. Changning white wheat was also significantly high. The results indicate that the probable origin of T. petropavlovskyi is divergence from a natural cross between T. aestivum and T. polonicum, via either spontaneous introgression or breeding effort.

Cytogenetic study and stripe rust response of the derivatives from a wheat - Thinopyrum intermedium - Psathyrostachys huashanica trigeneric hybrid.

To transfer multiple desirable alien genes into common wheat, we previously reported a new trigeneric hybrid synthesized by crossing a wheat - Thinopyrum intermedium partial amphiploid with wheat - Psathyrostachys huashanica amphiploid. Here, the meiotic behavior, chromosome constitution, and stripe rust resistance of F5 derivatives from the wheat - Th. intermedium - P. huashanica trigeneric hybrid were studied. Cytological analysis indicated the F5 progenies had chromosome numbers of 42-50 (average 44.96). The mean meiotic configuration was 1.28 univalents, 21.74 bivalents, 0.04 trivalents, and 0.02 tetravalents per pollen mother cell. In 2n = 42 lines, the average pairing configuration was 0.05 I + 19.91 II (ring) + 1.06 II (rod) + 0.003 IV, suggesting these lines were cytologically stable. Most lines with 2n = 43, 44, 46, 48, or 50, bearing a high frequency of univalents or multivalents, showed abnormal meiotic behavior. Genomic in situ hybridization karyotyping results revealed that 25 lines contained 1-7 Th. intermedium chromosomes, but no P. huashanica chromosomes were found among the 27 self-pollinated progenies. At meiosis, univalents (1-5) possessing Th. intermedium hybridization signals were detected in 19 lines. Bivalents (1-3) expressing fluorescence signals were observed in 12 lines. Importantly, 21 lines harbored wheat - Th. intermedium chromosomal translocations with various alien translocation types. Additionally, two homozygous lines, K13-668-10 and K13-682-12, possessed a pair of wheat - Th. intermedium small fragmental translocations. Compared with the recurrent parent Zhong 3, most lines showed high resistance to the stripe rust (Puccinia striiformis f. sp. tritici) pathogens prevalent in China, including race V26/Gui22. This paper reports a highly efficient technical method for inducing alien translocation between wheat and Th. intermedium by trigeneric hybridization. These lines might be potentially valuable germplasm resources for further wheat improvement.

A Multigenic Network of ARGONAUTE4 Clade Members Controls Early Megaspore Formation in Arabidopsis

The development of gametophytes relies on the establishment of a haploid gametophytic generation that initiates with the specification of gametophytic precursors. The majority of flowering plants differentiate a single gametophytic precursor in the ovule: the megaspore mother cell. Here we show that, in addition to argonaute9 (ago9), mutations in other ARGONAUTE (AGO) genes such as ago4, ago6, and ago8, also show abnormal configurations containing supernumerary gametophytic precursors in Arabidopsis thaliana. Double homozygous ago4 ago9 individuals showed a suppressive effect on the frequency of ovules with multiple gametophytic precursors across three consecutive generations, indicating that genetic interactions result in compensatory mechanisms. Whereas overexpression of AGO6 in ago9 and ago4 ago9 confirms strong regulatory interactions among genes involved in RNA-directed DNA methylation, AGO8 is overexpressed in premeiotic ovules of ago4 ago9 individuals, suggesting that the regulation of this previously presumed pseudogene responds to the compensatory mechanism. The frequency of abnormal meiotic configurations found in ago4 ago9 individuals is dependent on their parental genotype, revealing a transgenerational effect. Our results indicate that members of the AGO4 clade cooperatively participate in preventing the abnormal specification of multiple premeiotic gametophytic precursors during early ovule development in A. thaliana.

Beta-mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes.

In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1mM) and high (0.7mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recovered from slaughterhouse ovaries were used. Meiotic configuration after in vitro maturation (IVM) and early embryo production after intracytoplasmic sperm injection (ICSI) were considered as criteria for assessing nuclear and cytoplasmic maturation, respectively. A total of 1,076 oocytes were analysed over two experiments: 848 (control n=293, BME 0.1 n=270, BME 0.7 n=285) were stained with Hoechst 33342 and examined for nuclear stage after 26hr of IVM, and 228 MII oocytes were fertilized by ICSI (control n=83, BME 0.1 n=65, BME 0.7 n=80). Cleavage rates were determined after 60hr of culture. Unlike results obtained in other species, the addition of BME did not influence maturation rates (51.9% control vs 55.6% BME 0.1mM and 55.1% BME 0.7mM), nor cleavage rates after ICSI (38.6% vs 38.5% and 41.3%, respectively). In conclusion, the addition of BME at 0.1 and 0.7mM to the maturation medium, in our culture conditions, has no effect on nuclear and cytoplasmic maturation of equine oocytes.

Genomic affinities between Brassica napus and Raphanus raphanistrum as revealed by meiotic GISH

AbstractAn intergeneric hybrid between Brassica napus (AC, n = 19) and Raphanus raphanistrum (Rr, n = 9) was developed using the sequential ovary–ovule culture. Morphological, molecular, cytological studies and fluorescent genomic in situ hybridization were conducted to establish the hybridity and genomic relatedness. F1 hybrid plants were male sterile and morphologically intermediate between the two parents. Cytological analysis of the hybrid plants revealed 10II + 8I as the predominant meiotic configuration with the mean bivalent frequency of 8.83. A maximum of 13II were observed in 8.24% of the pollen mother cells (PMCs). GISH studies facilitated the identification of two allopairs, confirming homoeologous pairing between A/C and Rr genomes. This may have significant implications in terms of transgene flow and possible introgression into R. raphanistrum, which is a common canola weed in Canada and Australia. The intergeneric hybrid B. napus × R. raphanistrum so produced also has the potential to be used as a bridging species for the transfer of desirable genes to amphiploid crop Brassica species carrying A/C genomes.

Cytogenetic Behavior of Trigeneric Hybrid Progeny Involving Wheat, Rye and Psathyrostachys huashanica

Trigeneric hybrids are commonly used as bridges to transfer genes from some wild species to cultivated wheat and to measure the genomic interaction between donor species. We previously reported that trigeneric germplasms were produced by crossing wheat-Psathyrostachys huashanica amphiploids (PHW-SA, 2n = 8x = 56, AABBDDNsNs) with hexaploid triticale (Zhongsi 828, 2n = 6x = 42, AABBRR). In the present study, chromosome pairing behavior and the genome constitution of the F₄ progenies of wheat-rye-P. huashanica trigeneric hybrids were studied. Cytological analysis showed that the chromosome number of F₄ progenies ranged from 39 to 46, and 57.5% of them had 42 chromosomes. The mean meiotic configuration of F₄ lines was 1.71 univalents, 20.26 bivalents, 0.04 trivalents, and 0.001 quadrivalents per pollen mother cell. Among the lines with 2n = 42, the average pairing configuration was 1.21 univalents, 16.22 ring bivalents, 4.16 rod bivalents, and 0.01 trivalents. This result indicated that these lines were cytologically stable. Other lines with 2n = 39, 40, 41, 43, 44, 45, and 46, bearing a high number of univalents or multivalents, showed abnormal meiotic behavior. Genomic in situ hybridization (GISH) revealed that all F₄ lines had 11-14 rye chromosomes, but no P. huashanica chromosomes. The complete set of 14 rye chromosomes was found in 19 lines. At meiosis, GISH detected 1-6 univalents with hybridization signals of rye in 13 lines. Bivalents with fluorescence signals were identified in each line, ranging from 3 to 7. A quadrivalent with hybridization signals was observed in only 1 line, K13-714-8. Lagging chromosomes, chromosome bridges, micronuclei, and chromosome fragments hybridizing with the probe were not discovered in any of the lines. These results inferred that the behavior of rye chromosomes was normal during meiosis. In addition, 21 lines of 2n = 42 (91.3%) with 12 or 14 rye chromosomes, always contained 6 or 7 bivalents bearing fluorescence signals. This suggested that the rye chromosomes exhibiting complete pairing in these lines were cytologically stable during meiosis and may therefore be considered as new hexaploid triticales. Thus, these lines might be potential materials for further hexaploid triticale improvement.

Genetic differentiation within and away from the chromosomal rearrangements characterising hybridising chromosomal races of the western house mouse (Mus musculus domesticus).

The importance of chromosomal rearrangements for speciation can be inferred from studies of genetic exchange between hybridising chromosomal races within species. Reduced fertility or recombination suppression in karyotypic hybrids has the potential to maintain or promote genetic differentiation in genomic regions near rearrangement breakpoints. We studied genetic exchange between two hybridising groups of chromosomal races of house mouse in Upper Valtellina (Lombardy, Italy), using microsatellites. These groups differ by Robertsonian fusions and/or whole-arm reciprocal translocations such that F1 hybrids have a chain-of-five meiotic configuration. Previous studies showed genetic differentiation in two chromosomes in the chain-of-five (10 and 12) close to their centromeres (i.e. the rearrangement breakpoints); we have shown here that the centromeric regions of the other two chromosomes in the chain (2 and 8) are similarly differentiated. The internal chromosomes of the chain (8 and 12) show the greatest differentiation, which may reflect pairing and recombination properties of internal and external elements in a meiotic chain. Importantly, we found that centromeric regions of some non-rearranged chromosomes also showed genetic differentiation between the hybridising groups, indicating a complex interplay between chromosomal rearrangements and other parts of the genome in maintaining or promoting differentiation and potentially driving speciation between chromosomal races.

Resynthesis of Brassica napus through hybridization between B. juncea and B. carinata.

First report for the resynthesis of Brassica napus by recombining A and C genome from B. juncea and B. carinata , respectively. Also documents B genome introgressions in resynthesized B. napus. Resynthesis of Brassica napus (AACC) was achieved by hybridizing Brassica juncea (AABB) with Brassica carinata (BBCC). This was facilitated by spontaneous chromosome doubling in the F1 hybrid (ABBC) to yield octaploid (AABBBBCC), elimination of extra B genome chromosomes in the resulting octaploid and in subsequent selfed generations, aided with directed selection for fertile plants having B. napus morphology. Twenty-five plants with varying degrees of resemblance to natural B. napus were identified from 17 A5 progenies and assayed for cytogenetic stability and genetic diversity. Majority of these plants, except six (2n = 38) were hyperploids (2n = 40-56). The six plants with 2n = 38 were designated as derived B. napus types. These showed an expected meiotic configuration of 19II at metaphase-I, with 19-19 distribution at anaphase-I. Genotyping based on A and C genome specific primers confirmed genetic identity of six derived (2n = 38) B. napus plants with natural types whereas genotyping with B genome specific primers indicated introgression of B genome segments. This was also confirmed by genomic in situ hybridization (GISH). Strong signals of B genome probe were detected, proving hitherto unreported genetic exchanges between B and A/C chromosomes. These introgressions possibly occurred en route five generations of selfing. Derived plants yielded fertile hybrids in crosses with natural B. napus var. GSC 6. The selfed derived plants as evaluated in A6 plant to progeny rows were morphologically similar to natural B. napus, and meiotically stable. Agronomic assessment of these progenies revealed variation for key morpho-physiological traits. Of special interest were the progenies with plants having oil content exceeding 47% as against about 39-41% in existing cultivars.

211 PRODUCTION OF PROGESTERONE FROM CANINE CUMULUS CELLS MATURED IN VITRO WITH STEROIDS AND GONADOTROPIN

The canine reproductive physiology is distinct from other mammals, especially concerning the role of progesterone in ovulation, oocyte maturation, and fertilisation. In vitro maturation of canine oocytes is challenging mostly due to the culture conditions required for this species and the lack of knowledge about the factors involved in meiotic resumption and complete maturation. To obtain more information about this physiological phenomenon, the present study was designed to investigate the ability of canine cumulus cells to secrete progesterone in the maturation medium during in vitro culture for up to 96 h. Ovaries were collected from 18 bitches at different reproductive status (follicular, luteal, and anestrous stages; various breeds, age 1–7 years) by routine ovariohysterectomy and sliced in PBS-polyvinyl alcohol (PVA) to release the cumulus‐oocyte complexes (COC). A total of 896 COC grade I (2 or more dense layers of cumulus cells and darkly pigmented cytoplasm) were selected for culture. Oocytes were randomly allocated in 2 media consisting of TCM-199 (Sigma Chemical Co., St. Louis, MO, USA) with antibiotics, 10% FBS, 2.2 mg mL–1 sodium bicarbonate, and 20 μL mL–1 pyruvic acid (control) and control + hormones (25 IU mL–1 hCG (Sigma), 1 mg mL–1 progesterone (Sigma), 1 mg mL–1 estradiol (E2; Sigma). The COC were incubated at 38°C, 5% CO2 in air and cultured for 24, 48, 72, and 96 h. Maturation medium for in vitro culture of COC was collected at 0, 24, 48, 72, and 96 h and checked for progesterone content by radioimmunoassay (RIA). Maturation rates (meiotic configuration) were also evaluated at each culture period by using Hoechst 33342 (Sigma). Polynomial regression analysis was applied to assess variation of progesterone concentration in the maturation medium during the 96 h of IVM. Progesterone was detected in the control and hormonal media; however, in the control medium the concentration was <0.5 ng mL–1. In the hormonal medium, there was a decrease in progesterone concentration at 24 h in all groups (ranged from 461.8 to 587.2 ng mL–1) followed by a progressive increase until 72 h in follicular group (1371.4 ng mL–1) and until 96 h in luteal and anestrous groups (934.4 and 669.4 ng mL–1, respectively). Maturation rates (metaphase II oocytes) were higher at 72 h and decreased at 96 h in all groups. The results of this study suggest that hormonal supplementation is necessary for canine oocyte to resume meiosis and to cumulus cells be capable of producing progesterone. Moreover, progesterone secretion by cumulus cells seems to be related to the reproductive status of the donor bitch once higher concentration of progesterone was detected in medium from follicular group and lower in anestrous group.

Genomic affinities revealed by GISH suggests intergenomic restructuring between parental genomes of the paleopolyploid genus Zea.

The present work compares the molecular affinities, revealed by GISH, with the analysis of meiotic pairing in intra- and interspecific hybrids between species of Zea obtained in previous works. The joint analysis of these data provided evidence about the evolutionary relationships among the species from the paleopolyploid genus Zea (maize and teosintes). GISH and meiotic pairing of intraspecific hybrids revealed high genomic affinity between maize (Zea mays subsp. mays) and both Zea mays subsp. parviglumis and Zea mays subsp. mexicana. On the other hand, when Zea mays subsp. huehuetenanguensis DNA was probed on maize chromosomes, a lower affinity was detected, and the pattern of hybridization suggested intergenomical restructuring between the parental genomes of maize. When DNA from Zea luxurians was used as probe, homogeneous hybridization signals were observed through all maize chromosomes. Lower genomic affinity was observed when DNA from Zea diploperennis was probed on maize chromosomes, especially at knob regions. Maize chromosomes hybridized with Zea perennis DNA showed hybridization signals on four chromosome pairs: two chromosome pairs presented hybridization signal in only one chromosomal arm, whereas four chromosome pairs did not show any hybridization. These results are in agreement with previous GISH studies, which have identified the genomic source of the chromosomes involved in the meiotic configurations of Z. perennis × maize hybrids. These findings allow postulating that maize has a parental genome not shared with Z. perennis, and the existence of intergenomic restructuring between the parental genomes of maize. Moreover, the absence of hybridization signals in all maize knobs indicate that these heterochromatic regions were lost during the Z. perennis genome evolution.

Dynamic karyotype evolution and unique sex determination systems in Leptidea wood white butterflies.

BackgroundChromosomal rearrangements have the potential to limit the rate and pattern of gene flow within and between species and thus play a direct role in promoting and maintaining speciation. Wood white butterflies of the genus Leptidea are excellent models to study the role of chromosome rearrangements in speciation because they show karyotype variability not only among but also within species. In this work, we investigated genome architecture of three cryptic Leptidea species (L. juvernica, L. sinapis and L. reali) by standard and molecular cytogenetic techniques in order to reveal causes of the karyotype variability.ResultsChromosome numbers ranged from 2n = 85 to 91 in L. juvernica and 2n = 69 to 73 in L. sinapis (both from Czech populations) to 2n = 51 to 55 in L. reali (Spanish population). We observed significant differences in chromosome numbers and localization of cytogenetic markers (rDNA and H3 histone genes) within the offspring of individual females. Using FISH with the (TTAGG)n telomeric probe we also documented the presence of multiple chromosome fusions and/or fissions and other complex rearrangements. Thus, the intraspecific karyotype variability is likely due to irregular chromosome segregation of multivalent meiotic configurations. The analysis of female meiotic chromosomes by GISH and CGH revealed multiple sex chromosomes: W1W2W3Z1Z2Z3Z4 in L. juvernica, W1W2W3Z1Z2Z3 in L. sinapis and W1W2W3W4Z1Z2Z3Z4 in L. reali.ConclusionsOur results suggest a dynamic karyotype evolution and point to the role of chromosomal rearrangements in the speciation of Leptidea butterflies. Moreover, our study revealed a curious sex determination system with 3–4 W and 3–4 Z chromosomes, which is unique in the Lepidoptera and which could also have played a role in the speciation process of the three Leptidea species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0375-4) contains supplementary material, which is available to authorized users.

Mitotic and Meiotic Behavior of B Chromosomes in Crenicichla lepidota: New Report in the Family Cichlidae.

B chromosomes are additional genetic elements to the standard complement. They display distinctive features and have been found in 15% of eukaryote species. In this study, we analyzed 4 populations of Crenicichla lepidota from hydrographic system of Laguna dos Patos/RS (Brazil). All specimens showed 2n = 48 with 6m + 42st - a, FN = 54, with a secondary constriction on the first pair of the complement. Among the 18 samples analyzed, 6 individuals belonging to the Gasômetro and Saco da Alemoa populations presented 1-3 small-sized heterochromatic B chromosomes, with intra- and interindividual variation. Simple AgNORs coincident with 18S rDNA and CMA3 positive/DAPI negative sites were present in all populations. The extra chromosomes did not exhibit any 18S rDNA sites. The meiotic analyses showed heteropycnotic regions in leptotene and zygotene stages, which may be related to the presence of B chromosomes. During pachytene were found 24 bivalents and 1 spatially separated, as well as during metaphases I and diplotene, indicating that there is no association between B chromosomes and those of the A complement. During diakinesis, an unusual meiotic configuration was observed, revealing a proximity between the bivalent and chromosome B (univalent), that might be the result of a heterochromatin affinity between these chromosomes. In anaphase I, late migration of B chromosomes was detected. The low frequency of B chromosomes in the Cichlidae family and in Crenicichla suggests its recent origin in this group and may be ascribable to animal exposure to deleterious effects under certain environmental conditions. Moreover, this is the first report in C. lepidota.

Meiotic configuration and rDNA distribution patterns in SixRubustaxa

More than 200 Rubus species described so far have been included in subgenera Idaeobatus and Malachobatus. Tremendous amount of genetic diversity exists in Rubus due to inter- or intra-species hybridization, polyploidization, or agamospermy make it a typical taxonomically challenging but genetically intriguing genus among land plants. In present study, meiotic pairing behavior and the distribution patterns of 45S and 5S ribosomal DNA (rDNA) arrays were assessed by fluorescence in situ hybridization (FISH) in six Rubus taxa with ploidy ranging from diploid (2n=14) to octoploid (2n=56). At all ploidy levels, bivalent pairing is the most predominant form in meiotic configuration. Very few multivalents were observed in tetraploid or octoploid samples. FISH analysis revealed two 45S rDNA sites in each of the two diploid taxa. Each of the polyploids (2n=28) studied had the expected multiples of this number except the tetraploid R. setchuenensis (with only three sites). On the contrary, both diploid and tetraploid taxa had two 5S rDNA loci, while the octoploid R. buergeri carried only three sites. Both meiotic behavior observations and FISH results support that Rubus polyploids might be of allopolyploid in origin.

45 SPINDLE CONFIGURATION AND DNA FRAGMENTATION OF VITRIFIED BOVINE OOCYTES AFTER IN VITRO MATURATION WITH L-CARNITINE AND/OR RESVERATROL

After cryopreservation, oocytes may suffer morphological and functional damage, due to the high cytoplasm lipid content and to the reactive oxygen species formation. The aim of this work was to evaluate the spindle configuration and the DNA fragmentation of vitrified/warmed oocytes after in vitro maturation (IVM) in a media supplemented with l-carnitine and/or resveratrol, a lipolytic and antioxidant agent, respectively. The IVM viable COC with at least 3 cumulus cell layers and homogenous cytoplasm were randomly distributed into 4 groups: (1) control: conventional IVM media with TCM-199, epidermal growth factor, and 10% FCS; (2) L-CAR: control media supplemented with 0.6 mg mL–1 of l-carnitine; (3) RES: control media supplemented with 1 μM mL–1 of resveratrol; and 4) L+R: control media supplemented with 0.6 mg mL–1 of l-carnitine and 1 μM mL–1 of resveratrol. After 22 h of IVM, half of the COC from each group were vitrified and warmed, using the cryotop methodology. After warming, the oocytes were allowed to recover in their respective media for 2 additional hours. After 24 h of IVM, oocytes from all treatments were completely denuded and fixed and stained using specific fluorescent probes. The microtubule/chromosome configuration and the DNA fragmentation were analysed by immunocytochemistry under a fluorescent microscope (A.40FL, Carl Zeiss, Oberkochen, Germany). All statistical analyses were conducted with IBM SPSS 19 (IBM; Chicago, IL, USA). ANOVA was performed to analyse differences in meiotic spindle configuration, and the Chi-squared test was used for DNA fragmentation. The significance level was 5%. Although vitrification may cause severe oocyte damage, IVM with l-carnitine alone or in association with resveratrol was able to reduce the percentage of abnormal spindle configurations (Table 1), whereas the addition of resveratrol alone or its association with l-carnitine reduced DNA fragmentation of IVM oocytes after a vitrification/warming process. These results indicate the IVM supplementation with RES and/or L-CAR could modify oocyte composition, increasing its cryotolerance. However further studies are required to confirm the beneficial effect of these molecular interactions. Table 1.Evaluation of spindle configuration (Experiment 1) and apoptotic cell status (Experiment 2) of fresh or vitrified/warmed oocytes matured with RES and/or L-CAR

Characterization of intergeneric hybrid between common wheat andAegilops sharonensis(Eig) and transfer of alien high molecular weight glutenin subunits into wheat

Aegilops sharonensis (Sharon goatgrass) is a valuable source of novel high molecular weight glutenin subunits, resistance to wheat rust, powdery mildew, and insect pests. In this study, we successfully hybridized Ae. sharonensis as the pollen parent to common wheat and obtained backcross derivatives. F1 intergeneric hybrids were verified using morphological observation and cytological and molecular analyses. The phenotypes of the hybrid plants were intermediate between Ae. sharonensis and common wheat. Observations of mitosis in root tip cells and meiosis in pollen mother cells revealed that the F1 hybrids possessed 28 chromosomes. Chromosome pairing at metaphase I of the pollen mother cells in the F1 hybrid plants was low, and the meiotic configuration was 25.94 I + 1.03 II (rod). Two pairs of primers were screened out from 150 simple sequence repeat markers, and primer WMC634 was used to identified the presence of the genome of Ae. sharonensis. Sequencing results showed that the F1 hybrids contained the Ssh genome of Ae. sharonensis. The sodium dodecyl sulfate polyacrylamide gel electrophoresis profile showed that the alien high molecular weight glutenin subunits of Ae. sharonensis were transferred into the F1 and backcross derivatives. The new wheat- Ae. sharonensis derivatives that we have produced will be valuable for increasing resistance to various diseases of wheat and for improving the quality of bread wheat.

Molecular cytogenetic characterization of the dioecious Cannabis sativa with an XY chromosome sex determination system.

Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.

Deep cytogenetics analysis reveals meiotic recombination depletion in species of Senecio (Asteraceae).

BackgroundSenecio is the largest genus in the Asteraceae family growing in all environments around the world. It displays taxonomic and systematical difficulties. Cytogenetic knowledge of this genus is ancient, scarce and mainly restricted to chromosome number records.ResultsIn this study we analyzed chromosome number, meiotic configuration, bivalent morphology, meiotic behavior and pollen grain stainability on 100 accessions of 27 different polyploid Senecio L. sect Senecio entities. Median, standard deviation and mode were calculated for number and position of chiasmata and meiotic recombination was statistically evaluated. Although high frequency of multivalents and associated meiotic irregularities are expected in high polyploids, bivalents predominance and, consequently, regular meiosis were observed, with normal sporogenesis and high pollen grain stainability.ConclusionDepletion in the total chiasmata was significant only in some species but the terminal position was preferential in all the entities analyzed, indicating significant reduction in recombination. The regular meiosis observed suggest that intra and intergenomic reorganization process occur quickly and efficiently in this genus. Mechanisms of diploidization, common to all polyploids, are reinforced by the strong reduction in crossing-over rushing polyploids stabilization.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-20) contains supplementary material, which is available to authorized users.