FORMATION OF 2N MEGAGAMETOPHYTES IN DIPLOID TUBER-BEARING SOLANUMS

Development of megaspores and megagametophytes was analyzed for several diploid potato clones (Solanum spp.) that exhibit either high (HI) or low (LO) seed set when crossed as female with the tetraploid cultivated potato S. tuberosum Group Tuberosa. The objectives were to determine the relationship between ploidy and diam of nuclei and nucleoli, and to determine the mechanism(s) and frequencies of 2n megagametophyte formation. Sizes of nuclei and nucleoli were found to depend on ploidy. For HI clones, the distributions of sizes indicated that doubling occurred during meiosis, and that 30 to 50% of the megaspores and megagametophytes were 2n rather than haploid. Omission of the second meiotic division led to formation of second division restitution (SDR) 2n megagametophytes. Only one HI clone had abnormal meiosis I, in addition to omission of meiosis II in some meiocytes; this clone seemed to produce not only 1n and 2n, but also 4n megagametophytes. The results indicated that high crossability of the HI clones as female with tetraploids largely was due to formation of SDR 2n megagametophytes, a finding strongly supporting the hypothesis that sexual polyploidization is the driving force behind polyploidization of Solanums. The results contribute to increasing evidence that meiotic mutants and abnormalities play an important role in angiosperm evolution.     

Host Plant Specialization in the Sugarcane Aphid Melanaphis sacchari

Most aphids are highly specialized on one or two related plant species and generalist species often include sympatric populations adapted to different host plants. Our aim was to test the hypothesis of the existence of host specialized lineages of the aphid Melanaphis sacchari in Reunion Island. To this end, we investigated the genetic diversity of the aphid and its association with host plants by analyzing the effect of wild sorghum Sorghum bicolor subsp. verticilliflorum or sugarcane as host plants on the genetic structuring of populations and by performing laboratory host transfer experiments to detect trade-offs in host use. Genotyping of 31 samples with 10 microsatellite loci enabled identification of 13 multilocus genotypes (MLG). Three of these, Ms11, Ms16 and Ms15, were the most frequent ones. The genetic structure of the populations was linked to the host plants. Ms11 and Ms16 were significantly more frequently observed on sugarcane, while Ms15 was almost exclusively collected in colonies on wild sorghum. Laboratory transfer experiments demonstrated the existence of fitness trade-offs. An Ms11 isofemale lineage performed better on sugarcane than on sorghum, whereas an Ms15 lineage developed very poorly on sugarcane, and two Ms16 lineages showed no significant difference in performances between both hosts. Both field and laboratory results support the existence of host plant specialization in M. sacchari in Reunion Island, despite low genetic differentiation. This study illustrates the ability of asexual aphid lineages to rapidly undergo adaptive changes including shifting from one host plant to another.     

Distribution of signaling molecules involved in vasopressin-induced Ca2+ mobilization in rat hepatocyte multiplets.

In freshly isolated rat hepatocyte multiplets, Ca2+ signals in response to vasopressin are highly organized. In this study we used specific probes to visualize, by fluorescence and confocal microscopy, the main signaling molecules involved in vasopressin-mediated Ca2+ responses. V1a receptors were detected with a novel fluorescent antagonist, Rhm8-PVA. The Galphaq/Galpha11, PLCbeta3, PIP2, and InsP3 receptors were detected with specific antibodies. V1a vasopressin receptors and PIP2 were associated with the basolateral membrane and were not detected in the bile canalicular domain. Galphaq/Galpha11, PLCbeta3, and InsP3 receptors were associated with the basolateral membrane and also with other intracellular structures. We used double labeling, Western blotting, and drugs (cytochalasin D, colchicine) known to disorganize the cytoskeleton to demonstrate the partial co-localization of Galphaq/Galpha11 with F-actin.     

Genetic recombination in Sorghum bicolor x S. macrospermum interspecific hybrids

Sorghum has been improved by public and private breeding programs utilizing germplasm mostly from within the species Sorghum bicolor. Until recently, cross-incompatibilities have prevented hybridization of S. bicolor with most other species within the genus Sorghum. Utilizing germplasm homozygous for the iap allele, hybrids were readily produced between S. bicolor (2n = 20; AAB1B1) and S. macrospermum (2n = 40; WWXXYYZZ). These hybrids were intermediate to the parents in chromosome number (2n = 30) and overall morphology. Meiosis in both parents was regular; S. bicolor had 10 bivalents per pollen mother cell (PMC) and S. macrospermum had an average of 19.96 bivalents per PMC. Six hybrids were studied cytologically and meiosis was irregular, with the chromosomes associating primarily as univalents and bivalents. There was an average of 3.54 bivalents per PMC, with a range of 0-8 bivalents, most of which were rods (98%). Using FISH (fluorescent in situ hybridization), moderate levels (2.6 II per PMC) of allosyndetic recombination were observed. Genomic relationships were sufficient to assign S. macrospermum the genomic formula AAB1B1YYZZ (Y and Z remain unknown). Allosyndetic recombination in the interspecific hybrids indicates that introgression through genetic recombination should be possible if viable backcrosses can be recovered.     

A Detailed RFLP Map of Cotton, Gossypium Hirsutum X Gossypium Barbadense: Chromosome Organization and Evolution in a Disomic Polyploid Genome

We employ a detailed restriction fragment length polymorphism (RFLP) map to investigate chromosome organization and evolution in cotton, a disomic polyploid. About 46.2% of nuclear DNA probes detect RFLPs distinguishing Gossypium hirsutum and Gossypium barbadense; and 705 RFLP loci are assembled into 41 linkage groups and 4675 cM. The subgenomic origin (A vs. D) of most, and chromosomal identity of 14 (of 26), linkage groups is shown. The A and D subgenomes show similar recombinational length, suggesting that repetitive DNA in the physically larger A subgenome is recombinationally inert. RFLPs are somewhat more abundant in the D subgenome. Linkage among duplicated RFLPs reveals 11 pairs of homoeologous chromosomal regions-two appear homosequential, most differ by inversions, and at least one differs by a translocation. Most homoeologies involve chromosomes from different subgenomes, putatively reflecting the n = 13 to n = 26 polyploidization event of 1.1-1.9 million years ago. Several observations suggest that another, earlier, polyploidization event spawned n = 13 cottons, at least 25 million years ago. The cotton genome contains about 400-kb DNA per cM, hence map-based gene cloning is feasible. The cotton map affords new opportunities to study chromosome evolution, and to exploit Gossypium genetic resources for improvement of the world's leading natural fiber.     

Comparative mapping of bovine chromosome 13 by fluorescence in situ hybridization

We present chromosomal fluorescence in situ hybridization (FISH) results that both extend the HSA20/BTA13 comparative map as well as cytogenetically anchor two microsatellite markers. A bovine bacterial artificial chromosome (BAC) library was screened for conserved genes (type I loci) previously assigned to HSA10 or HSA20 and BTA13, and for microsatellites selected from two published BTA13 linkage maps. Clones from six out of nine comparative loci and both microsatellites were found represented in the BAC library. These BAC clones were used as probes in single colour FISH to determine the chromosome band position of each locus. As predicted by the human/bovine comparative map, all type I loci mapped to BTA13. Because single colour FISH analysis revealed that the loci were clustered within the distal half of BTA13, dual colour FISH was used to confirm the locus order. Established order was centromere- PRNP-(SODIL/AVP/OXT)-(BL42/GNAS1)-HCK-CSSM30 . The findings confirm the presence of a conserved HSA20 homologous synteny group on BTA13 distal of a HSA10 homologous segment.     

Genetic dissection of chromosome substitution lines of cotton to discover novel Gossypium barbadense L. alleles for improvement of agronomic traits

We recently released a set of 17 chromosome substitution (CS-B) lines (2n = 52) that contain Gossypium barbadense L. doubled-haploid line ‘3-79’ germplasm systematically introgressed into the Upland inbred ‘TM-1’ of G. hirsutum (L.). TM-1 yields much more than 3-79, but cotton from the latter has superior fiber properties. To explore the use of these quasi-isogenic lines in studying gene interactions, we created a partial diallel among six CS-B lines and the inbred TM-1, and characterized their descendents for lint percentage, boll weight, seedcotton yield and lint yield across four environments. Phenotypic data on the traits were analyzed according to the ADAA genetic model to detect significant additive, dominance, and additive-by-additive epistasis effects at the chromosome and chromosome-by-chromosome levels of CS-B lines. For example, line 3-79 had the lowest boll weight, seedcotton yield and lint yield, but CS-B22Lo homozygous dominance genetic effects on seedcotton and lint yield were nearly four times those of TM-1, and its hybrids with TM-1 had the highest additive-by-additive epistatic effects on seedcotton and lint yield. CS-B14sh, 17, 22Lo and 25 produced positive homozygous dominance effects on lint yield, whereas doubly heterozygous combinations of CS-B14sh with CS-B17, 22Lo and 25 produced negative dominance effects, suggesting that epistatic effects between genes in these chromosomes strongly affect lint yield. The results underscore the opportunities to systematically identify genomic regions harboring genes that impart agronomically significant effects via epistatic interactions. The chromosome-by-chromosome approach significantly complements other strategies to detect and quantify epistatic interaction effects, and the quasi-isogenic nature of families and lines from CS-B intermatings will facilitate high-resolution localization, development of markers for selection and map-assisted identification of genes involved in strong epistatic effects.     

FISH of a maize sh2-selected sorghum BAC to chromosomes of Sorghum bicolor.

Fluorescence in situ hybridization (FISH) of a 205 kb Sorghum bicolor bacterial artificial chromosome (BAC) containing a sequence complementary to maize sh2 cDNA produced a large pair of FISH signals at one end of a midsize metacentric chromosome of S. bicolor. Three pairs of signals were observed in metaphase spreads of chromosomes of a sorghum plant containing an extra copy of one arm of the sorghum chromosome arbitrarily designated with the letter D. Therefore, the sequence cloned in this BAC must reside in the arm of chromosome D represented by this monotelosome. This demonstrates a novel procedure for physically mapping cloned genes or other single-copy sequences by FISH, sh2 in this case, by using BACs containing their complementary sequences. The results reported herein suggest homology, at least in part, between one arm of chromosome D in sorghum and the long arm of chromosome 3 in maize.