Shallow genetic divergence and species delineations in the endemic Labeobarbus species flock of Lake Tana, Ethiopia.

To assess whether the species distinctions of Lake Tana's Labeobarbus spp. are supported by genetic information, microsatellite markers were used. A total of 376 Labeobarbus spp., belonging to 24 populations of 11 species from three regions of the lake (north, south and east), were sampled. Eight microsatellite markers were analysed. In general, differences between conspecific populations were smaller than differences between populations of different species. For six species, conspecific populations from different regions in the lake were consistently more similar than populations of other species from the same region. For four species this was not the case, while for one species two populations were similar, but different from the third population. River-spawning species appeared to be more distinct than presumed lake spawners. On the species level, there was a significant correlation between genetic and morphological differentiation, especially in morphological aspects associated with ecological functioning. This suggests that genetic differentiation arose together with adaptive radiation, although the overall genetic differentiation among the Lake Tana Labeobarbus spp. is small.

Effect of polymorphisms in the FASN, OLR1, PPARGC1A, PRL and STAT5A genes on bovine milk-fat composition.

The aim of our study was to estimate effects of polymorphisms in the ATP-binding cassette G2 (ABCG2), fatty acid synthase (FASN), oxidized low-density lipoprotein receptor 1 (OLR1), peroxysome proliferator-activated receptor-gamma coactivator-1alpha (PPARGC1A), prolactin (PRL) and signal transducer and activator of transcription 5A (STAT5A) genes on milk production traits and detailed milk-fat composition. Milk-fat composition phenotypes were available for 1905 Dutch Holstein-Friesian cows. First, the presence of each SNP in the Dutch Holstein-Friesian population was evaluated by direct sequencing of the PCR product surrounding the SNP in 22 proven Dutch Holstein-Friesian bulls. The ABCG2 SNP did not segregate in the bull population. Second, we genotyped the cows for the FASN(g.16024G>A), FASN(g.17924A>G), OLR1(g.8232C>A), PPARGC1A(c.1790+514G>A), PPARGC1A(c.1892+19G>A), PRL(g.8398G>A) and STAT5A(g.9501G>A) polymorphisms, and estimated genotype effects on milk production traits and milk-fat composition. FASN(g.17924A>G) and OLR(g.8232C>A) had a significant effect (P < 0.05) on milk-fat percentage. However, we were not able to confirm results reported in the literature that showed effects of all evaluated polymorphisms on milk-fat percentage or milk-fat yield. All polymorphisms showed significant effects (P < 0.05) on milk-fat composition. The polymorphisms in FASN and STAT5A, which had an effect on C14:0 and were located on chromosome 19, could not fully explain the quantitative trait locus for C14:0 that was previously detected on chromosome 19 in a genome-wide scan using linkage analysis.

The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium.

Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination. We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 (tt12), from a reduced seed dormancy screen. Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells. Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway. The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family. TT12 is expressed specifically in ovules and developing seeds. In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation. The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium.

Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis.

The testa of higher plant seeds protects the embryo against adverse environmental conditions. Its role is assumed mainly by controlling germination through dormancy imposition and by limiting the detrimental activity of physical and biological agents during seed storage. To analyze the function of the testa in the model plant Arabidopsis, we compared mutants affected in testa pigmentation and/or structure for dormancy, germination, and storability. The seeds of most mutants exhibited reduced dormancy. Moreover, unlike wild-type testas, mutant testas were permeable to tetrazolium salts. These altered dormancy and tetrazolium uptake properties were related to defects in the pigmentation of the endothelium and its neighboring crushed parenchymatic layers, as determined by vanillin staining and microscopic observations. Structural aberrations such as missing layers or a modified epidermal layer in specific mutants also affected dormancy levels and permeability to tetrazolium. Both structural and pigmentation mutants deteriorated faster than the wild types during natural aging at room temperature, with structural mutants being the most strongly affected.

Importance of the B2 domain of the Arabidopsis ABI3 protein for Em and 2S albumin gene regulation.

Genetic and molecular studies have shown that the Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3) protein plays a prominent role in the control of seed maturation. The ABI3 protein and its orthologues from various other plant species share four domains of high sequence identity, including three basic domains designated as B1, B2 and B3. The leaky abi3-1 mutation is a single amino acid substitution within the B3 domain. A new abi3 allele, abi3-7, was generated by mutagenizing abi3-1 seeds. The abi3-7 line contains, in addition to the abi3-1 mutation, a point mutation that converts residue Ala-458 into Thr within the B2 domain of the ABI3 protein. This Ala residue is absolutely conserved in all known ABI3 orthologues. Abi3-7 seeds display reductions in dormancy and in sensitivity to abscisic acid which are intermediate between those of the leaky abi3-1 and of the severe abi3-4 and abi3-5 mutants. Accumulation and distribution of At2S1 and At2S2 albumin mRNA as well as of AtEm1 and AtEm6 late embryogenesis-abundant proteins and mRNA have been analyzed. Both At2S1 and At2S2 mRNA are reduced in abi3-7, but distribution of At2S2 is spatially restricted. Accumulation of AtEm6 protein is more sensitive to abi3-7 mutation than AtEm1. However both mRNAs are considerably reduced in this mutant. Their distribution is also differentially affected. These results provide genetic evidence for the importance of the conserved B2 domain for ABI3 function in vivo.

Properties of proteins and the glassy matrix in maturation-defective mutant seeds of Arabidopsis thaliana.

In situ Fourier transform infrared microspectroscopy was used to study the heat stability of proteins and hydrogen bonding interactions in dry maturation-defective mutant seeds of Arabidopsis thaliana. alpha-Helical, turn and beta-sheet conformations were the major protein secondary structures in all of these seeds. On heating, intermolecular extended beta-sheet structures, typical of protein denaturation, were formed in abscisic acid-insensitive (abi3) and leafy cotyledon (lec) mutant seeds. Proteins in dry wild-type seeds did not denature up to 150 degrees C, but those in dry desiccation-sensitive, lec1-1, lec1-3 and abi3-5 seeds did at 68, 89 and 87 degrees C, respectively. In the desiccation-tolerant abi3-7 and abi3-1 seeds, denaturation commenced above 120 and 135 degrees C, respectively. Seeds of the aba1-1 abi3-1 double mutant showed signs of denaturation already upon drying. The molecular packing in the seeds was studied by observing the shift in the position of the OH-stretching vibration band with temperature. The maximal rate of change of this band with temperature was much higher in the desiccation-sensitive abi3-5, aba1-1 abi3-1, lec1-1, and lec1-3 mutant seeds than in the desiccation-tolerant wild-type, abi3-1, abi3-7, and lec2-1 seeds. We interpret this to mean that the molecular packing density is higher in dry desiccation-tolerant than in dry desiccation-sensitive seeds, which is associated with a higher or lower protein denaturation temperature, respectively. The results are discussed in relation to the physiological and biochemical characteristics of these mutant seeds.

QTL analysis of seed dormancy in Arabidopsis using recombinant inbred lines and MQM mapping.

The genetic differences for seed germination between two commonly used Arabidopsis thaliana ecotypes Ler and Col, both showing a low level of seed dormancy, were investigated. The analysis was performed with 98 recombinant inbred lines (RILs) derived from the cross between the two ecotypes, and these lines had previously been analysed for molecular marker composition by Lister and Dean (Norwich, UK). The analysis of germination was performed on seeds grown in three different maternal environments and each seed batch was tested in three different germination environments: in light, in darkness and in the presence of the gibberellin inhibitor paclobutrazol. Fourteen loci were identified using the multiple-QTL-model (MQM) procedure for mapping quantitative trait loci. At nine loci no significant interaction between the detection of the locus and environmental factors could be detected. However, three other distinct loci controlling the germination behaviour in the presence of the gibberellin inhibitor paclobutrazol had a much lower or no effect when germination was tested in water either in light or darkness. Two other loci affecting germination in darkness and/or light had practically no effect on germination in the presence of paclobutrazol.

Biochemical characterization of the aba2 and aba3 mutants in Arabidopsis thaliana.

Abscisic acid (ABA)-deficient mutants in a variety of species have been identified by screening for precocious germination and a wilty phenotype. Mutants at two new loci, aba2 and aba3, have recently been isolated in Arabidopsis thaliana (L.) Hynh. (K.M. Léon-Kloosterziel, M. Alvarez-Gil, G.J. Ruijs, S.E. Jacobsen, N.E. Olszewski, S.H. Schwartz, J.A.D. Zeevaart, M. Koornneef [1996] Plant J 10: 655-661), and the biochemical characterization of these mutants is presented here. Protein extracts from aba2 and aba3 plants displayed a greatly reduced ability to convert xanthoxin to ABA relative to the wild type. The next putative intermediate in ABA synthesis, ABA-aldehyde, was efficiently converted to ABA by extracts from aba2 but not by extracts from aba3 plants. This indicates that the aba2 mutant is blocked in the conversion of xanthoxin to ABA-aldehyde and that aba3 is impaired in the conversion of ABA-aldehyde to ABA. Extracts from the aba3 mutant also lacked additional activities that require a molybdenum cofactor (Moco). Nitrate reductase utilizes a Moco but its activity was unaffected in extracts from aba3 plants. Moco hydroxylases in animals require a desulfo moiety of the cofactor. A sulfido ligand can be added to the Moco by treatment with Na2S and dithionite. Treatment of aba3 extracts with Na2S restored ABA-aldehyde oxidase activity. Therefore, the genetic lesion in aba3 appears to be in the introduction of S into the Moco.

Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci.

Novel Arabidopsis mutants with lowered levels of endogenous abscisic acid (ABA) were isolated. These were selected in a screen for germination in the presence of the gibberellin biosynthesis inhibitor paclobutrazol. Another mutant was isolated in a screen for NaCl tolerance. The ABA-deficiency was caused by two monogenic, recessive mutations, aba2 and aba3, that were both located on chromosome 1. The mutants showed a phenotype that is known to be characteristic for ABA-deficiency: a reduced seed dormancy and excessive water loss, leading to a wilty phenotype. Double mutant analysis, combining different aba mutations, indicated the leaky nature of the mutations.

Arabidopsis mutants with a reduced seed dormancy.

The development of seed dormancy is an aspect of seed maturation, the last stage of seed development. To isolate mutants of Arabidopsis thaliana that are affected in this process, we selected directly for the absence of dormancy among freshly harvested M2 seeds. The screen yielded two mutants exhibiting a reduced dormancy, rdo1 and rdo2, that are specifically affected in dormancy determined by the embryo. The rdo1 and rdo2 mutants show normal levels of abscisic acid and the same sensitivity to abscisic acid, ethylene, auxin, and cytokinin as the wild type. The rdo2 mutant but not the rdo1 mutant has a reduced sensitivity to the gibberellin biosynthesis inhibitor tetcyclacis. Double-mutant analysis suggested that the RDO1 and RDO2 genes are involved in separate pathways leading to the development of dormancy. We assume that the RDO2 gene controls a step in the induction of dormancy that is most likely induced by abscisic acid and is expressed as an increase of the gibberellin requirement for germination.

A Seed Shape Mutant of Arabidopsis That Is Affected in Integument Development.

A seed shape mutant of Arabidopsis was isolated from an ethyl methanesulfonate-treated population. Genetic analysis revealed that the heart-shaped phenotype was maternally inherited, showing that this is a testa mutant. This indicated the importance of the testa for the determination of the seed shape. This recessive aberrant testa shape (ats) gene was located at position 59.0 on chromosome 5. A comparison was made between ovules and developing and mature seeds of the wild type and of the mutant using light and scanning electron microscopy. We showed that the mutant seed shape is determined during the first few days after fertilization, when the embryo occupies only a very small part of the seed. The integuments of ats ovules consisted of only three rather than five cell layers. In double mutants, the effect of ats was additive to other testa mutations, such as transparent testa, glabra (ttg), glabrous2 (gl2), and apetala2 (ap2). The ats mutation resulted in a reduced dormancy, which was maternally inherited. This effect of a testa mutation on germination was also seen in ttg seeds, in which the outer layer of the testa was disturbed. This indicated the importance of the testa as a factor in determining dormancy in Arabidopsis.

Acquisition of Desiccation Tolerance and Longevity in Seeds of Arabidopsis thaliana (A Comparative Study Using Abscisic Acid-Insensitive abi3 Mutants).

Two new abscisic acid (ABA)-insensitive mutants of Arabidopsis thaliana affected in the abi3 locus are described. These new mutants are severely ABA insensitive. Like the earlier described abi3-1 and the ABA-deficient and -insensitive double mutant aba,abi3, these new mutants vary in the extent of ABA-correlated physiological responses. Mutant seeds fail to degrade chlorophyll during maturation and show no dormancy, and desiccation tolerance and longevity are poorly developed. Carbohydrate accumulation as well as synthesis of LEA or RAB proteins are often suggested to be essential for acquisition of desiccation tolerance. In this work two points are demonstrated. (a) Accumulation of carbohydrates as such does not correlate with acquisition of desiccation tolerance or longevity. It is suggested that a low ratio of mono- to oligosac-charides rather than the absolute amount of carbohydrates controls seed longevity or stability to desiccation tolerance. (b) Synthesis of a few assorted proteins, which is responsive to ABA in the later part of seed maturation, is not correlated with desiccation tolerance or longevity.