A genome-wide survey of reproductive barriers in an intraspecific hybrid.

Genetic study of the reproductive barriers between related species plays an essential role in understanding the process of speciation. We developed a new method for mapping all possible factors causing deviations from expected Mendelian segregation ratios in F(2) progeny, which substantially contribute to reproductive isolation. A multiresponse nonlinear regression analysis of the allele frequencies of the markers covering an entire genome in the F(2) population was performed to estimate the map position and intensity of the reproductive barriers on each chromosome. In F(2) plants from a cross between a Japonica variety of rice, Nipponbare, and an Indica variety, Kasalath, the deviations of allele frequencies were well explained by 33 reproductive barriers. Of these, 15 reproductive barriers affected the allele transmission rate through the gametophyte and in 9 of these 15 cases, an Indica allele was transmitted at a higher frequency than a Japonica allele. The other 18 reproductive barriers altered the viability of the zygote via its genotype. Two zygotic reproductive barriers showed overdominance and 5 showed underdominance. The most pronounced reproductive barrier, mapped at 62.3 +/- 0.4 cM on chromosome 3, transmitted the Indica allele by 94% through the male gametophyte. The accuracy of the barrier position in the regression analysis was confirmed by progeny analysis. The regression analysis proved to be a powerful tool for detecting and characterizing every reproductive barrier, irrespective of whether it acted on the male or female gametophyte or the zygote.

A high-density rice genetic linkage map with 2275 markers using a single F2 population.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.

Detection of segregation distortions in an indica-japonica rice cross using a high-resolution molecular map.

We have constructed a high-resolution rice genetic map containing 1383 DNA markers covering 1575 cM on the 12 linkage groups of rice using 186 F2 progeny from a cross between a japonica variety, 'Nipponbare', and an indica variety, 'Kasalath'. Using this high-resolution molecular linkage map, we detected segregation distortion in a single wide cross of rice. The frequencies of genotypes for 1181 markers with more than 176 genotype data were plotted along this map to detect segregation distortion. Several types of distorted segregation were observed on 6 of the chromosomes. We could detect 11 major segregation distortions at ten positions on chromosomes 1, 3, 6, 8, 9, and 10. The strongest segregation distortion was at 107.2 cM on chromosome 3 and may be the gametophyte gene 2 (ga-2). The 'Kasalath' genotype at this position was transmitted to the progeny with about a 95% probability through the pollen gamete. At least 8 out of the 11 segregation distortions detected here are new. The use of the high-resolution molecular linkage map for improving our understanding of the genetic nature and cause of these segregation distortions is discussed.

A 300 kilobase interval genetic map of rice including 883 expressed sequences.

We have constructed a high resolution rice genetic map containing 1,383 DNA markers at an average interval of 300 kilobases (kb). The markers, distributed along 1,575 cM on 12 linkage groups, comprise 883 cDNAs, 265 genomic DNAs, 147 randomly amplified polymorphic DNAs (RAPD) and 88 other DNAs. cDNAs were derived from rice root and callus, analysed by single-run sequencing and searched for similarities with known proteins. Nearly 260 rice genes are newly identified and mapped, and genomic DNA and cloned RAPD fragments were also sequenced to generate STSs. Our map is the first significant gene expression map in plants. It is also the densest genetic map available in plants and the first to be backed up comprehensively by clone sequence data.

Hydrogen exchange of the tryptophan residues in bovine, goat, guinea pig, and human alpha-lactalbumin.

Hydrogen exchange of the individual tryptophan residues of bovine, goat, guinea pig, and human alpha-lactalbumin has been studied by both ultraviolet and NMR spectra. The assignment of the slowly exchanging imino proton resonances to the tryptophan residues (Trp26 and Trp60) was obtained by comparison of the nuclear Overhauser effect difference spectra of bovine, guinea pig, and human alpha-lactalbumin. Taking account of the thermal unfolding of each alpha-lactalbumin, the hydrogen exchange rates of the individual tryptophan residues are analyzed. The temperature dependence of the exchange rates classified their exchange mechanisms into two exchange processes: the "low activation energy process" and the "high activation energy process" which is associated directly with the global thermal unfolding of the protein. Trp26 of alpha-lactalbumin exchanges through the high activation energy process. The exchange behavior of Trp26 of guinea pig alpha-lactalbumin suggests a difference of the globally unfolded state of the protein from the other species. The exchange mechanism of Trp60 of human alpha-lactalbumin is the low activation energy process in contrast with those of the bovine and goat proteins, although their global thermodynamic properties are similar to each other. Trp104 and Trp118 of alpha-lactalbumin exchange through the low activation energy process, and the reaction rates are affected by the local structural differences around the tryptophan residues among these proteins. The results presented in this paper indicate that the hydrogen exchange rate through the low activation energy process provides the information only about the local nature of a protein while that through the high activation energy process provides the information about the global nature of a protein.

Innocuous character of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and EDTA as metal-ion buffers in studying Ca2+ binding by alpha-lactalbumin.

The binding of EGTA and EDTA to alpha-lactalbumin, first demonstrated by Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709) and afterwards regarded as a significant source of error in estimating the binding constant of Ca2+ to the protein, has been investigated by comparison of the thermal unfolding curves of the protein in the absence and presence of the chelators and also by measuring the NMR spectra of the protein, the chelators, and the protein-chelator mixtures. The unfolding curve in the presence of a large excess of each chelator has been found to be identical to that in the absence of chelator, indicating that there is essentially no interaction between the chelators and alpha-lactalbumin. The NMR results have also supported this conclusion, and the innocuous character of these chelators as metal-ion buffers in studying the Ca2+-binding properties of alpha-lactalbumin is demonstrated. In order to re-examine the binding constant for Ca2+ of alpha-lactalbumin without the aid of chelating metal-ion buffers, the thermal unfolding curve of the protein in the presence of 0.1 mM excess Ca2+ but without chelators has been compared with the unfolding curve in the absence of Ca2+ at a constant concentration of Na+ (0.010 or 0.10 M) at pH 7.0. The binding constant of alpha-lactalbumin can be calculated from the increment of melting temperature caused by the presence of Ca2+ and from the enthalpy and heat capacity changes in the unfolding. Because Ca2+ binding to the unfolded protein can be neglected under the conditions employed, the binding constant evaluated corresponds to the binding constant to protein that has native structure. The constant obtained is 3-5 X 10(9) M-1 after corrections for binding of Na+ to the protein and for ionic strength, and this shows excellent agreement with the corresponding value previously estimated (2.9 +/- 1.0 X 10(9) M-1), although the latter value was obtained in the presence of EDTA. The apparent Ca2+-binding constant that has been discussed in most previous studies, without taking account of the folding-unfolding equilibrium associated with the binding process, also depends on the concentration of monovalent cations such as Na+, and the present results lead to values of 1.5 X 10(8) and 8.7 X 10(6) M-1 at 0.01 and 0.1 M Na+, respectively.

Influence of Ca2+ binding on the structure and stability of bovine alpha-lactalbumin studied by circular dichroism and nuclear magnetic resonance spectra.

Both the Ca2+-bound and Ca2+-free forms of alpha-lactalbumin can assume essentially the same folded conformation as evidenced by similarity in their CD and proton n.m.r. spectra. Thermal unfolding followed by the aromatic CD has shown that the stability of the folded state is markedly enhanced by Ca2+ and that the stabilization is almost entirely entropic; addition of 0.1 mM Ca2+ shifts the transition temperature from 40 degrees to 62 degrees in 0.1M Na+ at pH 7.0. The enthalpy change of the unfolding, coincident between the two forms, is, however, significantly smaller than that known for lysozyme. The n.m.r. spectrum under the condition that both the forms of the protein are in the folded state reflects minor environmental changes of certain protons upon Ca2+ binding, and these changes are shown to afford useful probes for assessment of the location of the binding site. From the pH dependence and temperature dependence of the spectrum and also by using spin decoupling in the aromatic region (6.4-8.7 p.p.m.), it is shown that none of histidyl residues are affected and that at least two tryptophanyl ring protons experience environmental changes upon Ca2+ binding to the folded apo-protein. Effect of free excess Ca2+ on the spectrum has also shown that in native alpha-lactalbumin there is only one Ca2+-binding site that is detectable by the present method.