Successive Backcrosses Research Articles

Linkage Analysis of a Lateness Gene ef4 in Rice, Oryza sativa L.

A near isogenic line T65-LH2 bred from IR8 by five times of successive backcrossing with Taichung 65 (T65) as recurrent parent was found to harbor a lateness gene ef4(t). In the present study, we investigated the allelic relationship between ef4(t) and two earliness genes, Efx and Ef1, followed by linkage analysis to reveal the chromosomal location of ef4(t). T65-LH2 was crossed with two testers, T65-ER21 and T65-ER1 harboring the earliness genes, Efx and Ef1, respectively. F1 plants of the two crosses were raised to obtain F2 seeds. Moreover, the F1 plants were backcrossed with T65-LH2 as to obtain the B1F1 seeds. Heading time in F2 and B1F1 plants as well as parental lines were examined. F2 and B1F 1 plants of the crosses between T65-LH2 and T65-ER21 showed monogenic segregation while F2 and B1F 1 plants obtained from the cross between T65-LH2 and T65-ER1 exhibited digenic segregation. Thus, ef4(t) is allelic to Efx on chromosome 3 and independent of Ef1. Subsequently, linkage analysis of ef4(t) was carried out using three marker genes on chromosome 3, Hg, dl and bc1. T65-LH2 was crossed with the isogenic lines of T65 carrying those marker genes. Heading time and morphological characters in F2 plants and parental lines were observed. Results showed that ef4(t) was linked to Hg and dl with recombination values estimated at 33.2% and 16.8%, respectively, but segregated independently of bc1.

A brand-new bird: how two amateur scientists created the first genetically engineered animal

A brand-new bird: how two amateur scientists created the first genetically engineered animal

Location of New Gene for Late Heading in Rice, Oryza sativa L. Using Interchange Homozygotes

A near isogenic line T65-LH1 was developed from an upland rice variety R300 (Thailand) through eight successive backcrossing with Thaichung 65 (T65) as a recurrent parent. Previously unpublished study indicated T65-LH1 may harbor a recessive lateness gene which was likely located on the sixth chromosome. The present study was aimed at further identifying the lateness gene of this newly bred line by linkage study and allelism test. Linkage study was conducted using seven kinds of interchange homozygotes involved with the sixth chromosome. Individual interchange homozygous lines were crossed with T65-LH1, then, each F1 population was backcrossed to T65-LH1. Segregations for heading times in all B1F1 populations showed early and late types fitted to the 1: 1 expected ratio, suggesting that T65-LH1 carried a recessive lateness gene. Chi-square values for independence between the present gene and four breakpoints, 2-6, 6-7b, 6-7c and 6-10 were insignificant. However, those to three breakpoints, 4-6, 6-7 and 6-8 were significant at 1% level and the respective recombination values were estimated as 3.2%, 4.3% and 3.4%. This suggested the gene under study was located on the sixth chromosome. Subsequently, allelism test between the present gene and other three lateness genes, ef2(t), ef3(t) and ef4(t) was carried out. Crosses of T65-LH1 and three testers having each of those lateness genes were made. Segregations for heading times in F2 populations of those crosses fitted to the 9: 3: 3: 1, 12: 3: 1 and 9: 3: 3: 1 expected ratios. It suggested the present gene was independent from the lateness genes, ef2(t), ef3(t) and ef4(t). In conclusion, the lateness gene harbored by T65-LH1 was a new gene and designated as ef5. It was carried by the sixth chromosome.

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations.

The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F(1), BC(1)F(1) and BC(2)F(1)). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.

A new obesity-prone, glucose-intolerant rat strain (F.DIO).

Previous breeding for the diet-induced obese (DIO) trait from outbred Sprague-Dawley rats produced a substrain with selection characteristics suggesting a polygenic mode of inheritance. To assess this issue further, selectively bred DIO male rats were crossed with obesity-resistant inbred Fischer F344 dams. Male offspring were crossed twice more against female F344 dams. The resultant N3 (F.DIO) rats were then inbred three more times. On low-fat chow, 10-wk-old male and female DIO rats weighed 86 and 59% more than respective F344 rats. By the N3 (F.DIO) generation, they were only 12 and 10% heavier, respectively. After three additional inbreeding cycles, chow-fed F.DIO males had an exaggerated insulin response to oral glucose compared with F344 rats. After 3 wk on a 31% fat (high-energy) diet, male N3 F.DIO rats gained 16-20% more carcass and adipose weight with 98% higher plasma leptin levels, whereas F.DIO females gained 36-54% more carcass and adipose weight with 130% higher leptin levels than comparable F344 rats. After three inbreeding cycles, F.DIO males still gained more weight on high-energy diet and developed a threefold greater insulin response to oral glucose than F344 males. Preservation of the DIO and glucose intolerance traits through successive backcrosses and inbreeding cycles to produce the F.DIO strain lends further support to the idea that they inherited in a polygenic fashion.

Production of Intergeneric Hybrids between the C3-C4 Intermediate Species Diplotaxis tenuifolia (L.) DC. and Raphanus sativus L.

In intergeneric crossings between Diplotaxis tenuifolia (2n = 22, DtDt) and five cultivars of Raphanus sativus (2n = 18, RR), an intergeneric F1 hybrid was produced from the crossing of D. tenuifolia × R. sativus cv. ‘4-season leaf’ through ovary culture followed by embryo culture. The induced amphidiploid (2n = 40, DtDtRR) showed well-regulated meiotic features at PMCs and a high pollen fertility (75 %). Three BC1 hybrids with DtRR (2n = 29) or DtDtR (2n = 31) genome constitutions were obtained by the same embryo rescue procedure in the crossings of amphidiploid × R. sativus and D. tenuifolia × amphidiploid, respectively. In the successive backcrossings of two BC1 hybrids (DtRR, 2n = 29) to R. sativus, 102 BC2 hybrids were obtained by conventional pollination. In the reciprocal crossing of R. sativus × BC1 hybrids, 12 reciprocal BC2 hybrids were also produced without embryo rescue. The somatic chromosome number of 89 BC2 hybrids with D. tenuifolia cytoplasm and 12 reciprocal BC2 hybrids with R. sativus cytoplasm ranged from 2n = 18 to 2n = 23 that were estimated to carry 2n = 18 chromosomes of R. sativus and zero to five chromosomes of D. tenuifolia. Among them, 24.7 % of the BC2 hybrids and 41.6 % of the reciprocal BC2 hybrids were assumed to be monosomic addition lines (MALs, 2n = 19). The novel intergeneric hybrids obtained in this study could become useful materials for investigating the genetic effects on C3-C 4 intermediate traits at the genomic and chromosomal levels, as well as for estimating the performance of genetic improvement in Brassicaceae.

Proinsulin 2 knockout NOD mice: a model for genetic variation of insulin gene expression in type 1 diabetes.

Insulin is a major disease determinant in type 1 diabetes, type 2 diabetes, and related disorders. The role of variations in the expression of the insulin gene has been proposed in genetic susceptibility to the three pathological conditions in humans. In contrast to humans, rodents express two proinsulin isoforms. One isoform, proinsulin 1, is expressed exclusively in islets. The second, proinsulin 2, is expressed in islets and in other tissues, especially the thymus. We took advantage of the expression of these two isoforms to introduce a null proinsulin 2 allele in NOD mice and to evaluate the consequence of a variation of proinsulin 2 gene expression on the development of type 1 diabetes on the NOD genetic background. Heterozygote NOD mutant mice carrying a null proinsulin 2 mutation showed an increased incidence of type 1 diabetes at successive backcross generations. Plasma glucose and insulin levels were identical in prediabetic mutant and in wild-type mice at 4 weeks of age. Variation in insulin gene expression is hypothesized to interfere with diabetes development at both the islet and the thymus level.

Introgression of self-compatibility from Coffea heterocalyx to the cultivated species Coffea canephora.

Self-compatibility segregation was assessed in two successive backcross progenies originating from an interspecific cross between Coffea canephora (self-incompatible) and Coffea heterocalyx (self-compatible). After self- and cross-pollination, pollen tube behaviour in styles was observed under ultraviolet fluorescence microscopy and fruit-set was determined at harvesting time. Segregation ratios in the two progenies were consistent with monofactorial control of self-compatibility. Self-compatible plants exhibited higher fruit-set than self-incompatible ones in open-pollination conditions. Segregation of AFLP markers was scored in the first backcross progeny. By molecular linkage analysis, the S locus could be mapped to a short linkage group.

Destiny of a transgene escape from Brassica napus into Brassica rapa.

Transgenic Brassica napus can be easily crossed with wild Brassica rapa. The spread of the transgene to wild species has aroused the general concern about its effect on ecological and agricultural systems. This paper was designated, by means of population genetics, to study the fate of a transgene escape from B. napus to B. rapa. Three models were proposed to survey the change in gene frequency during successive backcross processes by considering selection pressures against aneuploids, against herbicide-susceptible individuals, and by considering A-C intergenomic recombination and the effect of genetic drift. The transmission rate of an A-chromosome gene through an individual to the next generation was 50%, irrespective of the chromosome number; while that of a C-chromosome transgene varied from 8.7% to 39.9%, depending on the chromosome number of the individual used in the backcross. Without spraying herbicide, the frequency of an A-chromosome gene was 50% in the BC(1) generation, and decreased by 50% with the advance of each backcross generation; that of a C-chromosome gene was around 39.9% in BC(1), 7.7% in BC(2), 1.2% in BC(3) and 0.1% in the BC(4) generation. Under the selection pressure against herbicide-susceptible individuals, the frequency of a transgene reached a stable value of about 5.5% within six generations of successive backcrossings. The effect of genetic drift and intergenomic exchange on gene transmission rate was discussed. It is suggested that the transgene integrated on a C-chromosome (or better on a cytoplasm genome) is safer than that on an A-chromosome. The transgenic cultivars should be cultivated rotationally by year(s) with other non-transgenic varieties in order to reduce the transfer of the transgene to wild B. rapa species.

The nuclear genome is involved in heteroplasmy control in a mitochondrial mutant strain of Drosophila subobscura.

Most (78%) mitochondrial genomes in the studied mutant strain of Drosophila subobscura have undergone a large-scale deletion (5 kb) in the coding region. This mutation is stable, and is transmitted intact to the offspring. This animal model of major rearrangements of mitochondrial genomes can be used to analyse the involvement of the nuclear genome in the production and maintenance of these rearrangements. Successive backcrosses between mutant strain females and wild-type males yield a biphasic change in heteroplasmy level: (a) a 5% decrease in mutated genomes per generation (from 78 to 55%), until the nuclear genome is virtually replaced by the wild-type genome (seven to eight crosses); and (b) a continuous decrease of 0.5% per generation when the nuclear context is completely wild-type. In parallel with these changes, NADH dehydrogenase activity, which is halved in the mutant strain (five subunits of this complex are affected by the mutation), gradually increases and stabilizes near the wild-type activity. A return to a nuclear context is accompanied by the opposite phenomena: progressive increase in heteroplasmy level and stabilization at the value seen in the wild-type strain and a decrease in the activity of complex I. These results indicate that the nuclear genome plays an important role in the control of heteroplasmy level and probably in the production of rearranged genomes.

Genetic and molecular characterization of mating type genes in Cochliobolus sativus

Genetic and molecular approaches were used to characterize the mating type (MAT) genes in Cochliobolus sativus. One hundred and four ascospore progeny derived from a cross of C. sativus isolates ND93–1 (MAT-1) X ND90Pr (MAT-2) were backcrossed with their parents to determine mating type, but only five progeny produced pseudothecia with asci and/or ascospores. When degenerate primers from the conserved high mobility group (HMG) protein domain encoded by the MAT-2 gene in Cochliobolus species were used in polymerase chain reaction (PCR) with genomic DNA of C. sativus as templates, an amplicon of predicted size was amplified only from MAT-2 isolates. The presence of a MAT-2 homolog in these MAT-2 isolates was confirmed by Southern hybridization with the HMG box as a probe. Additionally, the presence or absence of the HMG homolog in the progeny segregated in a 1:1 ratio, as expected for the single gene control of mating type. Using primers based on the conserved regions at the 5’ and 3’ flanks of the idiomorphs in the MAT genes of other Cochliobolus species, the full-length MAT-1 and MAT-2 idiomorphs were cloned by PCR from C. sativus isolates ND93–1 and ND90Pr, respectively. DNA sequence analysis indicated that these two idiomorphs are organized in a manner similar to their respective counterparts in other Cochliobolus species. DNA hybridization and PCR amplification analysis of 54 field isolates of C. sativus collected worldwide showed that both mating types exist in populations around the world. The low frequency of successful backcrosses of progeny to parents in the ND93–1 X ND90Pr cross, combined with the fact that many crosses between isolates of opposite mating type are unsuccessful, suggests that genetic factors other than MAT genes affect the fertility of the fungus.

Genetic and Molecular Characterization of Mating Type Genes in Cochliobolus sativus

Genetic and molecular approaches were used to characterize the mating type (MAT) genes in Cochliobolus sativus. One hundred and four asco- spore progeny derived from a cross of C. sativus iso- lates ND93-1 (MAT-1) X ND9OPr (MAT-2) were backcrossed with their parents to determine mating type, but only five progeny produced pseudothecia with asci and/or ascospores. When degenerate prim- ers from the conserved high mobility group (HMG) protein domain encoded by the MAT-2 gene in Coch- liobolus species were used in polymerase chain reac- tion (PCR) with genomic DNA of C. sativus as tem- plates, an amplicon of predicted size was amplified only from MAT-2 isolates. The presence of a MAT-2 homolog in these MAT-2 isolates was confirmed by Southern hybridization with the HMG box as a probe. Additionally, the presence or absence of the HMG homolog in the progeny segregated in a 1:1 ratio, as expected for the single gene control of mat- ing type. Using primers based on the conserved re- gions at the 5' and 3' flanks of the idiomorphs in the MAT genes of other Cochliobolus species, the full- length MAT-1 and MAT-2 idiomorphs were cloned by PCR from C. sativus isolates ND93-1 and ND9OPr, respectively. DNA sequence analysis indicated that these two idiomorphs are organized in a manner sim- ilar to their respective counterparts in other Cochliob- olus species. DNA hybridization and PCR amplifica- tion analysis of 54 field isolates of C. sativus collected worldwide showed that both mating types exist in populations around the world. The low frequency of successful backcrosses of progeny to parents in the ND93-1 X ND9OPr cross, combined with the fact that many crosses between isolates of opposite mating type are unsuccessful, suggests that genetic factors other than MAT genes affect the fertility of the fun- gus.

Size of donor chromosome segments around introgressed loci and reduction of linkage drag in marker-assisted backcross programs.

This article investigates the efficiency of marker-assisted selection in reducing the length of the donor chromosome segment retained around a locus held heterozygous by backcrossing. First, the efficiency of marker-assisted selection is evaluated from the length of the donor segment in backcrossed individuals that are (double) recombinants for two markers flanking the introgressed gene on each side. Analytical expressions for the probability density function, the mean, and the variance of this length are given for any number of backcross generations, as well as numerical applications. For a given marker distance, the number of backcross generations performed has little impact on the reduction of donor segment length, except for distant markers. In practical situations, the most important parameter is the distance between the introgressed gene and the flanking markers, which should be chosen to be as closely linked as possible to the introgressed gene. Second, the minimal population sizes required to obtain double recombinants for such closely linked markers are computed and optimized in the context of a multigeneration backcross program. The results indicate that it is generally more profitable to allow for three or more successive backcross generations rather than to favor recombinations in early generations.

Early‐bolting trait and RAPD markers in the specific monosomic addition line of radish carrying the e‐chromosome of Brassica oleracea

AbstractThe specific monosomic addition line of radish, Raphanus sativus, carrying the e chromosome of Brassica oleracea (2n = 19, e‐type MAL) with the genetic background of the late‐bolting cv.‘Tokinashi’ was produced by successive backcrossing of the original e‐type MAL of radish that showed early bolting in the genetic background of the cv. ‘Shogoin’. The early‐bolting trait specific to the e‐type MAL was constantly expressed in the backcrossed progenies (BC2, BC3 and BC4), whereas the reverted radish‐like plants (2n =18) were gradually converted to bolting as late as ‘Tokinashi’. The added e‐chromosome expressed an epistatic effect against the genome of Japanese radish. Its early‐bolting trait was dominant to the late‐bolting trait of ‘Tokinashi’ which may be under the control of a few genes. Moreover, e‐type specific RAPD markers detected in eight primers were invariably transmitted in the backcrossed progenies by ‘Tokinashi’. From the analysis of the characteristics to the e‐type MAL and e‐type specific RAPD markers, it is suggested that the e‐added chromosome of kale (B. oleracea) was transmitted from generation to generation without any recombination with the radish chromosome. The gene(s) for the early‐bolting trait detected in this study may be useful for breeding work in radish, especially in the tropical areas.

Modification of survival rate of mouse embryos developing in heterozygous females for ovum mutant gene.

The DDK syndrome (polar infertility) is caused by an incompatibility system due to the ovum mutant (Om) locus. For brevity, the following gene symbols are used in the present report: DDK allele, Om; C57BL/6Cr allele, +. In this investigation, we first attempted to introduce the Om allele of DDK strain into the genetic background of C57BL/6Cr strain. The attempt resulted in the production of no young at the third generation of successive backcrosses. Secondly, mating experiments were performed with heterozygous (Om/+) females having background genes of C57BL/6Cr and DDK strains in the ratios 1:1(B1D), 3:1(B3D), 7:1(B7D), and 15:1(B15D). The survival rate of the embryos as judged by the percentage number of live fetuses/number of corpora lutea at Day 12 of pregnancy was 41.3 +/- 3.2%, 27.3 +/- 3. 2%, 16.4 +/- 3.3%, and 11.3 +/- 3.2% (mean +/- SEM) in the B1D, B3D, B7D, and B15D females, respectively, when they were mated with C57BL/6Cr males. Furthermore, the increased embryonic mortality in the heterozygous (Om/+) females with more background genes of C57BL/6Cr strain was found to be due to a failure in blastocyst formation, as in the DDK syndrome. The parallelism between the proportion of C57BL/6Cr background genes and embryonic mortality has led to a hypothesis proposing the participation of a modifier gene, namely that a mechanism similar to allelic exclusion may be working in the synthesis of cytoplasmic factor of eggs and that only the Om allele is activated during oogenesis to produce DDK-type cytoplasmic factor in heterozygous (Om/+) females having a modifier gene in the homozygous state.

Introgression of crown rust resistance from Festuca spp. into Lolium multiflorum

AbstractCrown rust, Puccinia coronata Corda, causes one of the most damaging foliar diseases in Italian ryegrass, Lolium multiflorum Lam. For introgression of crown rust resistance, highly resistant hybrids of the crosses Festuca arundinacea (2n = 6x = 42) ×L. multiflorum (2n = 4x = 28) and reciprocally, L. multiflorum (2n = 2x = 14) ×Festuca pratensis (2n = 4x = 28) and subsequently resistant recombinant individuals were used as female parents and susceptible cultivars of Italian ryegrass as male parents in three successive backcrosses. The BC3 plants were selfed and crossed mutually. Uredospores of seven different crown rust isolates collected from plants of L. multiflorum, Lolium perenne, F. pratensis, F. arundinacea and L. multiflorum × F. pratensis hybrids were applied to identify the resistance or susceptibility of the parental species, backcrossed, selfed and intercrossed progenies. The various crown rust populations revealed a species‐specific capability to infect plants of the Lolium‐Festuca complex corresponding to the host species from which the spores originated. Selected BC9 plants, however, were found to be completely resistant to all crown rust populations tested. Successful introgression of the resistance was achieved from F. arundinacea as well as from F. pratensis. The resistance represents a dominant character, apparently based on a strong heterologous incompatibility between host and pathogen. In phenotype, bivalent formation during meiosis and in fertility, the novel germplasms are comparable with the L. multiflorum cultivars.

Successful first and second backcrosses of S. nigrum (+) S. tuberosum somatic hybrids with both Solanum parents

Somatic hybrids of Solanum nigrum (+) 2× potato were successfully crossed with S. nigrum and with potato. First and second backcross progeny with S. nigrum could easily be obtained. One of the BC1 genotypes was already self-fertile. Backcrosses with potato had a much lower success rate. Only pollinations with tetraploid potato resulted in seed-containing berries. Two BC1 genotypes were obtained after 4362 pollinations from which 505 ovules were cultured. The first BC1 genotype grew vigorously in vitro and in the greenhouse and flowered abundantly. The second BC1 showed many abnormalities and dropped its flowers before anthesis. The first BC1 was again crossed with tetraploid potato and in this generation also the success rate was low. Over 5000 pollinations resulted in 1750 berries from which over 3000 ovules were obtained. Twelve plants germinated from these ovules and they were not as vigorous in vitro and in vivo as the BC1 parent. Some of the BC2 genotypes were used for further backcrosses but no BC3 plants were obtained. BC1 and BC2 genotypes that resulted from the backcross program with potato were tested for resistance to Phytophthora infestans. The BC1 genotype was as resistant as the S. nigrum fusion parent, but among the eight BC2 genotypes scored six were resistant, whereas two genotypes showing lesions were susceptible.

511 Toward Mapping and Cloning Late Blight Resistance Derived from the Wild Solanum bulbocastanum using Potato + S. bulbocastanum Somatic Hybrids

Late blight of potato, caused by the fungal pathogen Phytophthora infestans, is of great economic significance and no important U.S. potato cultivars are reliably resistant. The diploid species Solanum bulbocastanum is highly resistant to late blight, even under extreme conditions, but is sexually incompatible with potato. We have generated potato + S. bulbocastanum somatic hybrids. These hexaploid hybrids are highly resistant to late blight and progeny from two successive backcrosses to cultivated potato are either fully resistant or susceptible. The advanced generations are morphologically similar to potato. We have initiated mapping efforts to identify bulbocastanum chromosomal regions responsible for late blight resistance. Using RFLPs, RAPDs, and AFLPs, we identified a single chromosomal region (i.e., single gene or group of linked genes) on chromosome 8 that accounts for 62.2% of the observed resistance. The tomato cDNA clone CT88 cosegregates with late blight resistance in our material and was used to isolate a homeologous BAC clone from a S. bulbocastanum library. Our current efforts include mapping resistance in both fusion-derived and diploid S. bulbocastanum materials via map merging. As markers linked to late blight resistance are identified, we will attempt to convert them to marker forms useful for large-scale breeding efforts. To date, we have successfully converted RAPD and RFLP markers to SCAR and CAPS marker forms. Finally, continued fine mapping and BAC clone characterization will enable future map-based cloning efforts.

Altered tissue-specific expression at the Wx gene of the opaque mutants in rice

Amylose content is a major determinant of the eating quality in rice. To elucidate the allelic diversity at the Waxy (Wx) gene which controls the amylose synthesis, two cultivated strains having opaque endosperms were studied. The gene responsible for opaque endosperms was introduced into the genetic background of the Japonica type of rice by successive backcrosses, and the two near-isogenic lines (NILs) were selected from the B5 generation. The genetic experiments revealed that an allele, Wxop, controls opaque endosperms which show chalky as wx endosperms in spite of the production of amylose. Immunoblotting analysis was carried out to compare the gene expression by using the NILs with 4 different alleles (Wxa, Wxb, Wxop and wx). The level of the gene product bound to starch granules was slightly lower in the NILs with Wxop than that with Wxb, showing a positive correlation with amylose content in the endosperm. Extracts from mature anthers indicated that the gene product was markedly reduced in the NILs with Wxop as well as that with wx, showing an altered expression in the tissue specificity in the Wxop lines. Sequence analysis suggested that the Wxop had been derived from Wxa, independently of the origin of Wxb. The importance of the gene regulation was discussed in relation to diversified phenotypes established during the domestication process.

Production of near-isogenic lines of common wheat for glaucousness and genetic basis of this trait clarified by their use.

Duplication of the genes for both wax production (W loci) and inhibition (proposed symbol, Iw loci) on chromosomes 2B and 2D hindered clarification of the allelic and linkage relationships between these loci in wheat. The near-isogenic lines of three genotypes, w1w1 w2w2, W1W1 w2w2 and w1w1 W2W2, for wax production (all iw1iw1 iw2 iw2 for wax inhibition) and two genotypes, Iw1Iw1 iw2 iw2 and iw1 iw1 Iw2Iw2, for wax inhibition (both W1W1 W2W2 for wax production) therefore were produced by ten backcrosses with Triticum aestivum cv. S615 (genotype W1W1 W2W2 iw1iw1 iw2iw2), or its equivalent w-S615 (w1w1 w2w2 iw1iw1 iw2iw2), as the recurrent parent. Segregation ratios between glaucous and non-glaucous plants in successive backcross generations or after selfing the backcrossed offspring indicated that all the wax-producing (W) and inhibiting (Iw) alleles were transmitted at equal frequencies to their respective recessive alleles, w and iw. Linkage analyses of W1, Iw1 and Ne2 (a gene for hybrid necrosis) on chromosome 2B and W2, Iw2 and C (a gene for compact ear) on chromosome 2D showed that the W1 and W2 loci respectively are located at sites different from the Iw1 and Iw2 loci. The presently accepted concept of multiple allelism for w, W and WI (wax inhibition) therefore is invalid. Rather two allelic sets, w and W, and iw and Iw, both of which are duplicated on chromosomes 2B and 2D, must be postulated for the glaucousness of wheat. The first genetic maps showing their linkage relationships to the centromere and the Ne2 and C loci have been constructed.