Keiichi Okazaki

Sequences of S-glycoproteins, products of the Brassica campestris self-incompatibility locus

The cruciferous genus Brassica has a self-incompatibility system that is under the sporophytic control of a single locus, S (ref. 1). Although evidence has accumulated that S-locus-specific proteins, termed S-glycoproteins2–4, are strongly associated with the expression of self-incompatibility4–8, their structures and functions are still unknown. This paper reports the sequences of the S-glycoproteins from three homozygotes of Brassica campestris. They show extensive homology. The published DNA sequence data for the S6-glycoprotein of B. oleracea9 can be revised to encode the total sequence of the glycoprotein on the basis of the sequences of S-glycoproteins of B. campestris. These S-glycoproteins have cysteine-rich clusters and 6–7 oligosaccharide chains. There is no homology between the S-glycoproteins of Nicotiana alata10 and those of B. campestris.

A Molecular Phylogeny of Lilium in the Internal Transcribed Spacer Region of Nuclear Ribosomal DNA

Abstract. Phylogenetic relationships among 55 species of Lilium, Cardiocrinum giganteum, and Nomocharis saluenensis were inferred from nucleotide sequence variations in the internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA. The phylogeny derived from ITS sequences estimated using maximum-likelihood methods indicated that (1) most of the species construct their own clade according to the classification based on morphological features at the section level; (2) section Daurolirion is not independent of Sinomartagon, and it is appropriate to integrate two sections as Sinomartagon; (3) it is appropriate that L. henryi and L. bulbiferum are classified into subsection 6a and Sinomartagon–Daurolirion, respectively; (4) subsection 6b is much closer to Sinomartagon than subsection 6a and Archelirion, and it arose directly from Sinomartagon; and (5) Lilium is much closer to Nomocharis than Cardiocrinum. Phylogenetic estimation using sequences of the ITS region is suitable at the levels of genus, section, and most of subsection.

Characterization of Brassica S‐haplotypes lacking S‐locus glycoprotein1

Self‐incompatibility (SI) in Brassica is regulated by a single multi‐allelic locus, S, which contains highly polymorphic stigma‐expressed genes, SLG and SRK. While SRK is shown to be the determinant of female SI specificity, SLG is thought to assist the function of SRK. Here we report that the SLG genes of self‐incompatible S 18 and S 60 homozygotes of Brassica oleracea have an in‐frame stop codon and a 23 bp deletion resulting in a frame‐shift, respectively. The finding that these SLG genes do not encode functional SLG proteins suggests that SLG is not essential for SI. The possible role of SLG in SI was discussed.

Identification of QTLs that control clubroot resistance in Brassica oleracea and comparative analysis of clubroot resistance genes between B. rapa and B. oleracea.

To perform comparative studies of CR (clubroot resistance) loci in Brassica oleracea and Brassica rapa and to develop marker-assisted selection in B. oleracea, we constructed a B. oleracea map, including specific markers linked to CR genes of B. rapa. We also analyzed CR-QTLs using the mean phenotypes of F3 progenies from the cross of a resistant double-haploid line (Anju) with a susceptible double-haploid line (GC). In the nine linkage groups obtained (O1-O9), the major QTL, pb-Bo(Anju)1, was derived from Anju with a maximum LOD score (13.7) in O2. The QTL (LOD 5.1) located in O5, pb-Bo(GC)1, was derived from the susceptible GC. Other QTLs with smaller effects were found in O2, O3, and O7. Based on common markers, it was possible to compare our finding CR-QTLs with the B. oleraceaCR loci reported by previous authors; pb-Bo(GC)1 may be identical to the CR-QTL reported previously or a different member contained in the same CR gene cluster. In total, the markers linked to seven B. rapaCR genes were mapped on the B. oleracea map. Based on the mapping position and markers of the CR genes, informative comparative studies of CR loci between B. oleracea and B. rapa were performed. Our map discloses specific primer sequences linked to CR genes and includes public SSR markers that will promote pyramiding CR genes in intra- and inter-specific crosses in Brassica crops. Five genes involved in glucosinolates biosynthesis were also mapped, and GSL-BoELONG and GSL-BoPro were found to be linked to the pb-Bo(Anju)1 and Bo(GC)1 loci, respectively. The linkage drag associated with the CR-QTLs is briefly discussed.

Induction of 2n pollen in tulips by arresting the meiotic process with nitrous oxide gas

Triploid tulips have agronomically desirable traits such as vigorous growth and large flower size, but only a portion of all cultivated tulips is triploid. To apply 2n pollen to polyploid breeding of tulips, the polyploidizing agent, nitrous oxide gas (N2O), was applied to bulbs. In tulips, meiosis in anthers occurs inside the bulbs from mid- to late-October. When meiosis in anthers (excised from bulbs) reached metaphase I, we treated other bulbs of the same clones with N2O for 24–48 h. Most of the treated plants produced pollen grains with a wide-ranging or bimodal size distribution, indicating a mixture of n, 2n and aneuploid pollen grains. The use of pollen containing a relatively high proportion of giant pollen grains tended to yield larger numbers of triploids in the progeny. The number of giant pollen grains could be increased when N2O-treated pollen grains were suspended in 10% sucrose and then sieved through a nylon mesh. Very few polyploids were observed in some cross combinations, even those involving pollen with a relatively high proportion of giant grains. Even so, this low polyploid yield most likely is due to a triploid block, because the capsules obtained in the crosses of the diploid×N2O-treated plants contained some abnormal seeds, which were mostly triploid. Embryo culture was useful in rescuing abnormal embryos. The present study reveals that 2n pollen can be produced at high frequency using N2O during tulip breeding.

Comparison of the Genome Structure of the Self-Incompatibility (S) Locus in Interspecific Pairs of S Haplotypes

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, both of which are encoded in the S locus. The nucleotide sequence analyses of many SRK and SP11/SCR alleles have identified several interspecific pairs of S haplotypes having highly similar sequences between B. oleracea and B. rapa. These interspecific pairs of S haplotypes are considered to be derived from common ancestors and to have maintained the same recognition specificity after speciation. In this study, the genome structures of three interspecific pairs of S haplotypes were compared by sequencing SRK, SP11/SCR, and their flanking regions. Regions between SRK and SP11/SCR in B. oleracea were demonstrated to be much longer than those of B. rapa and several retrotransposon-like sequences were identified in the S locus in B. oleracea. Among the seven retrotransposon-like sequences, six sequences were found to belong to the ty3 gypsy group. The gag sequences of the retrotransposon-like sequences were phylogenetically different from each other. In Southern blot analysis using retrotransposon-like sequences as probes, the B. oleracea genome showed more signals than the B. rapa genome did. These findings suggest a role for the S locus and genome evolution in self-incompatible plant species.

The origin of Darwin hybrid tulips analyzed by flow cytometry, karyotype analyses and genomic in situ hybridization

Chromosome morphology was studied in diploid cultivars of Tulipa fosteriana and T. gesneriana (2n = 2x = 24) and triploid Darwin hybrids (2n = 3x = 36) developed from interspecific crosses of T. gesneriana and T. fosteriana. Chromosomes were arranged in the karyotype according to decreasing total length. Based on our karyotypic analysis, we propose that median chromosomes may serve as markers for diploid genotypes. Discriminant analysis with respect to total chromosome length and short arm length showed a significant difference between the size of the larger median chromosomes of T. gesneriana and T. fosteriana Comparison of median chromosome length in Darwin hybrid tulips showed that two larger chromosomes and one smaller chromosome were derived from T. gesneriana and T. fosteriana, respectively. This finding was clearly and unambiguously confirmed by simultaneous hybridization of differentially labeled genomic probes of T. fosteriana and T. gesneriana to metaphase chromosomes of the triploid cultivar ‘Yellow Dover’, thereby enabling us to distinguish between the 24 chromosomes derived from T. gesneriana and 12 chromosomes derived from T. fosteriana. Thus, genomic in situ hybridization and median chromosome analyses can be useful to identify the genome constitution of triploid Darwin hybrid tulips. In addition, their hybridity was readily verified by flow cytometry using vegetative tissue of Darwin hybrid tulips. Our results clarify the process of Tulipa cultivar formation and will be useful for interspecific hybridization breeding.

Interspecific hybrids between Lilium nobilissimum and L. regale produced via ovules-with-placental-tissue culture

Reciprocal pollination was made between Lilium nobilissimum and L. regale. Pollen tubes reached the base of style within 144 h after pollination, but no mature seeds were obtained in either cross combination. Explants, or ovules-with-placental-tissue excised from each carpel 30 and 40 days after pollination (DAP), were cultured on a medium composed of major salts of B-5 macronutrient (Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Exp. Cell Res. 50, 151‐158), micronutrient, Fe-EDTA and vitamins of MS (Murashige, T., Skoog, F., 1962. Physiol. Plant. 15, 473‐497), 5% sucrose and 0.2% gellan gum. In L. regale Lilium nobilissimum, 3% of ovules excised at 30 DAP and 9% of those excised at 40 DAP developed into seedlings. In Lilium nobilissimum L. regale, only 3.6% of ovules excised 40 DAP developed into seedlings, and none of the ovules excised 30 DAP produced any seedlings. Bulbs of L. regale and hybrids transplanted to soil showed some resistance to bulb-rot, leaf-top scorch, browning spots and/or streaking on leaves, but those of Lilium nobilissimum were sensitive to these diseases. Flowering individuals were nearly intermediate between parents in their morphological characteristics. All flowering individuals (2na 24 chromosomes) were identified as hybrids based on karyotype, isozyme and random amplified polymorphic DNA analyses. # 2000 Elsevier Science B.V. All rights reserved.

Molecular cloning of Brassica rapa nitrilases and their expression during clubroot development.

SUMMARY Three isoforms of nitrilase were cloned from turnip, Brassica rapa L., and their expression during clubroot development caused by Plasmodiophora brassicae was investigated. The isoforms were designated BrNIT-T1, BrNIT-T2 and BrNIT-T4 based on homology to known nitrilases. BrNIT-T1 and BrNIT-T2 have 80% homology to three nitrilases from Arabidopsis thaliana (AtNIT1, AtNIT2 and AtNIT3). BrNIT-T4 showed 90% homology to AtNIT4. To confirm their enzyme activity, the recombinant proteins were expressed in Escherichia coli. The recombinant BrNIT-T1 and BrNIT-T2 but not BrNIT-T4 converted indole-3-acetonitrile to indole-3-acetic acid, an endogenous plant auxin, although kinetic analysis showed that indole-3-acetonitrile is a poor substrate compared with various aliphatic and aromatic nitriles. By contrast, the recombinant BrNIT-T4 specifically converted beta-cyano-l-alanine to aspartic acid and asparagine and these findings agree with the idea that it is involved in the cyanide detoxification pathway. Real-time PCR analysis clearly showed that these isoforms were differentially expressed during clubroot development. BrNIT-T1 transcripts were very low in non-infected roots but were enhanced up to 100-fold in infected roots exhibiting club growth. By contrast, BrNIT-T2 transcripts remained at a very low level during clubroot formation. All these results clearly indicate the specific involvement of BrNIT-T1 in clubroot formation. The BrNIT-T4 transcripts were substantially reduced in the clubroot-growing phase, but thereafter they increased rapidly to a level found in non-infected roots as the clubroot growth reached a plateau. These findings suggest the specific involvement of BrNIT-T4 in clubroot maturation. In fully developed clubs, the BrNIT-T1 and BrNIT-T2 transcripts also increased. Free indole-3-acetic acid (IAA) content increased in the early and the latest phase of infected roots compared with non-infected roots, but decreased substantially at the middle phase. Thus, free IAA may play a role in the initiation and maturation of clubroot. Total IAA content was significantly higher in infected roots than in non-infected roots throughout clubroot development and IAA conjugation/conjugate hydrolysis system as well as BrNIT-Ts appear to be involved in clubroot development.

Genetic mapping of a fusarium wilt resistance gene in Brassica oleracea

Fusarium wilt caused by Fusarium oxysporum f. sp. conglutinans is one of the most important diseases of Brassica crops, resulting in severe reductions in yield and quality. To characterize the inheritance pattern of fusarium resistance, a cross between a susceptible broccoli and a resistant cabbage was subjected to segregation analysis. Results indicated that resistance was controlled by a single dominant allele. This gene was named Foc-Bo1 and mapped to linkage group seven (O7) by both the segregation test and quantitative trait locus (QTL) analysis. The QTL on O7 was detected with a logarithm of odds score (LOD) of 19.5, which was above the threshold value with genome-wide 1% significance level (2.01). A minor QTL was also detected on O4 with a LOD score of 2.06. Inoculation tests indicated that stable expression of fusarium resistance at high temperatures required Foc-Bo1 homozygosity. The association between Foc-Bo1 and the closest simple sequence repeat marker (KBrS003O1N10) was analyzed in three F3 populations. Based on these studies, KBrS003O1N10 represents an effective marker-assisted selection (MAS) tool for breeding fusarium wilt resistance into Brassica oleracea crops. To our knowledge, this is the first paper to map the fusarium-resistance gene in Brassica species and to validate the effectiveness of MAS in improving fusarium resistance in these important plants.