SummaryReporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green¯uorescent protein (GFP) de novo, optimizing its codon usage to re¯ect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5¢-and 3¢-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a nonoptimized GFP cassette (GFPncb), also under the control of the rbcL 5¢-and 3¢-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate »80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5¢-and 3¢-UTRs, is suf®ciently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.

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Gametophytic self-incompatibility (SI), an important character for breeding seedless cultivars of Citrus, is known in pummelo, mandarin, and many hybrid cultivars with pummelo in their pedigrees. Only a little is known about the allelic variation in the self-incompatibility gene (S), S allele frequencies, and the genotypes of self-incompatible and semi-self-compatible cultivars. In this study, pollination of 'Banpeiyu' S 1 seedlings with 'Banpeiyu' and pollination between the S 1 seedlings were performed to determine homozygous S 1 seedlings for the S gene. Seventyeight Citrus accessions, including 55 pummelo accessions, were pollinated with each of two homozygous S 1 seedlings (S 1 S 1 and S 2 S 2 ). Pollen tube arrest in the style base of their pollinated pistils indicated that 23 accessions, including 'Banpeiyu', have an S 1 allele each and 16 accessions, including 'Banpeiyu', have an S 2 allele each. Frequency of accessions with S 1 allele was 29.9% (23 of 77 accessions examined) and S 1 allele frequency was 16.4% (23 of 140 alleles excluding S f allele). Frequency of accessions with S 2 allele was 21.3% (16 of 75 accessions examined) and S 2 allele frequency was 11.6% (16 of 138 alleles excluding S f allele). Pummelo accessions collected from Kagoshima Prefecture had S 1 alleles with two and half times higher frequency (56.3%) than that in all accessions examined. Of the 79 accessions, six accessions ('Banpeiyu', 'Iriki Buntan', 'Kaopang', Nagashima Buntan No. 6, Nagashima Buntan No. 7, and 'Soyu') were S 1 S 2 genotypes. The Citrus cultivars, whose S genotypes have been fully determined in this study, were

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Self-incompatibility (SI) in Citrus is known in cultivars and plants of pummelo, pummelo relative, and mandarin; however, the detailed mechanism of self-incompatibility and the allelic diversity of the SI gene (S) are not known because of the presence of barriers such as nucellar embryony, male sterility, and a long juvenile phase. Thus, there is little information on S genotypes of Citrus cultivars at present. In this study, the S genotypes of Citrus cultivars were estimated with the aid of allozymes produced by a glutamate oxaloacetate transaminase isozyme gene (Got-3), which appeared to link to the S gene. Of twenty-two F 1 progenies from eleven crosses and their reciprocal crosses with eight monoembryonic SI cultivars, including 'Banpeiyu' pummelo, three F 1 progenies from three crosses and three from reciprocal crosses showed segregation distortion for the Got-3 gene, suggesting the presence of the same S alleles in the cultivars used for the reciprocal crosses. Defining the 'Banpeiyu' genotype as S 1 S 2 , the genotype was estimated as S 1 S 3 for 'Tosa Buntan', S 4 S 5 for 'Hassaku', S 6 S 7 for 'Yuge-hyokan', and S 1 S 6 for 'Shishiyuzu'. Further allozyme analyses for segregation distortion in progenies from eight crosses with eight monoembryonic SI cultivars suggested the possibility that 'Hyuganatsu' has S 1 allele, while those from 15 crosses between the eight monoembryonic SI cultivars and five polyembryonic self-compatible cultivars suggested the possibility that 'Rough lemon' has S 1 allele. The estimated genotypes, consisting of two of the nine alleles (S 1 to S 8 and S f ) in these Citrus cultivars, may be useful for further estimation and determination of S genotypes in Citrus cultivars and plants.