Susumu Shiraishi

Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region

Abstract Using a fluorescence-based PCR-SSCP (single-strand conformation polymorphism), we verified imperfectibility in the paternal inheritance of chloroplast DNA (cpDNA) in Chamaecyparisobtusa (Cupressaceae) controlled crosses. An intraspecific sequence polymorphism of the intergenic spacer region between the trnD and trnY genes was utilized as a molecular marker. Of 361 progenies, in which the cpDNA haplotypes of their female and male parents were different, 352 (97.5%) possessed the same haplotypes as their male parents, and nine (2.5%) the same haplotypes as their female parents. The parentage of the nine progenies with female parental types was diagnosed using DNA fingerprinting based on fluorescence-based RAPD profiles. Their parentage showed convincing evidence of the low frequency of maternal inheritance. Moreover, heteroplasmy was observed in the open-pollinated seeds collected in a seed orchard. The confirmation of maternal plastid transmission in the full-sib families and the observation of heteroplasmy in seeds reveal that the paternal inheritance of cpDNA is not an exclusive phenomenon and that the mode of its inheritance is biparental in C. obtusa.

Realized genetic gain observed in second-generation seedling seed orchards of Acacia mangium in South Kalimantan, Indonesia

Realized genetic gains brought by the first-generation tree improvement of Acacia mangium were evaluated by analyzing the 1-year growth performance of growth (height, dbh) and form (stem straightness, multi-stem) traits using data collected from three second-generation orchards: groups B, C, and D. Each orchard was composed of improved families derived from open-pollinated seed of plus trees selected from the first-generation seed orchards and newly added unimproved families collected from the same geographic region. The number of improved families and that of the unimproved in each orchard were 31 and 23 for group B, 28 and 39 for group C, and 24 and 30 for group D. Gains were calculated as percentage increase of improved families over unimproved ones. Improved families performed better than unimproved ones in 11 out of 12 comparison cases: four traits each in the three orchards. Averages of the realized genetic gain across the three seedling seed orchards were 3.1%, 5.2%, 4.3%, and 0.5% for height, dbh, stem straightness, and multi-stem, respectively. Except for multi-stem, the performances of improved families were significantly better than those of unimproved families in all the three orchards for dbh and in two out of three orchards for height and stem straightness.

Isolation and characterization of tetranucleotide microsatellite loci in Pinus massoniana (Pinaceae)

PREMISE OF THE STUDY Tetranucleotide microsatellite markers were developed for the first time in Pinus massoniana Lamb. to facilitate studies of population and conservation biology in this species. METHODS AND RESULTS Ten tetranucleotide microsatellite primer pairs were developed using dual suppression PCR. Seven, six, and eight of the primer pairs exhibited cross-species transferability to P. thunbergii, P. densiflora, and P. luchuensis, respectively. The number of alleles ranged from 1 to 31 per locus across four pine species. CONCLUSIONS Considering its advantage over dinucleotide microsatellites in generating fewer artifacts arising from stutter bands, this tetranucleotide microsatellite panel will facilitate future population and conservation biological studies in P. massoniana. Six to eight markers can also be used in studies of three congeneric species.

Screening of heterozygous DNA markers in shiitake (Lentinula edodes) using de-dikaryotization via preparation of protoplasts and isolation of four meiotic monokaryons from one basidium

A suitable screening method for heterozygous DNA markers in shiitake,Lentinula edodes (Berk.) Pegler, is reported. Monokaryons were derived from a dikaryon by de-dikaryotization via protoplast formation. Compatibility of the monokaryons was determined by pairwise culture on agar plates. We selected the primers to amplify polymorphic fragments among the original strain (Hokken600∶H600) and two monokaryons (H600PP-39 and H600PP-67) showing compatibility. A total of 135 fragments were selected as specific random amplified polymorphic DNAs (RAPDs) resulting from 56 primers of the 147 primers tested. Furthermore, we tested whether the polymorphic fragments segregated into 2∶2 among four strains isolated from a basidium. Most of the polymorphic fragments (about 97.8%) showed 2∶2 segregation among the four strains. We concluded that the polymorphic fragments were heterozygous if they were detected in either of the monokaryons (H600PP-39 and H600PP-67) and segregated to 2∶2 among four meiotic strains (H600B-1,-2, -3, and -4). A total of 132 heterozygous DNA markers were therefore selected from a dikaryon of shiitake (Hokken600∶H600).

Hybrid origin of Athrotaxis laxifolia (Taxodiaceae) confirmed by random amplified polymorphic DNA analysis

Random amplified polymorphic DNA (RAPD) and single-strand conformation polymorphism (SSCP) analyses were employed for investigating genetic relationships of three Athrotaxis D.Don species. Twenty-nine RAPD primers produced 103 polymorphic bands. Principal component analysis revealed the genomic differentiation among three Athrotaxis species. Mean genetic distance (mean d) between A. selaginoides D.Don and A. cupressoides D.Don was 0.89. Mean d values were reduced to 0.42/0.54 between A. laxifolia Hook. and A. selaginoides/A. cupressoides, respectively. Intraspecific mean d of A. selaginoides and A. cupressoides were, respectively, 0.03 and 0.11. These values indicated that A. laxifolia, which is regarded as a hybrid between A. selaginoides and A. cupressoides, is genetically intermediate between A. selaginoides and A. cupressoides. This genetic characteristic and previously reported morphological characteristics suggest the hybrid origins of A. laxifolia. The genomic composition of A. laxifolia was estimated by the number of bands specific to A. selaginoides or A. cupressoides in order to determine the genomic contribution of these two species to its proposed hybrid, A. laxifolia. All of the five individuals investigated herein possessed genomes derived almost evenly from A. selaginoides and A. cupressoides. Furthermore, the pollen donor of A. laxifolia was determined by SSCP analysis of the atpA gene on chloroplast DNA. Because all of the five A. laxifolia possessed the A. selaginoides-type chloroplast genome, A. laxifolia would be a hybrid of A. selaginoides as a paternal parent and A. cupressoides as a maternal parent.

Polymorphism analysis of fagaceae and DNA-based identification of fagus species grown in Japan based on the rbcl gene

Fagaceae species in Japan were identified by restriction fragment length polymorphism (RFLP) and sequence comparison of a region ofrbcL. Of nine restriction endonucleases used for digestion, three (MspI,RsaI,HaeIII) produced different restriction patterns in Fagaceae. Digestion byMspI yielded four patterns: Fagus species,Castanea crenata, Pasania glabra, and others. Digestion byRsaI andHaeIII afforded two patterns:Fagus species and others. These facts indicate thatCastanea crenata andPasania glabra can be identified byMspI restriction patterns ofrbcL. Sequence comparison of a region of therbcL gene among 20 species of Fagaceae showed that: (1) they could be divided into seven groups; (2) there is a site mutation betweenFagus crenata andF. japonica. The latter indicates that the wood of both Fagus species are identifiable at the species level, which is not the case using conventional methods. This result indicates the possibility of wood identification based on DNA polymorphism in Fagaceae at the intrageneric level.

RAPD Variation among Quercus Species Distributed in Temperate Deciduous Forests of the Hiruzen Mountains

Species of the genus Quercus are widespread in the northern hemisphere. In eastern Asia, Quercus serrata Thunb., Q. mongolica Fisch. ex Ledeb. var. crispula (Blume), Q. dentata Thunb. and Q. aliena Blume, which are classified into the section Prinus, are distributed in temperate deciduous forests of north-eastern China, the Korean Peninsula, and the Japanese Islands. Several presumptive hybrids of these Quercus species have been recorded in the same locations. These species of the section Prinus and the presumptive hybrids are very important for economic utilization and investigations of ecology, forest genetics, and species evolution. In the vicinity of the Hiruzen mountains, rising over Tottori and Okayama Prefectures, Japan, Q. serrata, Q. mongolica var. crispula, and Q. dentata are distributed together in temperate deciduous forests at altitudes from 550 m to 650 m. Quercus takatorensis Makino and Q. anguste-lepidota Nakai, which are presumed to be the intermediate types of the three Quercus species, are commonly observed in the same forests. Hashizume et al. (1994) reported that the morphological measurements and structures of leaves, cupules, and acorns of the presumptive intermediate types indicated intermediate characteristics of the three Quercus species. In fact, the flowering season of the three Quercus species overlaps each year in May, suggesting the possibility of cross pollination among them in the Hiruzen Forest of Tottori University Forests (Lee, unpublished data). However, there is no direct evidence to prove natural interspecific hybridization occurring among the species of the section Prinus. In general, genetic markers have many potential applications in forest genetics and tree breeding research, including genetic diversity, population structure, phylogeny, mating systems, and tree classification. The lack of suitable markers has hindered the direct analysis of genetic structures in plant species. Isozymes have been useful to estimate genetic diversity of plant populations (Hokanson et al., 1993; Hyun et al., 1987). The isozyme analysis has an advantage in its simplicity and rapidity, however, orffy a limited number of polymorphic loci are available for use as genetic markers. Recently, a method for comparing random amplified poly1Present address: 5-72-4, Tachikawa-cho, Tottori 680,

Isolation and characterization of tetranucleotide microsatellite markers for Pinus merkusii

Pinus merkusii is an important tree native to Southeast Asia. Thirteen tetranucleotide microsatellite makers were developed from this species using a dual-suppression polymerase chain reaction technique. Of these markers, nine loci were polymorphic. The mean number of alleles per locus was 3.18 (range 2–5) and mean expected heterozygosities were 0.525 (range 0.384–0.712). These microsatellite markers will be available to assess the genetic diversity, mating patterns and population structure which will facilitate conservation and management strategies.

Diagnosis of interspecific hybrids between Acacia mangium and A. auriculiformis using single nucleotide polymorphism (SNP) markers

Abstract This paper describes a diagnostic system to verify interspecific hybrids between Acacia mangium and A. auriculiformis using single nucleotide polymorphism (SNP) markers. Forty-eight DNA fragments were selected based on random amplified polymorphic DNAs (RAPD) amplified across 48 individuals from each parental species, and were transformed into 44 sequence-characterized amplified region (SCAR) markers. Five SNP markers that generated species-specific alleles for each species were selected from the 28 sequenced SCARs. A multiplex single nucleotide primer extension (SNuPE) analyses of the five SNPs using 40 A. mangium, 40 A. auriculiformis and 16 Acacia hybrids showed high discrimination power. This diagnostic system, with high discriminatory ability, provides a highly reliable and fast method for identifying interspecific hybrids of A. mangium and A. auriculiformis.

Improved AFLP protocol using dual-suppression PCR and its application to species with large genomes.

To improve the amplified fragment length polymorphism assay, dual‐suppression PCR was introduced into the preamplification step of the assay. The dual‐suppression PCR blocked completely the amplification of fragments with the same sequence (Bsp1407I‐Bsp1407I or NlaIII‐NlaIII) at both ends and amplified selectively fragments with different adaptor sequences (Bsp1407I‐NlaIII) at each end. Two protocols, referred to as A and B, were established for species with medium‐ and large‐sized genomes, respectively. Both protocols incorporated the dual‐suppression PCR. Protocol A resulted in high‐quality electrophoretic profiles for black cottonwood and rice, which have medium‐sized genomes. In protocol B, an intensely selective PCR step was added to protocol A. Protocol B yielded profiles for Japanese black pine and Japanese cedar that were improved significantly relative to protocol A: the number of strong peaks increased and that of low peaks decreased. Japanese black pine and Japanese cedar have large genomes. The optimal profiles were generated with a total of eight or nine selective nucleotides.