Maki Saito

The construction of a high-density linkage map for identifying SNP markers that are tightly linked to a nuclear-recessive major gene for male sterility in Cryptomeria japonica D. Don

BackgroundHigh-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection (MAS). MAS is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. Our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene (ms1) for MAS in C. japonica, a species that is important in Japanese afforestation but which causes serious social pollinosis problems.ResultsWe constructed a high-density saturated genetic linkage map for C. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism (SNP) markers, most of which were genotyped using the GoldenGate genotyping assay. A total of 1261 markers were assigned to 11 linkage groups with an observed map length of 1405.2 cM and a mean distance between two adjacent markers of 1.1 cM; the number of linkage groups matched the basic chromosome number in C. japonica. Using this map, we located ms1 on the 9th linkage group and constructed a partial linkage map around the ms1 locus. This enabled us to identify a marker (hrmSNP970_sf) that is closely linked to the ms1 gene, being separated from it by only 0.5 cM.ConclusionsUsing the high-density map, we located the ms1 gene on the 9th linkage group and constructed a partial linkage map around the ms1 locus. The map distance between the ms1 gene and the tightly linked marker was only 0.5 cM. The identification of markers that are tightly linked to the ms1 gene will facilitate the early selection of male-sterile trees, which should expedite C. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity.

Inheritance of the Trait of Male Sterility in Cryptomeria japonica

Matings of male-sterileC. japonica and fertile eliteC. japonica, as well as backcross seedlings of male-sterile trees, were carried out to clarify the genetic trait of male sterility of theC. japonica. The seeds from male-sterileC. japonica were germinated in an incubator and grown them in the greenhouse between 1994 and 1997. The seedlings were treated with 100 ppm gibberellin at early July 1994 and early July 1995 to promote the formation of male flowers. In the middle of January 1995, the male flowers of all seedlings were examined under the microscope to confirm the production of pollen. In January 1996, pollen did not developed in to the male flowers from the seedlings between the fertiled elite and backcrossC. japonica. In January 1997, all seedlings of eliteC. japonica produced pollen in their male flowers; however, pollen did not developed in 55 out of 120 backcrossed seedlings. This evidence suggests that the heredity pattern of male sterility inC. japonica is nuclear male sterility controlled by a pair of recessive genes.

Cytological and Genetical Studies on Male Sterility in Cryptomeria japonica D. Don

Genetic male sterility is a useful trait in plant breeding, especially in angiosperm crops such as corn, onion and carrot. We found a male sterile sugi (Cryptomeria japonica D. Don) tree in Toyama, Japan. Pollen of sugi is one of the major causes of pollinosis in Japan. We carried out this research in an attempt to make clear the characteristics and inheritance of this male sterility. Microsporogenesis of the male sterile tree proceeded meiosis, however, the microspores collapsed after they were separated from pollen tetrads in locules, resulting in complete male sterility. Most likely, ethylene evolution was responsible for male sterility expression. Full seed setting in the male sterile tree indicated normal macrosporogenesis. Seeds obtained from crossing between male sterile and normal lines showed relatively high level of germination and their seedlings grew vigorously. The somatic chromosome numbers of 241 germinated seeds, derived from the male sterile tree, were mostly 22, euploid. These results indicated that male sterile tree was different from other similar previously reported trees with low pollen fertility, resulting from triploid or trisomics. Probably, male sterility in sugi is either nuclear genetic male sterility or cytoplasmic male sterility.

Plus tree of Cryptomeria japonica D. Don with a heterozygous male-sterility gene

To find plus tree clones of Cryptomeria japonica that are heterozygous for a male-sterility gene (Aa), we crossed a homozygous male-sterile tree (aa) with 63 clones. Male sterility in this case is controlled by a recessive allele at a single gene locus and is expressed only in homozygotes. All F1 seedlings obtained by crossing the male-sterile mother tree and 62 out of the 63 clones produced pollen. In contrast, F1 seedlings obtained from the crossing between the male-sterile mother tree and a plus tree clone, Ohara 13, produced 64 male-sterile individuals and 52 fertile individuals. The segregation ratio fitted the expected 1 : 1 ratio according to a chi-square test. These results clearly demonstrate that the Ohara 13 clone is heterozygous for a male-sterility gene.

Effects of Stand Density on the Productivity of Cryptomeria japonica Male Flowers

We have studied male flower production and relative illumination in clonal sugi (Cryptomeria japonica) forests planted at densities of 1,500/ha, 3,000/ha, and 5,000/ha, to clarify the effects of forest management on male flower production. Both the relative illumination and male flower production decrease as the stand density increases. The lowest position at which male flowers are formed decreases as the density of the stand decreases. High thinning intensity promotes male flower production and light pruning is ineffective to reduce male flower production. Therefore, traditional management methods used in sugi forests (i.e. planting at high density, frequent light thinning, intensive pruning and short rotation) are ideal strategies for limiting male flower production.

Selections of Male Sterile Sugi (Cryptomeria japonica D. Don) Trees from Open Pollinated Seedlings in a Seed Orchard.

スギ花粉症対策の育種母材料として利用するため,3年生の10,902本のタテヤマスギ(実生苗)から雄性不稔スギを探索した。その結果,雄性不稔と思われるスギを二個体選抜することができた。これら二個体のスギの雄花は,外見上正常なスギと変わりがなく,春先の開花期には花軸を伸長させるが花粉を全く飛散させなかった。さらに,電子顕微鏡で調べてみると花粉同士が崩れた形で融合しており,正常な花粉はほとんど認められなかった。これらの個体は染色数に異常がなかったことから,花粉形成に関与する遺伝子の突然変異(雄性不稔遺伝子)によって雄性不稔性は引き起こされていると思われた。また,今回の調査結果では5,451分の1の頻度で雄性不稔個体が出現した。この方法は3年生の実生苗を材料に用いるので少ない栽培面積と短い生育期間で済み,雄性不稔スギを選抜するのに有効な手法であると考えられた。

Study of sugi (Cryptomeria japonica D. Don) with male-sterility gene using CAPS markers

The frequency of open-pollinated male-sterile seedlings from a male-sterile mother tree in Cryptomeria japonica D. Don was determined in 765 open-pollinated seedlings. Pollen was not formed by 29 out of the 765 seedlings. To find pollen donor candidates for 29 male-sterile seedlings, a male-sterile tree was crossed with five individuals in the neighborhood of the mother tree. Analysis of microspores in male flowers of F1 seedlings revealed that one of the five individuals had a heterozygous male-sterility gene. Paternity tests were conducted on the 29 male-sterile seedlings using eight kinds of CAPS markers in C. japonica. The pollen parents of 29 male-sterile seedlings were not only a tree in the neighborhood of the mother tree, but also other trees. Since it was found that the pollen parents of four of the male-sterile seedlings were not in the study plot, it was inferred that C. japonica trees that possess the male-sterility gene grow in other places and may be widely distributed.