Labor-saving Hydroponic Technology for Cultivating Male Sterile Japanese Cedar (<i>Cryptomeria japonica</i> D. Don) Container Seedlings Using Fallow Fields

This study aimed to obtain information from several embryogenic cell (EC) genotypes analyzing the factors that affect somatic embryogenesis (SE) initiation in sugi (Cryptomeria japonica, Cupressaceae) to apply them in the improvement of protocols for efficient induction of embryogenic cell lines (ECLs). The results of several years of experiments including studies on the influence of initial explant, seed collection time, and explant genotype as the main factors affecting SE initiation from male-fertile, male-sterile, and polycross-pollinated-derived seeds are described. Initiation frequencies depending on the plant genotype varied from 1.35 to 57.06%. The best induction efficiency was achieved when seeds were collected on mid-July using the entire megagametophyte as initial explants. The extrusion of ECs started approximately after 2 weeks of culture, and the establishment of ECLs was observed mostly 4 weeks after extrusion on media with or without plant growth regulators (PGRs). Subsequently, induced ECLs were maintained and proliferated on media with PGRs by 2–3-week-interval subculture routines. Although, the initial explant, collection time, and culture condition played important roles in ECL induction, the genotype of the plant material of sugi was the most influential factor in SE initiation.

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Fine mapping of the male-sterile genes (MS1, MS2, MS3, and MS4) and development of SNP markers for marker-assisted selection in Japanese cedar (Cryptomeria japonica D. Don)

PLoS ONE ◽

10.1371/journal.pone.0206695 ◽

2018 ◽

Vol 13 (11) ◽

pp. e0206695 ◽

Cited By ~ 8

Author(s):

Yoichi Hasegawa ◽

Saneyoshi Ueno ◽

Asako Matsumoto ◽

Tokuko Ujino-Ihara ◽

Kentaro Uchiyama ◽

...

Keyword(s):

Fine Mapping ◽

Marker Assisted Selection ◽

Cryptomeria Japonica ◽

Japanese Cedar ◽

Snp Markers ◽

Male Sterile

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Identification and genetic diversity analysis of a male-sterile gene (MS1) in Japanese cedar (Cryptomeria japonica D. Don)

Scientific Reports ◽

10.1038/s41598-020-80688-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yoichi Hasegawa ◽

Saneyoshi Ueno ◽

Fu-Jin Wei ◽

Asako Matsumoto ◽

Kentaro Uchiyama ◽

...

Keyword(s):

Cryptomeria Japonica ◽

Lipid Transfer Protein ◽

Pollen Dispersal ◽

Japanese Cedar ◽

Lipid Transfer ◽

Male Sterile ◽

Causative Gene ◽

Transfer Protein ◽

The Japanese Archipelago ◽

And Function

AbstractIdentifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

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Identification and genetic diversity analysis of a male-sterile gene (MS1) in Japanese cedar (Cryptomeria japonica D. Don)

10.1101/2020.05.09.085464 ◽

2020 ◽

Author(s):

Yoichi Hasegawa ◽

Saneyoshi Ueno ◽

Fu-Jin Wei ◽

Asako Matsumoto ◽

Kentaro Uchiyama ◽

...

Keyword(s):

Cryptomeria Japonica ◽

Lipid Transfer Protein ◽

Pollen Dispersal ◽

Japanese Cedar ◽

Lipid Transfer ◽

Male Sterile ◽

Causative Gene ◽

Transfer Protein ◽

The Japanese Archipelago ◽

And Function

AbstractIdentifying causative genes for a target trait in conifer reproduction is challenging for species lacking whole-genome sequences. In this study, we searched for the male-sterility gene (MS1) in Cryptomeria japonica, aiming to promote marker-assisted selection (MAS) of male-sterile C. japonica to reduce the pollinosis caused by pollen dispersal from artificial C. japonica forests in Japan. We searched for mRNA sequences expressed in male strobili and found the gene CJt020762, coding for a lipid transfer protein containing a 4-bp deletion specific to male-sterile individuals. We also found a 30-bp deletion by sequencing the entire gene of another individual with the ms1. All nine breeding materials with the allele ms1 had either a 4-bp or 30-bp deletion in gene CJt020762, both of which are expected to result in faulty gene transcription and function. Furthermore, the 30-bp deletion was detected from three of five individuals in the Ishinomaki natural forest. From our findings, CJt020762 was considered to be the causative gene of MS1. Thus, by performing MAS using two deletion mutations as a DNA marker, it will be possible to find novel breeding materials of C. japonica with the allele ms1 adapted to the unique environment of each region of the Japanese archipelago.

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Marker-Assisted Selection for Pollen-Free Somatic Plants of Sugi (Japanese Cedar, Cryptomeria japonica): A Simple and Effective Methodology for Selecting Male-Sterile Mutants With ms1-1 and ms1-2

Frontiers in Plant Science ◽

10.3389/fpls.2021.748110 ◽

2021 ◽

Vol 12 ◽

Author(s):

Momi Tsuruta ◽

Tsuyoshi E. Maruyama ◽

Saneyoshi Ueno ◽

Yoichi Hasegawa ◽

Yoshinari Moriguchi

Keyword(s):

Marker Assisted Selection ◽

Cryptomeria Japonica ◽

Japanese Cedar ◽

Causative Mutation ◽

Efficient Production ◽

Male Sterile ◽

Plant Materials ◽

Embryogenic Cells ◽

Embryogenic Cell ◽

Somatic Plants

Pollen allergy caused by sugi (Japanese cedar, Cryptomeria japonica) is a serious problem in Japan. One of the measures against pollinosis is the use of male-sterile plants (MSPs; pollen-free plants). In this context, the development of a novel technique for the efficient production of sugi MSPs, which combines marker-assisted selection (MAS) with somatic embryogenesis (SE), was recently reported by our research group. To improve the efficiency of MSP production, in this paper we report improved MAS for male-sterile individuals from embryogenic cells, cotyledonary embryos, and somatic plants of sugi using a newly developed marker in the form of the causative mutation of MS1 itself, selecting individuals with ms1-1 and ms1-2 male-sterile mutations. We also describe simplified methods for extracting DNA from different plant materials and for MAS using LAMP diagnostics. Finally, we show that MAS can be efficiently performed using the one-step indel genotyping (ING) marker developed in this study and using InstaGene for DNA extraction. The combination of SE and 100% accurate marker selection during the embryogenic cell stage enables the mass production of MS1 male-sterile sugi seedlings.

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CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

Scientific Reports ◽

10.1038/s41598-021-95547-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yoshihiko Nanasato ◽

Masafumi Mikami ◽

Norihiro Futamura ◽

Masaki Endo ◽

Mitsuru Nishiguchi ◽

...

Keyword(s):

Genome Editing ◽

Cryptomeria Japonica ◽

Fluorescent Protein ◽

Japanese Cedar ◽

Targeted Mutagenesis ◽

Embryogenic Tissue ◽

Breeding Period ◽

Single Mutation ◽

Knock Out ◽

Guide Rna

AbstractCryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

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