Genetic gains in wood property can be achieved by indirect selection and nondestructive measurements in full-sib families of Japanese cedar (Cryptomeria japonica. D. Don) plus tree clones

Along with progress in sequencing technology and accumulating knowledge of genome and gene sequences, molecular breeding techniques have been developed for predicting the genetic potential of individual genotypes and for selecting superior individuals. For Japanese cedar (Cryptomeria japonica (L.f.) D.Don), which is the most common coniferous species in Japanese forestry, we constructed a custom primer panel for target amplicon sequencing in order to simultaneously determine 3034 informative single nucleotide polymorphisms (SNPs). We performed primary evaluation of the custom primer panel with actual sequencing and in silico PCR. Genotyped SNPs had a distribution over almost the entire region of the C. japonica linkage map and verified the high reproducibility of genotype calls compared to SNPs obtained by genotyping arrays. Genotyping was performed for 576 individuals of the F1 population, and genomic prediction models were constructed for growth and wood property-related traits using the genotypes. Amplicon sequencing with the custom primer panel enables efficient obtaining genotype data in order to perform genomic prediction, manage clones, and advance forest tree breeding.

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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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