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

Abstract

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.

An Improved and Simplified Propagation System for Pollen-Free Sugi (Cryptomeria japonica) via Somatic Embryogenesis

“…Plant materials for the propagation of pollen-free sugi were obtained by artificial crossings between a male-sterile tree ( ms1 / ms1 ) as a seed parent and a pollen donor with heterozygous sterile allele ( Ms1 / ms1 ; Maruyama et al, 2020 ; Tsuruta et al, 2021a ). To ensure seed production, gibberellin (GA 3 ) treatments were applied to the branches to promote flowering before the artificial crossings.…”

“…Using 5 to 10 mg of the maintained ECs, DNA was extracted using InstaGene Matrix (Bio-Rad, Hercules, CA, United States). The MS1 genotype of the ECL was determined using PCR amplification of the marker directly developed for the gene that causes male sterility ( Tsuruta et al, 2021a ), followed by electrophoresis on 1.5% agarose gel. Each reaction condition is described in Table 3 .…”

“…To date, only 23 male-sterile sugi individuals have been reported across the entire country (Saito, 2010); the majority of these male-sterile trees possess MALE STERILITY 1 (MS1), a principal causative gene of male sterility in sugi (Moriguchi et al, 2020), which is associated with a developmental abnormality in the pollen tetrad period caused by one recessive allele ms1 (Taira et al, 1999;Miura et al, 2011). At present, seeds for the propagation of C. japonica MSPs are produced by artificial crossing between a male-sterile tree (ms1/ms1), as a seed parent, and a pollen donor (an elite tree selected based on growth performance and morphological traits) with heterozygous sterile allele (Ms1/ms1; Maruyama et al, 2020;Tsuruta et al, 2021a).…”

“…The development of this novel technique for the efficient production of sugi MSPs was first reported by our research group ( Maruyama et al, 2020 ). Then, we released improved methodologies for SE initiation ( Maruyama et al, 2021a ), somatic embryo production, and somatic plant regeneration ( Maruyama et al, 2021b ; Tsuruta et al, 2021b ), as well as for the early effective selection of male-sterile ECLs ( Hasegawa et al, 2021 ; Tsuruta et al, 2021a ).…”

Plant Regeneration and In Vitro Growth Performance of Male-Sterile Somatic Plantlets of Sugi (Japanese Cedar, Cryptomeria japonica) Derived from Different Embryogenic Cell Lines

“…Finally, 19 male-sterile ECLs were selected based on the genotype determined from the presence of male-sterile-and/or wild-type-specific amplified fragments [4]. The genotypes and male-sterile discrimination of these lines were subsequently confirmed with a more accurate genetic marker, which was developed on the causable mutation itself [24].…”