Abstract

BackgroundSomatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons. This capability for apomixis is attractive in agricultural production systems using hybrid seeds, and many studies have been performed to elucidate the molecular mechanisms of various types of apomixis. To identify the gene responsible for somatic embryogenesis in citrus, a custom oligo-DNA microarray including predicted genes in the citrus polyembryonic locus was used to compare the expression profiles in reproductive tissues between monoembryonic and polyembryonic varieties. The full length of CitRKD1, which was identified as a candidate gene responsible for citrus somatic embryogenesis, was isolated from satsuma mandarin and its molecular function was investigated using transgenic ‘Hamlin’ sweet orange by antisense-overexpression.ResultsThe candidate gene CitRKD1, predominantly transcribed in reproductive tissues of polyembryonic varieties, is a member of the plant RWP-RK domain-containing protein. CitRKD1 of satsuma mandarin comprised two alleles (CitRKD1-mg1 and CitRKD1-mg2) at the polyembryonic locus controlling embryonic type (mono/polyembryony) that were structurally divided into two types with or without a miniature inverted-repeat transposable element (MITE)-like insertion in the upstream region. CitRKD1-mg2 with the MITE insertion was the predominant transcript in flowers and young fruits where somatic embryogenesis of nucellar cells occurred. Loss of CitRKD1 function by antisense-overexpression abolished somatic embryogenesis in transgenic sweet orange and the transgenic T1 plants were confirmed to derive from zygotic embryos produced by self-pollination by DNA diagnosis. Genotyping PCR analysis of 95 citrus traditional and breeding varieties revealed that the CitRKD1 allele with the MITE insertion (polyembryonic allele) was dominant and major citrus varieties with the polyembryonic allele produced polyembryonic seeds.ConclusionCitRKD1 at the polyembryonic locus plays a principal role in regulating citrus somatic embryogenesis. CitRKD1 comprised multiple alleles that were divided into two types, polyembryonic alleles with a MITE insertion in the upstream region and monoembryonic alleles without it. CitRKD1 was transcribed in reproductive tissues of polyembryonic varieties with the polyembryonic allele. The MITE insertion in the upstream region of CitRKD1 might be involved in regulating the transcription of CitRKD1.

Highlights

  • Somatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons

  • 391 probes including multiple probes for each newly predicted open reading frame (ORF) and 29,148 probes for mRNA loci of the clementine mandarin genome were mounted on the custom oligo-DNA microarray, in which 50 genes overlapped between the polyembryonic locus and mRNA loci of the clementine genome sequence

  • NP-ORF44 was selected as a candidate for the gene that regulates somatic embryogenesis out of 79 NP-ORFs in the polyembryonic locus region

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Summary

Introduction

Somatic embryogenesis in nucellar tissues is widely recognized to induce polyembryony in major citrus varieties such as sweet oranges, satsuma mandarins and lemons. Somatic embryogenesis in nucellar tissues of citrus species is an apomictic system and genetically uniform clones with the same genotype as the maternal plant can feasibly be produced by sowing the seeds despite their highly heterozygous genomes [16] This capability for apomixis is attractive in agricultural production systems using hybrid seeds, and many researchers have investigated the molecular mechanisms of various types of apomixis [20]. Among the various types of apomixis, citrus apomixis, in which a somatic embryo develops in nucellar tissues, is classified as sporophytic apomixis [37] Major citrus varieties, such as satsuma mandarin (Citrus unshiu Marc.), sweet orange (C. sinensis (L.) Osbeck), grapefruit (C. paradisi Macfad.), and ponkan mandarin (C. reticulata Blanco), generally develop one or more somatic embryos that are genetically identical to the mother tree in addition to or instead of a zygotic embryo in the seed. Polyembryony is useful in rootstock propagation and genetically uniform rootstocks can feasibly be prepared solely by sowing seeds despite the highly heterozygous genomes of citrus species

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