Abstract
BackgroundIn recent years, the development of structural genomics has generated a growing interest in obtaining haploid plants. The use of homozygous lines presents a significant advantage for the accomplishment of sequencing projects. Commercial citrus species are characterized by high heterozygosity, making it difficult to assemble large genome sequences. Thus, the International Citrus Genomic Consortium (ICGC) decided to establish a reference whole citrus genome sequence from a homozygous plant. Due to the existence of important molecular resources and previous success in obtaining haploid clementine plants, haploid clementine was selected as the target for the implementation of the reference whole genome citrus sequence.ResultsTo obtain haploid clementine lines we used the technique of in situ gynogenesis induced by irradiated pollen. Flow cytometry, chromosome counts and SSR marker (Simple Sequence Repeats) analysis facilitated the identification of six different haploid lines (2n = x = 9), one aneuploid line (2n = 2x+4 = 22) and one doubled haploid plant (2n = 2x = 18) of 'Clemenules' clementine. One of the haploids, obtained directly from an original haploid embryo, grew vigorously and produced flowers after four years. This is the first haploid plant of clementine that has bloomed and we have, for the first time, characterized the histology of haploid and diploid flowers of clementine. Additionally a double haploid plant was obtained spontaneously from this haploid line.ConclusionThe first haploid plant of 'Clemenules' clementine produced directly by germination of a haploid embryo, which grew vigorously and produced flowers, has been obtained in this work. This haploid line has been selected and it is being used by the ICGC to establish the reference sequence of the nuclear genome of citrus.
Highlights
In recent years, the development of structural genomics has generated a growing interest in obtaining haploid plants
The first haploid plant of 'Clemenules' clementine produced directly by germination of a haploid embryo, which grew vigorously and produced flowers, has been obtained in this work. This haploid line has been selected and it is being used by the International Citrus Genomic Consortium (ICGC) to establish the reference sequence of the nuclear genome of citrus
The recovery of haploid and double haploid plants from gametic embryogenesis enables homozygous lines to be isolated in a single step, whereas only nearhomozygous genotypes can be obtained through several generations of selfing in classical genetic approaches [1]
Summary
The development of structural genomics has generated a growing interest in obtaining haploid plants. The recovery of haploid and double haploid plants from gametic embryogenesis enables homozygous lines to be isolated in a single step, whereas only nearhomozygous genotypes can be obtained through several generations of selfing in classical genetic approaches [1]. Such traditional methods are extremely difficult to implement in woody species, such as citrus, which are highly heterozygotic and have long juvenile phases, requiring several decades to obtain a near-homozygous plant. In the near future, haploid and double haploid plants will play an increasingly important role in whole genome sequencing (WGS) projects, where homozygosity is a particular advantage. The WGS of grapevine was made from a near-homozygous line obtained after six successive self-pollinated generations [14,15]
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