A pure line derived from a self-compatible Chrysanthemum seticuspe mutant as a model strain in the genus Chrysanthemum

Highlights

• Cultivated chrysanthemum is one of the most industrially important flowers.

• It is hexaploid and self-incompatible, making genetic analysis difficult.

• We isolated a self-compatible mutant of the diploid species Chrysanthemum seticuspe.

• We bred the pure line Gojo-0 through repeated selfing and selection.

• Gojo-0 could be a model Chrysanthemum strain for molecular genetic studies.

Abstract

Asteraceae is the largest family of angiosperms, comprising approximately 24,000 species. Molecular genetic studies of Asteraceae are essential for understanding plant diversity. Chrysanthemum morifolium is the most industrially important ornamental species in Asteraceae. Most cultivars of C. morifolium are autohexaploid and self-incompatible. These properties are major obstacles to the genetic analysis and modern breeding of C. morifolium. Furthermore, high genome heterogeneity complicates molecular biological analyses. In this study, we developed a model strain in the genus Chrysanthemum. C. seticuspe is a diploid species with a similar flowering property and morphology to C. morifolium and can be subjected to Agrobacterium-mediated transformation. We isolated a natural self-compatible mutant of C. seticuspe and established a pure line through repeated selfing and selection. The resultant strain, named Gojo-0, was favorable for genetic analyses, including isolation of natural and induced mutants, and facilitated molecular biological analysis, including whole genome sequencing, owing to the simplicity and homogeneity of its genome. Interspecific hybridization with Chrysanthemum species was possible, enabling molecular genetic analysis of natural interspecific variations. The accumulation of research results and resources using Gojo-0 as a platform is expected to promote molecular genetic studies on the genus Chrysanthemum and the genetic improvement of chrysanthemum cultivars.

Introduction

Asteraceae is the largest family of angiosperms, including 1,600–1,700 genera and ca. 24,000 species, comprising one-tenth of the angiosperms [1]. Interestingly, this large number of species with diverse characteristics is thought to have only diverged within the last 40–50 million years [2]. Thus, Asteraceae is an important group for the study of plant diversity. Anthemideae, a tribe in Asteraceae, consists of 111 genera and 1,800 species [3], and includes some industrially important species, such as Tanacetum cinerariifolium (Trevir.) Sch. Bip., from which the insecticide pyrethrin is produced [4], and Artemisia annua L., which is a raw material in the most important commercial antimalaria drug, artemisinin [5]. The cultivated chrysanthemums, Chrysanthemum morifoliumm Ramat, one of the most important ornamental flowers in the world, also belongs to the Anthemideae [3]. The cultivated chrysanthemum is thought to originate in East Asia from hybrids between wild Chrysanthemum species [[6], [7], [8], [9], [10]]. Species in the genus Chrysanthemum are classified into four groups according to their morphological characteristics, particularly those affecting synflorescence and the capitulum, a single flower-like inflorescence consisting of florets and involucre [11]. Species in the Indicum group have capitula consisting of female ray florets with yellow ligules at the margin and bisexual disc florets without ligules in the center. Their capitula essentially form a corymbose synflorescence. Species in the Makinoi group have similar capitula to those in the Indicum group, except that the ligules are white. Their synflorescences are solitary to lax corymbose. The capitula of species in the Zawadskii group are similar to those of the Makinoi group, including the color of ligules, but they are more solitary. Furthermore, Zawadskii group species develop radical leaves with short internodes, and retain them for longer than the species in other groups. Species in the Ajania group are sometimes classified into the genus Ajania, which is characterized by disciform capitula [12], but several lines of evidence suggest that at least some of the Ajania species (e.g., the Ajania group) belong to the genus Chrysanthemum [11]. As autopolyploidization has repeatedly occurred during the evolution of Chrysanthemum; each group contains polyploid species, with up to decaploids in some groups [13]. Thus, the genus Chrysanthemum is a good example for investigating the evolutionary mechanism of polyploidization. Interestingly, interspecific crosses between species with significantly different morphology is possible in Chrysanthemum, which enables the genetic analysis of natural variations between different species [[14], [15], [16], [17], [18]]. Although cultivated chrysanthemums is one of the most popular ornamental flowers in the world, genetic analysis has not been fully applied to elucidate the mechanisms of important agricultural and ornamental traits. Two properties of cultivated chrysanthemums have mainly prevented molecular genetic analyses. First, the majority of cultivated chrysanthemums are autohexaploid, which makes genetic analysis complicated, because at least three homeologs with redundant functions may exist in their genomes. In addition, the large genome size (˜10 Gbps) due to its polyploidy has also been problematic [19]. Second, Chrysanthemum species have self-incompatibility [20], which maintains a highly heterozygous genome over generations and complicates molecular biological/genetic analyses, including whole genome sequencing. Self-incompatibility also makes it difficult to obtain homozygotes of recessive mutations and isolate induced mutants. Although recent advances in next-generation sequencing techniques have enabled the development of DNA markers for distinguishing homeologous chromosomes in the autopolyploid genome of C. morifolium, their application to genetic analysis is still limited [21,22].

Wild chrysanthemum species often have similar properties to cultivated chrysanthemums. Chrysanthemum seticuspe (Maxim.) Hand.-Mazz. is one of these species, which has similar growth and flowering properties, and flower morphology (We use this name in the broad sense, including forma seticuspe, Japanese name “Kamome-giku”, and f. boreale (Makino) H.Ohashi et Yonek., Japanese name “Kikutani-giku”). In particular, because C. seticuspe is a diploid species and is easy to cultivate, it could be a good reference for cultivated chrysanthemums and the model species for the genus Chrysanthemum. Indeed, C. seticuspe has been used in analysis of the regulation of flowering in chrysanthemums, including molecular identification of the florigen gene CsFTL3 [23] and the anti-florigen gene AFT [24]. This information greatly contributed to the understanding of the flowering physiology of C. morifolium [25].

Similar to other Chrysanthemum species, C. seticuspe is self-incompatible, which has impeded the establishment of C. seticuspe as a model species. Several studies on self-compatibility have been reported in C. morifolium [[26], [27], [28], [29], [30], [31]], but stable self-compatible strains have not been reported. In contrast, we have isolated AEV02, a self-compatible mutant strain of C. seticuspe [32]. Self-compatibility of AEV02 was retained over six generations. We developed a pure line, named Gojo-0, from the progeny of AEV02 through repeated selfing and selection. Gojo-0 has overcome the problems that make genetic analysis difficult in Chrysanthemum: polyploidy, self-incompatibility, and high heterogeneity of the genome. C. seticuspe can be transformed via Agrobacterium-mediated leaf disc methods, and the whole-genome sequence of XMRS10, another selfed progeny of AEV02, is now available [33]. Thus, Gojo-0 is a promising model strain for molecular genetic analysis of the genus Chrysanthemum.