使用SSR荧光标记对须鸢尾品种的再次开花特性进行遗传多样性和关联分析

《Technology in Horticulture》：Genetic diversity and association analysis of the reblooming trait in bearded iris cultivars using SSR fluorescent markers

绣球菊品种的SSR分子标记遗传分析及重瓣特性关联研究。通过转录组筛查获得29,800个SSR位点，筛选出8个高多态性核心标记（PIC平均0.6228），构建了17个品种的分子指纹和聚类树（UPGMA法），揭示9个重瓣品种与8个单瓣品种的遗传分化（K=4）。关联分析发现P2S19标记与重瓣性状显著相关（p<0.05）。该研究建立了绣球菊种质资源分子鉴定体系，为重瓣性状的分子育种提供了标记资源。

　　
ornamental plants have long been a focus of horticultural research due to their aesthetic and ecological value. Among these, bearded iris (*Iris germanica*) has emerged as a model species for studying flowering regulation and genetic diversity. The study presented here investigates the genetic basis of reblooming—a trait that allows plants to flower twice within a single year—a feature highly desired for extending visual display periods in gardens and landscapes. By integrating transcriptomic analysis, SSR marker development, and association genetics, this research provides novel insights into the molecular mechanisms underlying reblooming and establishes a practical framework for iris breeding.

The study begins with a comprehensive analysis of SSR (Simple Sequence Repeat) loci within the bearded iris transcriptome. Over 29,800 SSR sites were identified across 100,391 unigenes, revealing a repeat density of 29.68%. This high frequency of SSRs underscores their potential as molecular markers for genetic studies. The distribution of repeat motifs showed dinucleotides (AG/CT and AT/AT) as the most abundant, followed by trinucleotides like AAG/CTT and AGG/CCT. The average SSR length was 15.05 bp, with shorter repeats (less than 15 bp) comprising 62.89% of all loci. These findings align with broader trends in plant genomics, where SSRs are frequent and serve as reliable markers for diversity studies.

A key challenge in horticultural breeding is the delayed phenotypic expression of traits in hybrid progeny. For iris, traditional breeding cycles require three years to observe flowering, complicating early-stage trait selection. To address this, the study employed SSR markers for high-throughput genotype screening. From 110 designed primers, eight were selected as core markers based on amplification efficiency, product size (150-500 bp), and melting temperature (60°C). These primers were fluorescently labeled for capillary electrophoresis, enabling precise detection of 43 alleles across 17 cultivars. The polymorphic information content (PIC) of these markers averaged 0.6228, indicating high allelic diversity—a critical factor for marker-assisted breeding.

The genetic diversity of the 17 iris cultivars was analyzed using eight SSR markers. Population parameters such as Nei's genetic distance (mean 1.3203) and Shannon's information index (mean 1.3203) revealed significant variation. The observed heterozygosity (Ho) ranged from 0.2280 to 0.8077, while expected heterozygosity (He) varied between 0.1500 and 0.8095, suggesting non-random mating and possible selection pressures. These metrics are consistent with prior studies on iris germplasm, where artificial hybridization and selective breeding have enriched genetic diversity.

Cluster analysis based on SSR profiles divided the cultivars into four groups. Notably, Group I contained nine reblooming cultivars, while Groups II-IV included once-flowering types. This clustering strongly correlated with phenotypic data, as reblooming cultivars consistently showed larger flower diameters (10.3-13.8 cm) and greater plant heights (42.3-99 cm) compared to once-flowering counterparts (8.6-11.7 cm and 60.0-77.7 cm). The independent clustering of 'Fufted Cloud' (CC16) highlighted its unique genetic background, possibly shaped by distinct breeding histories or environmental adaptations.

Association analysis identified P2S19 as the first SSR marker significantly linked to reblooming status (p < 0.05). This marker, with dinucleotide repeats (AC) and a 173 bp amplicon, showed differential inheritance patterns between reblooming and non-reblooming cultivars. While mixed linear models (MLMs) accounted for population structure and kinship effects, generalized linear models (GLMs) better detected marker-trait associations in small populations. The discrepancy between GLM and MLM results underscores the importance of model selection in marker studies, particularly when sample sizes are limited.

The study's practical implications are significant for iris breeding. By establishing molecular fingerprints and cluster dendrograms, researchers can now reliably distinguish reblooming cultivars during early growth stages. This reduces reliance on time-consuming field observations and allows breeders to prioritize hybrids with desired traits. The identified P2S19 marker could serve as a genetic anchor for futureGWAS (Genome-Wide Association Studies) to pinpoint candidate genes involved in flowering regulation. Such knowledge would enable targeted breeding strategies, accelerating the development of new reblooming cultivars.

However, the research also highlights limitations inherent in current methodologies. The sample size of 17 cultivars, while sufficient for initial exploration, may underrepresent the genetic diversity of global iris germplasm. Future studies should incorporate larger, more geographically diverse collections to refine clustering accuracy and marker efficacy. Additionally, the absence of significant SSR associations with flower diameter or plant height suggests these traits involve complex polygenic networks or interactions with environmental factors not captured in this analysis.

The broader significance of this work extends to ornamental plant breeding in general. SSR markers, with their high co-dominant inheritance and stability across environments, offer a cost-effective solution for genetic mapping and trait prediction. The integration of transcriptomic SSR discovery with capillary electrophoresis demonstrates a scalable approach for marker development in perennials. This methodology could be adapted to other long-lived horticultural species, such as peonies or roses, where phenotypic traits are similarly delayed and environment-sensitive.

Another critical contribution is the identification of P2S19 as a potential regulator of flowering rhythm. While the exact genetic mechanism remains unclear, the marker's association with reblooming status suggests it may be located near genes involved in vernalization or photoperiodic responses. This finding aligns with previous research on *Iris* species, where specific loci have been linked to flowering time under controlled conditions. Future work could combine P2S19 with additional markers to construct a high-density genetic map, facilitating precise gene isolation.

From an applied perspective, the study provides actionable tools for breeders. The eight core SSR markers, with average PIC exceeding 0.6, offer robust discriminatory power for cultivar identification. This is particularly valuable in the commercial sector, where mislabeling or hybrid contamination can lead to significant losses. The cluster dendrogram further serves as a genetic reference for breeders to select parental lines with complementary traits.

The research also advances theoretical understanding of trait inheritance. The observed correlation between SSR markers and reblooming status supports the hypothesis that specific SSR regions are associated with flowering time control. This could involve regulatory elements, transcription factors, or epigenetic modifiers. The fact that only one marker showed significant association in this small population suggests that reblooming is governed by multiple loci, with P2S19 representing one of the most influential.

In conclusion, this study bridges molecular genetics and horticultural breeding by establishing a reliable SSR-based workflow for iris cultivar analysis. The identified markers and clustering patterns provide a foundation for marker-assisted selection, while the P2S19 association highlights a potential target for future genetic engineering. The methodological framework presented—transcriptomic SSR discovery, high-throughput genotyping, and association modeling—offers a replicable model for studying开花调控 (flowering regulation) in other ornamental species. Future expansions of this research to include more cultivars, combined with next-generation sequencing and epigenetic analysis, will further elucidate the genetic architecture of reblooming and enable more precise breeding strategies, ultimately contributing to sustainable horticultural practices and biodiversity conservation.