**4. Discussion**

The elaboration of the key regulatory mechanism underlying one or several traits as well as the fast selection of elite progenies is crucial for plant breeding. When obtaining a certain mutant, the identification of the allele(s) related to the phenotype is usually performed by the forward genetics, in which F2 rough mapping provides an approximate location of the mutation causative allele(s) on chromosomes without the requirement of a large amount of samples and high-throughput sequencing, narrowing down the targets for further fine mapping. In the last decades, the development of PCR-based markers such as RAPD, SSR and amplified fragment length polymorphisms (AFLPs) have fulfilled the shortage of map-based cloning [34]. In the field of ornamental breeding, these sequence-related amplified polymorphisms (SRAP) markers have been applied to the vase life-associated or disease-resistant QTL mapping and analyse chrysanthemum, carnation and lily, to cite a few [35].

Nevertheless, the development of such markers is labour intensive, and their application is limited in certain situations, since they are usually not genome-wide. Earlier, benefiting from the availability of an annotated reference genome and sequenced accessions, genetic markers based on InDels have been developed in *Arabidopsis*, accelerating the identification of the mutated allele(s) [36]. With the booming of sequencing technology and the following drop in sequencing expense, plentiful plant genomes were released for crops and horticultural plants [37–40]. The resequencing-based InDel makers were then developed in cotton [41], rice [42], Brassica [43], buckwheat [44], jute [45], melon [46], chickpea [47], cucumber [48], etc., used for research such as disease-related gene identification or accession discrimination. However, the systematic development of such markers has not been reported in ornamental species.

In this study, we constructed a genome-wide InDel marker system for *G. paniculata* through genome resequencing. Similar to the early report in jute [45], InDels detected in the *G. paniculata* genome are quite abundant, but most of them are shorter than 5 bp, which makes them hard to use as markers. Regardless, the number of the ~5% InDels that are longer than 10 bp is as large as 68302, equivalent to 91 InDels per Mb, which is more than needed. To meet the demand for mapping (1 maker/2 Mb), 409 InDels distributed on 17 chromosomes were selected, and the relative primers were then designed. Of these, 289 can discriminate alleles from 2 wild types donating the genome sequence data, coming to a success rate of 70.6%. Although we expected to obtain an available marker every 2 Mb, the outcome was barely satisfactory. There were usually missing available InDel markers in the middle (calculated by physical distance) of the chromosome, such as Chr. 4, 7 and 14 (Figure 3). The same situation happened during the development of InDel markers in rice [42] and *Capsicum* spp. [49]. It might be dissolved by adding other molecular markers when mapping a certain QTL, or the InDels shorter than 10 bp can also be developed as markers based on a high-resolution melting curve, as reported [50]. Since the discrimination of genetic resources and extension of the application of markers are crucial in the breeding process [44], we then detected the polymorphisms of the designed InDel markers in four best-selling cultivars. Over 170 makers were available to differentiate each commercial cultivar and WT-P, whereas only less than 50 markers worked for discrimination between the 4 commercial cultivars. It makes sense, since all the commercial cultivars bloom with white flowers and may share more common genetic information rather than WT-P.

Used not only as the filler flower but also as a preserved flower which decorates the environment after colourful staining, the status of cut flower *G. paniculata* is rising, leading to a massive demand for the innovation of this species. Molecular genetics play a more and more important role in floricultural breeding, in which a molecular marker system covering the whole genome is the basis for genetic molecular research in the era of genomics. However, it is still a gap for *G. paniculata*. Here, we provide the first genetic map of *G. paniculata* in this study, consisting of a comprehensive set of InDel markers for the molecular research of *G. paniculata*. The success in our case also implies that the development of InDel markers covering the whole genome is cost- and labour-effective with a high success rate, deserving to be applied in other ornamental species for which cross-breeding is the main method for cultivar innovation.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae8100921/s1, Table S1: Primers used in this study.

**Author Contributions:** C.J. and F.L. conceived and designed the research; B.L. performed the experiments and analysed the data; C.J. and F.L. wrote the manuscript and revised the manuscript; J.R. and C.Y. provided the wildtype plants and commercial cultivars. F.L. supervised the project. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China (31960608), Yunnan Fundamental Research Projects (202101AT070147) and High-level Talent Introduction Program of Yunnan Province—Industrial Talent Special Project (YNQR-CYRC-2020-004).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

