Development Of High-Throughput SSR And Construction Of Linkage Map In Diploid Roses

Roses are a series of varieties with continuous flowering, which developed by hybrid-ization repeatedly within Rosa L. Due to their unique spiritual and cultural connotations as well as aesthetic features, roses for centuries have been the most important and popu-lar ornamental flowers and landscape plants. In China, rose culture owns a long history.In this study, we employed old garden roses Rosa chinensis ’Pallida’ owns the habits of continuous flowering and thorn, a thornless horticultural variety Rosa wichuriana’Basye’s thornless’with continuous flowering trait as the parents to develop a F1genetic segregating population. We constructed a Simple Sequence Repeat linkage map by the F1population.In addition, base on the good microsynteny between Rosa sp and Fragaria vesca, We characterized high-throughput SSR markers using woodland strawberry genome sequence. We try to find a method that aim to develop SSR markers targets a specific linkage group. The map of diploid roses is not only useful for revealing the molecular mechanisms of the superior traits and Marker-Assisted Selection, but also providing a framework for Rose Genome Project. Five parts of results achieved as following:1. A diploid roses experimental population development. Two varieties hold different habits in flowering time and stem thorn were employed to construct an experimental population. As a result, A total283individuals of F1were achieved, and157individuals with stem thornless, the remains are not.2. SSR markers Introduction and the polymorphisms evaluation.60primer pairs of SSR markers are obtained from INRA(National Agricultural Institute of France), of which20were EST-SSR markers, and40were Genome-SSR markers.Total29primer pairs displayed polymorphic between the parents. Meanwhile, we selected the polymorphisms of30EST-SSR markers developed by Flower Reseach Institutue (FRI), and thirteen loci are polymorphic.3. High-throughput SSR markers development. As a reference, woodland strawberry genome sequence was employed to develop SSR markers, we identified a total of60040SSR loci by MicroSAtellite (MISA) software, of which21,693one single nucleotide repeat loci,28524double bases repeat loci,7575tri-nucleotides repeat loci,710four nucleotides repeat loci,865five nucleotides repeat loci and673six bases repeat loci.Moreover, we detected7461SSR loci base on the fifth chromosome sequence of the woodland strawberry genome, and3230primer pairs of the SSR designed using primer3software.4. Cross-species transferability and polymorphisms of the new SSR markers investigation. We randomly selected100primer pairs of SSR designed from the fifth chromosome sequence of the woodland strawberry genome, and the Cross-species transferability and polymorphisms were evaluated within eight species(varieties) belonging to four sections or groups in roses. They are Rosa omeiensis and Rosa mairei belong to celery leaf group, Rosa lucidissima and Rosa odorata belong to Rose Group, Rosa xanthina and Rosa banksiae var normalis belong to Wood Hong Group, Rosa wichuriana’Basye’s thornless1belongs to aggregate column groups, and the old garden roses Rosa chinensis ’Pallida’.The results showed that37of100primer pairs amplified successfully, and the remains63primer pairs could not amplified. The ratios of the transferability of the37primers pairs are between67.6%-81.1%, with an average of72.3%.26loci displayed polymorphism, accounting for70.3%of the total amplified loci. Alleles of the single maker distributed in the range of2-6,and with a mean of2.7.5. A molecular genetic linkage map of diploid roses construction based on SSR markers. We employed164F1as mapping population, and based on the pseuo-test crossing strategy in Joinmap4.0software. We constructed a genetic map which covered34SSR loci. Seven linkage groups obtained respectively. The total genetic distance of the map is285.7cM, and the average distance between markers was10.58cM. Maximum distance between markers was21.5cM, and the minimum spacing was4.4cM. The average length of a single linkage group was40.81cM. the maximum with69.1cM, and the minimum length was15.6cM. For the seventh linkage group, the average distance between two markers reached4.5cM. This study laid a foundation for using this genetic segregating population to locate thornless loci.