In order to optimize the chromosome preparation of Primulina (Gesneriaceae), evaluate the effects of various factors on chromosome preparation, and perform karyotype analysis of three Primulina species, providing technical support for chromosome research and hybrid breeding in this genus.

Three Primulina species were used as experimental materials to investigate the effects of different sampling sites, plant maturity, pretreatment methods and durations, dissociation time, and preparation techniques on chromosome preparation. The chromosome preparation protocol was optimized, and karyotype analysis was conducted based on the refined method.

1) The third leaf from each plant was used for leaf cutting hydroponics, with optimal chromosome preparation achieved when the new root tips reached 0.5-1.0 cm in length, allowing clear observation of cell division stages. Pretreatment with 0.02% colchicine for 3 hours yielded the best results, producing well-dispersed and appropriately concentrated chromosomes. For acid hydrolysis, the optimal dissociation occurred with 1 mol/L HCl + 45% acetic acid at 60 ℃ for 4 minutes. The most effective enzyme digestion formula was a mixture of 2.5% cellulase and pectinase, with a 5 minute dissociation time. Both methods produced clear, well-dispersed chromosomes. 2) All three Primulina species are diploid, with a chromosome number of 2n=36 and a basic number of X=18. The karyotype formula of Primulina eburnea is 2n=2x=36=28m+6sm+2st, with a satellite on chromosome 1. The relative chromosome length ranges from 1.12% to 4.27%. The chromosome composition is 8L+5M2+19M1+4S, and the karyotype asymmetry coefficient is 58.70%, classified as type “2B”. The karyotype formula of Primulina pungentisepala is 2n=2x=36=30m+6sm, with a satellite on chromosome 11. The relative chromosome length ranges from 1.89% to 4.22%. The chromosome composition is 3L+11M2+19M1+3S, and the karyotype asymmetry coefficient is 57.10%, also classified as type “2B”. The karyotype of Primulina ‘Spring of Beilin’ has a chromosome number of 2n=36, with a basic number of X=18, and the karyotype formula is 2n=2x=36=32m+4sm. The relative chromosome length ranges from 1.94% to 3.98%. The chromosome composition is 3L+14M2+17M1+2S, with a karyotype asymmetry coefficient of 56.60%, also classified as type “2B”.

The chromosome preparation protocol for the genus Primulina was successfully optimized using three Primulina species. The study concludes that these species are diploid, possess small chromosomes, and have karyotypes classified as relatively symmetric.

In order to explore hybrid affinity and the causes of hybrid incompatibility within Primulina.

4 wild species and 2 cultivars were used as materials for conventional hybridization. After screening the parents for pollen vitality and stigma receptivity, hybridization work was carried out to preliminarily determine the affinity relationship of the hybrid combinations. The development of the ovary after pollination was tracked and observed, combined with experiments of pollen tube fluorescence observation, to explore the affinity and hybridization barriers of the interspecific hybridization in Primulina.

(1) Pollen viability and stigma receptivity within the Primulina was not absolutely causally related to hybrid affinity reaction. (2) The ovary swelled rate of all 16 hybrids was above 65.00%, and the seed setting rate was obviously different. When P. eburnea, P. pungentisepala, P. macrorhiza, and P. fimbrisepala were used as the female samples, the seed setting rates of each hybrid reached 75.55%, 78.44%, 46.67%, 57.78%, respectively, and showed high affinity, whereas the combination with P. ‘Spring of Beilin’, P. ‘Four Season’ as the female sample also showed the highest seed setting rate of only 6.67% and weaker affinity. (3) Fluorescence observations were conducted on the pollen tube behavior of five hybrid combinations with weak affinity. It was found that there was all combinations exhibited a certain degree of pre-fertilization barrier, which was manifested as pollen tube callus reaction, pollen tube tip swelling, twisting and rupture. However, all combinations eventually had pollen tubes extending into the embryo sac. Judging from the number of pollen tubes reaching the embryo sac, the amount of pollen from three native species such as P. eburnea was sufficient for fertilization, but the fruit set rate was still low.

Crosses within the genus Primulina show some affinity, but some combinations fail to produce seeds. This is not caused by limited pollen germination or pollen tube growth prior to fertilization, but may result in abortion due to metabolic disturbances occurring during the later development of zygotes or young embryos.

Rosa persica is the only single-leaf species of Rosa genus, which has been classified as the second class of national endangered plants. The development of efficient molecular markers can provide important data support for the analysis of the genetic diversity of R. persica populations and the study of the growth pattern of clones, as well as theoretical guidance for the conservation of genetic resources of its populations.

SSR loci with 1-6 nucleotide repeats in the whole genome sequence of R. persica were searched and analyzed by Krait v1.3 software, and then primers were designed by Primer Premier3.0 software. Sixteen samples of R. persica were selected to test the validity and polymorphism of the primers by agarose gel electrophoresis and capillary electrophoresis. Finally, the data of amplification products of high polymorphism primers were analyzed by POPGENE32 software and PowerMarker3.25 software for genetic parameters, and the 16 R. persica samples were analyzed by clustering using the software NTSYS-pc 2.10.

A total of 142 083 SSR loci were identified on the genome sequence of R. persica, with the largest number of dinucleotide repeat types, accounting for 46.95%. The units with the highest percentage from mononucleotide to hexanucleotide repeat types were A/T, AT/AT, AAG/CTT, AAAT/ATTT, AAAAT/ATTT and AAAAAG/CTTTTTT, which fully indicated that A/T were the dominant unit. The SSR sequence lengths of R. persica genome varied from 12 to 1 026 bp, and the different nucleotide repeat types all showed the pattern of the longer length and the lower number, and the number of SSR loci with interval lengths of 10-15 bp was the largest, accounting for 47.42%. Among the 140 pairs of primers synthesized, 112 pairs of primers could obtain clear bands, and the primer efficiency was 80%; finally, 14 pairs of primers with high polymorphism and good stability were screened out and 58 alleles were detected in 16 R. persica samples. The PIC value ranged from 0.314 3 to 0.675 9. The average allele number (Na), effective alleles (Ne), shannon diversity index (I) and polymorphic information content (PIC) were 4.142 9, 2.576 9, 1.080 8 and 0.529 2, respectively. Pearson correlation analysis showed that there was no significant correlation between the PIC values of the screened primers and the lengths of the SSRs. Cluster analysis revealed that the 14 pairs of primers screened were able to better differentiate R. persica based on different populations, with genetic similarity coefficients ranging from 0.45 to 0.86.

The large-scale development of SSR markers using the whole genome of R. persica is rich in number and diverse in type, and the highly polymorphic SSR loci screened will play an important role in the study of the genetic diversity of R. persica populations.