Most Fabaceae have nitrogen fixation abilities and are valuable forage and medicinal resources. However, cytogenetic data of many Fabaceae species are unclear. Karyotypes reveal cytological characteristics and are crucial to understanding the organization and evolution of chromosomes in species. Oligo-FISH can reveal genetic composition and karyotype variation patterns with rapid and efficient results. Karyotype analysis of five Fabaceae species by oligonucleotide probes showed that: Robinia pseudoacacia, karyotype formula 2n = 2x = 20m + 2sm, cytotype 2B, arm ratio 3.4821, eight chromosomes distributed 5S rDNA signal. The karyotype formula of Robinia pseudoacacia ‘idaho’ was 2n = 2x = 20m + 2sm, cytotype 1A, arm ratio 1.8997, and 5S rDNA signal was distributed on six chromosomes. Karyotype of Robinia pseudoacacia f. decaisneana 2n = 2x = 20m + 2sm, cytotype 1B, arm ratio 2.0787, the distribution of eight chromosomes with 5S rDNA signal. Karyotype formula of Styphnolobium japonicum 2n = 2x = 14m + 12sm + 2st, cytotype 2B, arm ratio 2.6847, two chromosomes have 5S rDNA signal. Amorpha fruticose karyotype 2n = 2x = 38m + 2sm, cytotype 1B, arm ratio 3.2058, four chromosomes possessed 5S rDNA signal. Both ends of all species’ chromosomes have (AG3T3)3 signals. The results of this study provide chromosome numbers and a physical map, contributing to the construction of the Oligo-FISH barcode and providing molecular cytogenetics data for Fabaceae.

Bletilla spp.Rchb. F. is a traditional Chinese medicinal material. In this study, Bletilla striata (Thunb. ex A. Murray) Rchb F, Bletilla formosana (Hayata) Schltr, and Bletilla ochracea Schltr were collected to analyze the genetic diversity of 16 materials using specific site-amplified fragment sequencing (SLAF-seq) and fluorescence in situ hybridization (FISH). The results showed that the phylogenetic tree of the single-nucleotide polymorphism (SNP) data rendering system was correlated with the shape and geographical distribution of the material. The results of the population structural analysis showed that all the materials containing yellow labellum came from the same ancestor. The results of the principal component analysis were able to preliminarily judge the genetic distance and provided a reference for the selection of hybrid parents. The FISH analysis showed that the chromosomes of B. striata were 2n = 32 and the chromosomes of the B. striata (safflower) mutant were 2n = 34 and the chromosomes of B. ochracea and B. formosana were 2n = 34–36. The (AG3T3)3 non-terminal signal was different from the 5S rDNA signal. These results revealed that the 16 materials had rich genetic diversity, which can provide molecular and cytogenetic data for the study of the genus and its relatives and serve as a reference for the breeding of new genus varieties and improve breeding efficiency and cost.