Banana (Musa spp.) is one of the most economically important horticultural crops. There are many types of banana, with differing ploidy (usually diploid, triploid, or tetraploid) and genome types (most containing the A or/and B genome). Currently, observation and genome type detection are commonly used to identify banana germplasm resources. However, observation is tedious, while genome type detection cannot distinguish categories below genome types. It is, therefore, urgent to establish a simple and effective method for identifying banana germplasm resources. This study sequenced and analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 62 banana germplasm resources and found that the sequencing peaks, especially the 20 bp region near the 420-bp position (referred to as the 420-bp region), exhibited relatively recognizable and repeatable polymorphism characteristics. Using the 420-bp region as a marker, we were able to quickly distinguish bananas belonging to different genome type groups or different subgroups in the same genome type group. Moreover, it appeared that Sanger sequencing of ITS could be used to identify hybrid banana offspring. In general, ITS sequencing simplifies the classification of banana germplasm resources and has potential application in several areas of Musa improvement.

Many types of banana (Musa spp.)—one of the most economically important horticultural crops—exist, and their ploidy (diploid, triploid, and tetraploid, usually) and genome types (containing A or/and B genome in most cases) differ. At present, observation and genome type detection are commonly used to identify banana germplasm resources. However, the former is tedious, while the latter cannot distinguish categories below genome types. It is therefore urgent that a simple and effective method for identifying banana germplasm resources is established. We sequenced and analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 62 banana germplasm and found that the sequencing peaks, especially the 20 bp region near the 420 bp position (referred to as the “420 bp region”), exhibited relatively recognizable and repeatable polymorphism characteristics. Using the “420 bp region” as a marker, we were able to quickly distinguish bananas belonging to different genome type groups (comprising the AA, AB, AAA, ABB, AAB, ABBB, AAAB, and AAAA groups) or different subgroups in the same genome type group (for example, the Cavendish, Gros Michel, Red, Lakatan, and Ibota Bota subgroups in the AAA group; and the Pisang Raja, Plantain, Pome and Silk subgroups in the AAB group). Moreover, it appeared that Sanger sequencing of ITS could be used for identifying banana hybrid offspring. These results demonstrated that the banana ITS region contains rich genetic information that is useful for genotyping. In general, ITS sequencing simplifies the classification of banana germplasm resources and has potential application in several areas of Musa improvement.