Identification of Banana Hybrid and Selfing Offspring Based on Internal Transcribed Spacer（ITS）Sanger Sequencing

doi:10.13560/j.cnki.biotech.bull.1985.2024-0501

Abstract

Abstract:

【Objective】The aim of this study is to utilize the Internal Transcribed Spacer（ITS）region as a molecular marker in identifying newly developed hybrid and selfing offspring banana progeny, thereby laying the foundation for advancements in banana breeding and genetics research.【Method】DNA of parents and their suspected offspring was extracted, then PCR amplifification and Sanger sequencing were conducted, and sequence alignment was performed using software “DNAMAN” and “SnapGene” to analyze the polymorphism of ITS, thereby determining the hybrid and selfing offspring. The plant was classified as a positive plant when the ITS sequence of the suspected offspring plant showed differences at one or more base sites compared with the parent; conversely, if no such differences were found, the plant was determined to be a false positive plant.【Result】The study encompasses three sets of putative progeny derived from cultivated banana crossed with wild banana（‘Guang Fen No. 1’ × ‘Tian Ye BB No. 1’）, selfed offspring of a cultivated variety（‘Fen Za No.1’）, and a cross between two wild bananas（‘Musa acuminata No. 1’ × ‘Musa acuminata No. 2’）. Applying the established banana ITS Sanger sequencing protocol, the notable disparities linked to the ITS sequences of these candidate offspring compared to their parents were identified. Phenotypic observations further confirmed variances in the offspring plants, suggesting they were positive plants. In the hybrid offspring of ‘Guang Fen No.1’ and ‘Tian Ye BB No.1’, polymorphisms in the “410 bp region” were observed in the ITS Sanger sequencing profiles, accompanied by distinct changes in bunch characteristics. Among four selfed offspring of ‘Fen Za No.1’, polymorphic peaks in the “50 bp” and “410 bp regions” were detected, with two lines exhibiting distinctive leaf traits. For the ten seedlings resulting from the ‘Musa acuminata No. 1’ × ‘Musa acuminata No. 2’ cross, significant differences in the sequencing profiles were noted at “115 bp” “431 bp” and “452 bp” relative to their parents.【Conclusion】The ITS Sanger sequencing approach emerges as a complementary tool to phenotypic assessments and SSR-based identification methods, facilitating rapid, flexible, and convenient material authenticity confirmation in distinguishing banana hybrid or selfed offspring. This methodology aids breeders and researchers in efficiently confirming their materials accuracy in breeding and genetic studies.

Key words: banana, germplasm resources, hybrid, selfing, molecular identification, ITS

ZENG Hong-yun, XU Lin-bing, WU Yuan-li, HUANG Bing-zhi. Identification of Banana Hybrid and Selfing Offspring Based on Internal Transcribed Spacer（ITS）Sanger Sequencing[J]. Biotechnology Bulletin, 2024, 40(12): 53-60.

Figures/Tables 3

Fig. 1 Identification of hybrids from the cross between ‘Guang Fen No. 1’ and ‘Tian Ye BB1 No. 1’ A: Field traits. B: Sanger sequencing chromatogram of the “420 bp region” of ITS

Fig. 2 Identification of selfing offspring of ‘Fen Za No.1’ A: Field characteristics. B: Sanger sequencing chromatogram of the “50 bp region” of ITS. C: Sanger sequencing chromatogram of the “420 bp region” of ITS

Fig. 3 Identification of hybrids derived from the cross between ‘Musa acuminata No.1’ and ‘Musa acuminata No.2’ A: Alignment of ITS sequences. B: Sanger sequencing chromatogram of the “110 bp region” of ITS. C: Sanger sequencing chromatogram of the “440 bp region” of ITS

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