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Seminar: Rob Suppa: Chloroplast Genome Sequencing in Cercis canadensis and Ploidy Manipulation in Wild Peanut Species

July 25, 2023 | 9:00 am - 10:00 am

Chloroplast Genome Sequencing in Cercis canadensis and Ploidy Manipulation in Wild Peanut Species
Robert Suppa, MS Seminar
Tuesday, July 25, 2023, 9:00 am
(Under the direction of Dr. Hsuan Chen, Chair)

Location: 121 Kilgore / Hybrid
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Meeting ID: 938 4605 6772
Passcode: 082115

Eastern redbud, Cercis canadensis L., is a small tree with various ornamental traits. Two cultivars, ‘Floating Clouds’ and ‘Silver Cloud’, display visually appealing green and white variegated leaves. Variegated leaf is a pattern of leaf color variation caused by mutations in the nuclear, mitochondrial, or chloroplast genome. These mutations have been discovered to interfere with chloroplast biogenesis, photosynthetic machinery, or pigment synthesis pathways, resulting in color variations. Previous research has indicated ‘Silver Cloud’ inherits variegation through a nuclear recessive gene, while ‘Floating Clouds’ inherits variegation maternally. This pattern of maternal inheritance suggests that the mutation causing the variegated leaf in ‘Floating Clouds’ should be present in either the chloroplast or mitochondrial genome. The objective of this study was to search for a candidate gene(s) that could be involved in maternally inherited variegation in the chloroplast genome. Chloroplast DNA of the two variegated cultivars was extracted and sequenced by the PacBio Sequel IIe System and compared to a published chloroplast genome of a non-variegated cultivar C. canadensis ‘Forest Pansy’ to identify candidate genes. Polymorphisms were identified using the variant detector FreeBayes and visualized with IGV to determine their location in the genome. One SNP leading to an amino acid change was found at amino acid position 236 of the NADH dehydrogenase subunit H (NCBI: YP_009734414.1). Because of this gene’s involvement with photosystem I, this mutation was determined to be the best potential candidate gene for the ‘Floating Clouds’ variegated leaf trait.

Peanuts, Arachis hypogaea L., are a widely cultivated crop in the US, and wild species, Arachis spp., have been known to contain a wide diversity of functional genes. However, the major obstacle preventing introgression of those genes into cultivar germplasm is their difference in ploidy. Wild peanut species are diploids with various genome types (A, B, D, F, G, or K), while the commonly cultivated species is an allotetraploid (AABB). Doubling the diploid wild species genome is the first step to overcoming the hybrid barriers. In this study, four wild species, A. cardenasii (AA), A. correntina (AA), A. diogoi (AA), and A. magna (BB), were used for developing genome doubling methods. In the first part of this research, three mitotic inhibitors, colchicine, oryzalin, or trifluralin, were applied to the shoots of young seedlings by solid agar treatment. The results of this methodology showed that Arachis seedlings are extremely sensitive to colchicine, mildly responsive to oryzalin, and there was no observed response to trifluralin. Oryzalin was determined to be the optimal chemical for solid agar treatment producing two tetraploid and many mixoploid individuals; however, all tested tetraploid seedlings reverted to the diploid state after three months. In the second part of this research, pre-germinated seedlings were immersed in liquid solutions containing colchicine or oryzalin for various periods: six hours, 12 hours, and 24 hours. As a result, the six-hour colchicine treatment on pre-germinated seedlings demonstrated the most success in generating tetraploids, with 4.3% of treated seeds testing as having two tetraploid results. This protocol is recommended as an optimal protocol for wild peanut species genome doubling.


July 25, 2023
9:00 am - 10:00 am
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Rachel McLaughlin
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121 Kilgore Hall
2721 Founders Drive
Raleigh, NC 27606 United States
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