Methods of analysis of chloroplast genomes of C3, Kranz type C4 and Single Cell C4 photosynthetic members of Chenopodiaceae

Richard M Sharpe; Bruce Williamson-Benavides; Gerald E Edwards; Amit Dhingra

doi:10.1186/s13007-020-00662-w

Methods of analysis of chloroplast genomes of C₃, Kranz type C₄ and Single Cell C₄ photosynthetic members of Chenopodiaceae

Plant Methods. 2020 Aug 31:16:119. doi: 10.1186/s13007-020-00662-w. eCollection 2020.

Authors

Richard M Sharpe^#¹, Bruce Williamson-Benavides^#^{1

2}, Gerald E Edwards^{2

3}, Amit Dhingra^{1

2}

Affiliations

¹ Department of Horticulture, Washington State University, Pullman, WA 99164 USA.
² Molecular Plants Sciences, Washington State University, Pullman, WA 99164 USA.
³ School of Biological Sciences, Washington State University, Pullman, WA 99164 USA.

^# Contributed equally.

Abstract

Background: Chloroplast genome information is critical to understanding forms of photosynthesis in the plant kingdom. During the evolutionary process, plants have developed different photosynthetic strategies that are accompanied by complementary biochemical and anatomical features. Members of family Chenopodiaceae have species with C₃ photosynthesis, and variations of C₄ photosynthesis in which photorespiration is reduced by concentrating CO₂ around Rubisco through dual coordinated functioning of dimorphic chloroplasts. Among dicots, the family has the largest number of C₄ species, and greatest structural and biochemical diversity in forms of C₄ including the canonical dual-cell Kranz anatomy, and the recently identified single cell C₄ with the presence of dimorphic chloroplasts separated by a vacuole. This is the first comparative analysis of chloroplast genomes in species representative of photosynthetic types in the family.

Results: Methodology with high throughput sequencing complemented with Sanger sequencing of selected loci provided high quality and complete chloroplast genomes of seven species in the family and one species in the closely related Amaranthaceae family, representing C₃, Kranz type C₄ and single cell C₄ (SSC₄) photosynthesis six of the eight chloroplast genomes are new, while two are improved versions of previously published genomes. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality and repeat region sequences. Comparison of the chloroplast genomes with previously sequenced plastid genomes revealed similar genome organization, gene order and content with a few revisions. High-quality complete chloroplast genome sequences resulted in correcting the orientation the LSC region of the published Bienertia sinuspersici chloroplast genome, identification of stop codons in the rpl23 gene in B. sinuspersici and B. cycloptera, and identifying an instance of IR expansion in the Haloxylon ammodendron inverted repeat sequence. The rare observation of a mitochondria-to-chloroplast inter-organellar gene transfer event was identified in family Chenopodiaceae.

Conclusions: This study reports complete chloroplast genomes from seven Chenopodiaceae and one Amaranthaceae species. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality, and repeat region sequences. Therefore, the use of high throughput and Sanger sequencing, in a hybrid method, reaffirms to be rapid, efficient, and reliable for chloroplast genome sequencing.