Complete chloroplast genome of a novel chlorophyll-deficient mutant (clm) in sweetpotato (Ipomoea batatas L.)

Abstract The complete chloroplast genome of a novel chlorophyll-deficient mutant (clm) and its wild type (WT) in sweetpotato (Ipomoea batatas L.) was sequenced. The complete chloroplast genome of clm and WT was 161,393 bp and 161,429 bp in length, containing a large single copy (LSC) region of 87,561 bp and 87,597 bp, respectively, a small single copy (SSC) region with the same length of 30,890 bp and a pair of inverted repeat regions (IRs) with the same length of 12,052 bp. Both of them contained 132 genes including 87 protein-coding sequences, 37 tRNA, and eight rRNA. Comparing to the WT, four SNPs and three INDELs were detected and only one INDEL in the exon affecting the translation of rpoA gene. Phylogenetic analysis showed that clm and WT were closely related to Ipomoea tabascana. The complete chloroplast genome of clm and its WT will play a role in understanding the molecular mechanism of chlorophyll deficiency and developing molecular markers in sweetpotato.

The sweetpotato (Ipomoea batatas L.) is generally vegetatively propagated by means of roots or cuttings. The sports (vegetative mutations) are fairly universal in sweetpotato cultivation (Gustafsson and Gadd 2009). As one kind of common mutations in sweetpotato, although the chlorophyll-deficient mutant was found as early as 1952 (Kuwata 1950), its physiological and molecular mechanisms have not been studied in detail. The chlorophyll-deficient mutant is a valuable material for research in chlorophyll biosynthesis, chloroplast development and structure function, photosynthesis mechanism, gene function, as well as ornamental plant breeding (Frank and Chitwood 2016). Here, we first sequenced and characterized the complete chloroplast genome of a novel chlorophyll-deficient mutant (clm) and its wild type (WT) in sweetpotato (Ipomoea batatas L.), and analyzed their phylogenetic relationship in Ipomoea genus.
The fresh leaves of clm and WT were collected in Guangzhou, Guangdong Province, China (N23 39 0 41.36 00 ; E113 44 0 04.18 00 ). Voucher specimens and DNA were deposited in the Crops Research Institute, Guangdong Academy of Agricultural Sciences (voucher number: CRI20191024). The total genomic DNA of clm and WT was extracted using the Plant Genomic DNA Kit (Tiangen, Beijing, China). The whole genome sequencings were conducted by Nanjing Genepioneer Biotechnologies Inc. (Nanjing, China) using Illumina Hiseq 2500 system. The raw reads were filtered by Trimmomatic v0.32 (Bolger et al. 2014) and mapped to Xushu 18 chloroplast genome (Yan et al. 2015) as the reference by Bowtie2 v2.35 (Langmead and Salzberg 2012). The obtained high-quality chloroplast reads were assembled in chloroplast genome by SPAdes v3.10 (Bankevich et al. 2012). The final annotations were performed using GeSeq (Tillich et al. 2017) and Blast search.
The complete chloroplast genome of clm (accession number MW122506) and WT (accession number MW122507) was 161,393 bp and 161,429 bp in length, containing a large single copy (LSC) region of 87,561 bp and 87,597 bp, respectively. Both of them had a small single copy (SSC) region with the same length of 30,890 bp and a pair of inverted repeat regions (IRs) with the same length of 12,052 bp. The annotation results showed that both the chloroplast genome of clm and WT contained 132 genes, including 87 mRNA, 37 tRNA, and eight rRNA. The structure variation between clm and WT chloroplast genome was screened by MAFFT v7.450 (Katoh and Standley 2013) and perl script. Comparing to WT, four SNPs in ycf1 gene and three INDELs in rpoA gene were identified in clm. But there was only one INDEL variation affecting gene expression. The rpoA protein was shortened by the alternation in exon locus of rpoA gene, which may lead to the phenotype of chlorophyll-deficient.
To figure out the phylogenetic status of clm and its WT in Ipomoea genus, the maximum-likelihood tree was constructed based on eight complete chloroplast genomes of Ipomoea species from GenBank, and Cressa cretica and Cuscuta reflexa were used as the outgroup. These 12 whole chloroplast genomes were aligned using MAFFT v7.450 (Katoh and Standley 2013) and the maximum-likelihood tree was constructed using RAxML v8.2.10 (Stamatakis 2014) with 1000 bootstrap value (Figure 1). The result of phylogenetic analysis showed that the clm mutant and its WT were clustered together, and close to Ipomoea tabascana. It was further confirmed that I. tabascana could be a recent hybrid between I. batatas and I. trifida, and there were two I. batatas lineages due to chloroplast capture (Muñoz-Rodr ıguez et al. 2018). The complete chloroplast genome of clm and its WT will play an important role in understanding the molecular mechanism of chlorophyll deficiency and developing molecular markers in sweetpotato.

Disclosure statement
No potential conflict of interest was reported by the author(s).