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Genomic Signature for Stem Swollen of Kohlrabi Morphotype in Brassica oleracea
Plant Breed. Biotech. 2021;9:45-54
Published online March 1, 2021
© 2021 Korean Society of Breeding Science.

Hyunjin Koo1, Hyeonah Shim1, Sampath Perumal2, Ho Jun Joh1, Tae-Jin Yang1*

1Department of Agriculture, Forestry and Bioresources, Plant Genomics & Breeding Institute, College of Agriculture & Life Sciences, Seoul National University, Seoul 08826, Korea
2Agriculture and Agri-Food Canada, Saskatoon, SK S7N 0X2, Canada
Corresponding author: Tae-Jin Yang, tjyang@snu.ac.kr, Tel: +82-2-880-4547, Fax: +82-2-873-2056
Received November 3, 2020; Revised November 21, 2020; Accepted January 12, 2021.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Brassica oleracea contains various morphotypes within the species, but genomic signatures differentiating each morphotype have been poorly understood of. Here, we utilized whole genome sequence data of 44 B. oleracea collections including those of seven different morphotypes such as cabbage, broccoli, cauliflower, kailan, kale, brussels sprout, and kohlrabi to elucidate the genomic signature of B. oleracea morphotypes. Molecular structure analysis divided the 44 B. oleracea lines into two groups: group I represents broccoli, cauliflower, kailan; group II represents other B. oleracea subspecies. Kohlrabi has admixed genomic structure through genetic admixture analysis. Based on the population stratification result, we have investigated genetic signatures that offer the possible evolutionary processes for the kohlrabi morphotype. Several statistical analyses were implemented to identify selective regions and explore 45 candidate loci that may contribute to stem swollen in kohlrabi. Above all, we identified two kohlrabi-unique genes, LOC106333915 and LOC106308097, showing kohlrabi-unique non-synonymous mutations, which might be candidate genes for stem swollen in kohlrabi.
Keywords : Brassica oleracea, Comparative analysis, Morphotype, Selective signature, Whole genome sequencing
INTRODUCTION

Brassica oleracea (CC) is a member of three diploid Brassica species from the classical triangle of U that includes Brassica rapa (AA) and Brassica nigra (BB), and three allotetraploids Brassica juncea (AABB), Brassica napus (AACC), and Brassica carinata (BBCC). B. oleracea species are economically important crops for their contribution to the human food supply. (Nagaharu et al. 1935; Cheng et al. 2014; Liu et al. 2014). Most B. oleracea have high levels of carotene, tocopherol, vitamin and ascorbate that function as protective antioxidants (Packer et al. 1979; Liebler and McClure 1996; Kurilich et al. 1999; Kopsell and Kopsell 2006). Moreover, Glucosinolates (GSLs), found within B. oleracea, are well known as the major class of secondary metabolites with beneficial effects on human health such as preventing cancer (Sotelo et al. 2014). It is interesting to note that despite being the same species, members of B. oleracea display extremely wide ranges of distinctive phenotypes that are shown in cabbage, cauliflower, brussels sprout, kailan, kale, broccoli, and kohlrabi (Kennard et al. 1994). Such phenotypes later developed into distinct traits in B. oleracea species shown as leaf bud in cabbage, flower buds in broccoli and cauliflower, and tuberous stems in kohlrabi (Björkman and Pearson 1998; Maggioni et al. 2010).

Discovery on selection signature offers information about the evolutionary processes related to morphotype and functional roles. Previous studies were conducted to unravel the genomic diversity assumed to be affecting the trait (Cheng et al. 2019; Hou et al. 2019). We focused on the stem swollen trait in kohlrabi by comparing to other B. oleracea subspecies. Crops with tuberous stems such as asparagus, lettuce, mustard and potato, are morphologically diverse. Tubers are rich in antioxidants, minerals, essential amino acids, and in particular, many types of vitamins. Although a wide range of studies on the genes controlling tuber formation have been conducted, there is still a lack of information on genetic features affecting the stem swelling trait (Xu et al. 2008; Abelenda et al. 2011). Kohlrabi, an edible food source known as a cold weather plant, has tuberous stems (Shams 2012). Previously, Cheng et al. (2016) conducted morphotype domestication study with tuber formation on B. rapa turnips and B. oleracea kohlrabies (Cheng et al. 2016). They have discovered some loci under selection in turnips and kohlrabies, revealing sub-genome parallel selection and convergent domestication (Cheng et al. 2016). Unlike the previous study by Cheng et al. (2016), in which signals were detected by conducting case and control studies, our aim is to identify the signal based on the result divided by population stratification. Whole genome scan of B. oleracea may assist in unique discovery on the genetic features affecting stem swollen in kohlrabi.

In this study, we focused on investigating positively selected genes for tuberous stem development in B. oleracea. We performed several statistical analyses to explore selective signature related to stem swollen in kohlrabi using 44 B. oleracea genotyping data.

MATERIALS AND METHODS

Re-sequencing and variant calling

We used whole genome sequence data of 44 B. oleracea accessions (Perumal et al. 2020). Raw paired-end (PE) sequences were filtered based on the criteria for sequence quality and length using Trimmomatic-0.33. Filtered reads were aligned to the reference sequences of B. oleracea TO1000 (Parkin et al. 2014) using Bowtie-2.2.5. Read grouping and filtering of potential PCR duplicates were done using Picard (http://picard.sourceforge.net). Local realignment was conducted to correct misalignments caused by the presence of indels using Genome Analysis Toolkit (GATK) ‘RealignerTargetCreator’ and ‘IndelRealigner’ (McKenna et al. 2010). Finally, ‘HaplotypeCaller’ and ‘SelectVariants’ arguments of GATK were used for SNP calling. To avoid possibilities of false positives and filter variants, ‘VariantFiltration’ argument was used to reduce false positive calls with the following options: (1) a phred-scaled quality score < 30; (2) SNPs with variant call confidence (QUAL) < 30; and (3) SNPs with strand bias score from Fisher’s Exact test (FS) > 200. A total of 5,668,402 raw SNPs were collected for inspection. SNPs with call rates of < 90% and minor allele frequency (MAF) of < 0.05 were filtered using PLINK (Purcell et al. 2007). Blastall was utilized to remove SNPs that are located in paralogous sequences. Following the variant filtering process, missing alleles were imputed using BEAGLE with default parameters (Browning and Browning 2016).

Phylogenetic analysis and population stratification

Phylogenetic analysis was performed by SNPhylo software using the bootstrap value of 1000 (Lee et al. 2014b). ADMIXTURE program was used to conduct population stratification analysis (Alexander et al. 2009). The population structure analysis was performed with 1,110,638 markers for K values from 1 to 7. We calculated eigenvectors which indicate the proportion of variance explained by SNPs using Genome-wide Complex Trait Analysis (GCTA) for principal component analysis (PCA) (Yang et al. 2011).

Identification of kohlrabi-specific enriched variants

SNP annotations of all B. oleracea were obtained using the SnpSift and SnpEff software (Cingolani et al. 2012; Husson et al. 2018). In SnpSift, we conducted Cochran–Armitage trend test and Fisher’s exact test, which are implemented for analyzing variants count data. Bonferroni correction was used to correct the multiple testing errors, according to the method previously introduced. We focused on SNPs located in the coding region of genes, excluding variants located in non-coding regions. Furthermore, synonymous SNPs which are not likely to affect protein function were filtered out. To identify genetic variants related to the changes in the protein structure and the loss of function, we used non-synonymous variants with high or moderate impact. At first, we tried to identify kohlrabi-specific genes based on the existence of mutually exclusive non-synonymous SNPs present in kohlrabi cultivars at orthologous regions compared to other B. oleracea cultivars. However, this method was too strict to identify the mutually exclusive non-synonymous variants. Therefore, we selected non-synonymous mutations in four or more of the five kohlrabi cultivars with lower than original criteria.

Statistics to detect selective sweep regions in kohlrabi

To infer positive signatures related to stem swollen in kohlrabi, we employed three methods. First, the XP-EHH method, which calculates cross-population extended haplotype homozygosity, was used to identify selective sweeps using XPEHH software (Sabeti et al. 2007). This method detects haplotypes which have escalated in frequency to the spot of complete fixation. We divided the genome into non-overlapping segments of 10-kb windows and selected the maximum XP-EHH score in each segment to represent each window. In order to determine the empirical P-value, genomic windows were binned in increments of 500 SNPs. When a window contains more than 1000 SNPs, we merged all the windows into one bin. We determined an empirical P-value for each window following the method of previous studies (Granka et al. 2012; Kim et al. 2017). All regions with P-values less than 0.01 were considered as positively significant regions in kohlrabi cultivars. We additionally conducted CLR statistics, which measures selective sweeps involved in modeling the multi-locus allele frequency differentiation between two populations, with non-overlapping sliding windows of 10 kb and the maximum number of SNPs within each window as 600 using the XP-CLR software package (Chen et al. 2010). The regions with the XP-CLR values in the top 1% of the empirical distribution were considered as candidate sweep regions (Lee et al. 2014a). In order to validate the positive signature regions using the above two statistics-based methods, we conducted Tajima’s D analysis using VCFtools v0.1.13 (Danecek et al. 2011; Korneliussen et al. 2013). Tajima’s D statistics is used to identify positively signature regions going to fixation, which results in a negative value of Tajima’s D. For our purposes, we calculated the Tajima’s D values using a window size of 10 kb.

Characterization of candidate genes under selection

Gene ontology (GO) analysis was carried out to understand the biological functions using the Blast2GO tool (Conesa et al. 2005). First, the genes were annotated against the non-redundant protein database downloaded from NCBI with an E-value threshold of 10-3 using local BLASTX. Using the annotation information, GO terms in three categories (biological process, cellular component, and molecular function) were identified via Blast2GO. Visualization of the integration of GO terms were conducted using the ClueGo plugin of Cytoscape v3.4.0 (Bindea et al. 2009).

RESULTS

Sequencing and identification of SNPs

All high-quality genome sequence data of 44 B. oleracea accessions were genotyped based on the reference sequence of B. oleracea TO1000 (Parkin et al. 2014; Perumal et al. 2020). Most of the SNPs (68%) were located in 5-kb upstream and downstream regions of genes while the remaining SNPs were present in intergenic regions (10.9%), exon (15%) and intron (4.06%) regions.

Phylogenetic analysis and population stratification

In the phylogenetic analysis, the general structure of the tree was categorized into two groups: one group - broccoli, cauliflower, kailan; the others – kohlrabi, cabbage, kale and brussels sprout (Supplementary Fig. S1a). We conducted population structure analysis with the genotyping data to investigate population stratification before deciphering selective signal. Population stratification analysis was conducted through admixture analysis with the number of ancestral populations ranging from 1 to 7 on the entire dataset using filtered variants. The admixture of each subspecies following the analysis with the ancestral population of two is presented in Fig. 1a. As shown in the figure, kohlrabi samples have highly admixture genetic structure. Moreover, we have found the minimum cross-validation (CV) error at K = 3, suggesting that the population can be divided into three clusters (Supplementary Fig. S2). Fig. 1b represents the admixture of cultivars, assuming three ancestral population (K = 3). We could categorize the population into group I, group II, and kohlrabi as K. To further understand relationship of admixture, we performed PCA analysis using the genotype data. The first eigenvector divided group I from group II, and kohlrabi and group II were separated by the second eigenvector (Supplementary Fig. S1b).

Figure 1. The population structure of 44 B. oleracea accessions when the number of assumed ancestral population of (a) K = 2 and (b) K = 3.

Selective signature in the kohlrabi

To identify the signature of positive selection in kohlrabi against other groups, we used three statistical methods in order to obtain more powerful results than the results obtained from the test alone (XP-CLR, XP-EHH and Tajima’s D). First, we conducted XP-CLR analysis to identify the genomic regions where the alterations in allele frequency at the locus appeared very quickly between two populations. All windows above the top 1% of the empirical distribution were considered as significant regions. 445 positively selected genes were observed in both tests between kohlrabi against group I and group II (Fig. 2a). Next, we used the XP-EHH statistic to compare kohlrabi with group I and group II that resulted in 443 and 441 genes detected as advantageous selected signatures, respectively (Fig. 2b). Lastly, in order to confirm the selective signature within subspecies, Tajima’s D analysis was performed. The regions detected as negative value through this analysis implies a significant signal from neutrality and reveals the selective state of alleles within the kohlrabi population compared to group I and group II counterparts. We selected candidate regions from detected signals obtained through more than two statistical methods from the comparison of K-group I and K-group II. Finally, we detected 45 candidate genes cross-validated from the comparison between K and group I with the comparison between K and group II (Supplementary Table S1). Among the 45 putative genes, 12 were not characterized in terms of their functions. Among the other 33 genes, LOC106303849 and LOC106317031 genes were reported to take part in the regulation of cell elongation and expression levels of tuberization, respectively (Roumeliotis et al. 2013; Zhang et al. 2017).

Figure 2. Manhattan scatter plot for stem swollen traits using two statistical analyses. Asterisk indicates missense variant among the positive selection genes. (a) Results of XP-CLR analyses and (b) Results of XP-EHH with kohlrabi against group I or group II for detection of positive selection signature.

Two kohlrabi-unique candidate genes for stem swollen

In order to detect kohlrabi-specific genes, we conducted a variant enrichment analysis. Among the missense variants, six SNPs were co-located within two selective sweep genes, LOC106333915 and LOC106308097 (Fig. 3). LOC106333915 and LOC106308097 were encoded as polyribonucleotide nucleotidyltransferase 2 and OTU domain-containing protein, respectively. LOC106333915 had five kohlrabi specific mutations. The first variant was a non-synonymous mutation (p.Asp396Asn) in exon 2. The second missense mutation (p.Pro287Leu) was observed in exon 4 and was found to be mutually exclusive in kohlrabi. The third and fourth variants located in exon 5 were non-synonymous mutations p.Ser248Asn and p.Thr235Ala, respectively. The fifth and last variant found in this gene was a missense mutation (p.Glu197Lys) in exon 7. In LOC106308097, a missense variation (p.Met86Val) in the second exon was a mutually exclusive variant in kohlrabi compared to the others (Fig. 3, Supplementary Fig. S3).

Figure 3. Kohlrabi-specific candidate regions detected by variant analysis. The allele type of missense variants of kohlrabi and other subspecies are marked respectively for genes, (a) LOC106333915 and (b) LOC106308097.

Gene Ontology analysis related to candidate genes under selection

To observe how candidate genes function in biological systems, we have additionally conducted GO enrichment analysis based on the common genes which were selected in the selective sweep analysis. The significant hits were discovered based on the E-valued threshold as 10-3 (Fig. 4). Details on the functions obtained in this study are shown in Supplementary Table S2. As a result of GO analysis, we discovered that the genes under selective signature were associated with various GO terms. Among various biological process functions including oxidation-reduction process, protein phosphorylation, DNA repair, transmembrane transport and response to oxidative stress were observed to be over-represented. We could also detect the molecular functions including ATP binding, protein kinase activity, protein binding, heme binding, nucleic acid binding, zinc ion binding, catalytic activity and DNA binding. As cellular component functions, integral to membrane and membrane were identified in high frequency. Especially, LOC106303849 is involved in drug transmembrane transport, drug transmembrane transporter activity, antiporter activity, as well as membrane. LOC106317031 was assigned to integral to membrane and transmembrane transport functions. Five terms (oxidoreductase activity acting on paired donors with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water, oxidation-reduction process, protein kinase activity, protein phosphorylation, and ATP binding) are associated with LOC106308097.

Figure 4. Gene ontology analysis of 33 positively selected genes related to stem swollen in B. oleracea population. The most prominent gene ontology term for each group is highlighted in colors, and the gene ontology terms marked in circles are related to stem swollen characteristics.
DISCUSSION

Population stratification in B. oleracea

To observe the ancestral history of the population, we performed phylogenetic analysis based on the whole genome data. Our results indicated that there are two large structures in B. oleracea: group I – broccoli, cauliflower and kailan, and group II – kohlrabi, cabbage, brussels sprout, and kale. As we have seen in the previous phylogenetic analysis, kohlrabi and other B. oleracea did not correctly divide into two groups; therefore, prior to carrying out statistics analyses, we took more consideration on the genetic admixture pattern within the population. In this study, we conducted population structure analysis and PCA analysis. As a result of these analyses, we could observe the two distinct groups with kohlrabi having admixed genetic structure. The genetic structure of kohlrabi was observed to be in an intermediate position between group I and group II.

Positive selection signature in kohlrabi

We performed selective sweep analysis in kohlrabi compared to group I and group II. Overall, 33 candidate overlapping genes were identified as positively selected in kohlrabi when compared to other subspecies. In addition, we also discovered 12 genic regions that underwent selection which were not functionally characterized. Although there has been no experimental validation process, the powerful signal of positive selection was detected near these genes. As mentioned above in the result section, LOC106303849 and LOC106317031 were described as the regulator gene for cell elongation and tuberization expression levels, respectively. Based on GO analysis, we could detect GO terms related to the candidate genes located in the selective sweep region. Among various GO terms, integral to membrane and trans-membrane transport assigned to LOC106317031 were observed. This is important to note as such function was also elucidated in another tuber plant: potato. Trans-membrane domains are highly enriched in potato, PIN proteins which arbitrate auxin efflux during plant development. Several reports highlighted that auxin affects development of tuber and swelling stolon in potato (Kloosterman et al. 2008; Roumeliotis et al. 2013). Our results suggest that these genes may become an evidence of selection for stem swollen in kohlrabi which tend to have its own tuberization phenomenon not seen in other sub-species.

To provide more support on our hypothesis, we investigated genes with the existence of missense variants leading to change in the amino acid sequence specifically found in kohlrabi. Initially, we annotated the variants located in the overlapping genes to retain the impact and the additional information using SnpEff software. As a result of this analysis, we observed six non-synonymous variants located in two genes (LOC106333915 and LOC106308097) encoding polyribonucleotide nucleotidyltransferase and OTU domain-containing protein, respectively. According to the previous study, OTU domain protein has been shown to have multiple roles in controlling the activity of deubiquitination. In the expression analysis in Arabidopsis, OTU domain proteins were involved in hormone signaling, cell expansion and plant growth through histone modification. This independent study elucidated that OTU domain protein has a major role in cell proliferation (Granka et al. 2012; Isono and Nagel 2014; March and Farrona 2018). Cell proliferation and expansion have been reported to have a crucial role in stem development (Maeda et al. 2014). Furthermore, potato is a well-known tuber plant, with its tuber maturation stage being identified with lots of cell divisions and expansions (Xu et al. 1998). Cheng et al. (2016) also found a gene family related to functions such as transport, expansion, root development and cell division, which is predicted to be contributing to convergent domestication of kohlrabi tuber formation (Cheng et al. 2016). These gene families are strongly consistent with the functional role of our candidate genes. Moreover, we could discover five GO terms associated with OTU domain protein. Especially, “protein kinase activity” and “protein phosphorylation” were known as a key regulatory factor in the accumulation of storage protein in potato and innate to inner membrane of potato tuber mitochondria as mentioned in previous studies (Struglics et al. 1999; Struglics et al. 2000; Bernal et al. 2019). Based on these findings, we could suggest that protein interactions with the OTU domain protein might also have effect on the selection for tuber development of kohlrabi distinct from other B. oleracea subspecies. Although selective sweep signal near the polyribonucleotide nucleotidyltransferase encoding gene has many non-synonymous mutations, it was difficult to figure out the critical role in tuber formation. In order to support this study, it is necessary to functionally validate these results through further study. From this study, we have tried to detect the specific genetic signature in the 2-kb flanking region of the genes that underwent selective sweep in order to figure out the factors that could influence gene regulation. However, we were unable to detect genetic signatures, which calls for further investigation.

In this study, we conducted several genomic studies using 44 accessions of B. oleracea which includes seven different morphotypes. Based on genetic variations, we have inferred the phylogenetic relationship of the 44 accessions. Furthermore, our analysis identified several candidate genes that are associated with stem swollen in kohlrabi through exploring the signal of positive selection. This study might have contributed to the deep under-standing of how genetic variation affects phenotypic diversity.

Supplementary Materials
pbb-9-1-45-supple.zip
ACKNOWLEDGEMENTS

This research was supported by “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01311901)”, Rural Development Administration, Republic of Korea.

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