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Article

Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.)

by
Yongfu Tao
1,
Barbara George-Jaeggli
1,2,
Marie Bouteillé-Pallas
1,
Shuaishuai Tai
3,
Alan Cruickshank
2,
David Jordan
1 and
Emma Mace
1,2,*
1
Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Hermitage Research Facility, Warwick, QLD 4370, Australia
2
Agri-Science Queensland, Department of Agriculture and Fisheries (DAF), Hermitage Research Facility, Warwick, QLD 4370, Australia
3
BGI Genomics, BGI-Shenzhen, Shenzhen 518083, China
*
Author to whom correspondence should be addressed.
Genes 2020, 11(7), 806; https://0-doi-org.brum.beds.ac.uk/10.3390/genes11070806
Submission received: 24 June 2020 / Revised: 9 July 2020 / Accepted: 13 July 2020 / Published: 16 July 2020
(This article belongs to the Section Plant Genetics and Genomics)
Browse Figures
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Abstract

:
C4 photosynthesis has evolved in over 60 different plant taxa and is an excellent example of convergent evolution. Plants using the C4 photosynthetic pathway have an efficiency advantage, particularly in hot and dry environments. They account for 23% of global primary production and include some of our most productive cereals. While previous genetic studies comparing phylogenetically related C3 and C4 species have elucidated the genetic diversity underpinning the C4 photosynthetic pathway, no previous studies have described the genetic diversity of the genes involved in this pathway within a C4 crop species. Enhanced understanding of the allelic diversity and selection signatures of genes in this pathway may present opportunities to improve photosynthetic efficiency, and ultimately yield, by exploiting natural variation. Here, we present the first genetic diversity survey of 8 known C4 gene families in an important C4 crop, Sorghum bicolor (L.) Moench, using sequence data of 48 genotypes covering wild and domesticated sorghum accessions. Average nucleotide diversity of C4 gene families varied more than 20-fold from the NADP-malate dehydrogenase (MDH) gene family (θπ = 0.2 × 10−3) to the pyruvate orthophosphate dikinase (PPDK) gene family (θπ = 5.21 × 10−3). Genetic diversity of C4 genes was reduced by 22.43% in cultivated sorghum compared to wild and weedy sorghum, indicating that the group of wild and weedy sorghum may constitute an untapped reservoir for alleles related to the C4 photosynthetic pathway. A SNP-level analysis identified purifying selection signals on C4 PPDK and carbonic anhydrase (CA) genes, and balancing selection signals on C4 PPDK-regulatory protein (RP) and phosphoenolpyruvate carboxylase (PEPC) genes. Allelic distribution of these C4 genes was consistent with selection signals detected. A better understanding of the genetic diversity of C4 pathway in sorghum paves the way for mining the natural allelic variation for the improvement of photosynthesis.

1. Introduction

C4 photosynthesis has independently evolved in more than 60 different plant taxa [1]. The main driver for this convergent evolution is the tendency of Ribulose-1,5-bisphosphate carboxylase (Rubisco), which catalyzes the net fixation of carbon dioxide (CO2) to also catalyze an unfavorable oxygenation reaction. This reaction produces toxic phosphoglycolate which has to be converted to useful metabolites requiring substantial metabolic energy [2,3]. This wasteful use of CO2 is termed photorespiration. Photorespiration becomes a major constraint of photosynthesis in situations where CO2 to O2 ratios are low and temperatures are high. The evolution of C4 photosynthesis coincided with declining atmospheric CO2 concentrations [1,4] as a mechanism to avoid photorespiration by concentrating CO2 in the vicinity of ribulose-1,5-bisphosphate carboxylase (Rubisco).
In the majority of C4 plants, this is achieved via spatial separation of the initial CO2 fixation and the Calvin–Benson–Bassham cycle in two different cell types, most often mesophyll cells and bundle sheath cells [5]. CO2 concentration in C4 bundle sheath cells is up to 10-fold higher than that found in C3 mesophyll cells [6]. At higher temperatures, C4 photosynthesis is not only more efficient compared with C3 photosynthesis in terms of reducing energy losses from photorespiration, but due to the improved efficiency of this pathway, it renders plants more nitrogen- and water-use efficient [7,8]. C4 plants are more productive than C3 plants in areas with high light intensities, warm temperatures, and low rainfall, such as the sub-tropical and tropical areas around the globe.
Many of the major crops that originated from warm and dry regions of the world, such as maize, sorghum, millet, sugarcane, miscanthus, and switchgrass, use the C4 pathway [9]. C4 crops account for an estimated 23% of global primary production [10]. Improved photosynthetic capacity has been suggested as the next frontier in lifting crop productivity [11]. The C4 photosynthesis pathway is a good starting point to improve photosynthetic capacity and resource efficiency in crop plants. Attempts are currently being undertaken to integrate characteristics of the C4 pathway into C3 crops [7,12,13,14].
However, possibly due to multiple independent evolutions of C4 photosynthesis in different plant taxa [1], large variation also exists among C4 species in terms of the biochemical pathway. It has long been known that three major biochemical subtypes—nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME), nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) and phospho-enol-pyruvate carboxykinase (PCK)—exist among C4 species [15]. More recently, it has been suggested that mixtures among them exist [16] and that the subtypes vary in their performance under different environmental conditions, e.g., low light [17]. Especially among the grasses, which all of the C4 cereals belong to, differences in pathway and performance are likely to exist, as C4 photosynthesis has evolved at least 25 times in this group of plants [18]. Exploring such variation may provide avenues to further improve C4 photosynthetic efficiency [9].
Sorghum is an NADP-ME subtype C4 crop well-known for its adaption to drought and high temperatures. It provides staple food for over 500 million people in the semi-arid tropics of Africa and Asia; in addition to being an important source of feed, fiber, and biofuel. Due to these characteristics, it is expected to play an increasingly important role in meeting the challenges of feeding the world’s growing population under the threat of global warming. Substantial variation in photosynthesis and related traits has been revealed in sorghum [19,20,21,22,23], indicating existence of genetic variation of underlying genes. However, this variation has not yet been studied.
The recent assembly of whole-genome sequences for a wide range of wild and cultivated sorghum species [24,25,26] provides an excellent opportunity to explore genetic diversity of genes related to the C4 photosynthetic pathway. Several high-throughput comparative transcriptomics and evolutionary studies using C3 and C4 phylogenetically related species and cell-specific gene expression have elucidated the key genes and regulatory networks that underpin the C4 photosynthetic pathway [5,27,28,29,30,31,32,33,34,35,36,37]. In the present study, we explored the genetic variation in genes that have previously been identified as core C4 genes, mined their allelic diversity and investigated signatures of selection during domestication in sorghum.

2. Materials and Methods

Identification of C4 Gene Families

This study focuses on 8 key proteins in the NADP-ME photosynthetic pathway in sorghum (Figure 1). A total of 9 genes encoding these proteins with expression and evolutionary evidence supporting their involvement in NADP-ME pathway (hereafter, referred as C4 genes), and their non-C4 isoforms in sorghum were extracted from two previous studies [38,39] (Table 1). These non-C4 isoforms are homologous of C4 genes but there was no evidences supporting their involvement in the NADP-ME photosynthetic pathway. Homology between these sorghum C4 genes and their non-C4 isoforms was further verified via a local blast strategy. Protein sequences of these 9 core C4 genes were extracted from the sorghum reference genome V3.1 and were blasted against the reference genome. Blast hits of each gene were filtered using the criteria: E-value <−10, sequence identity >60%, and alignment length >80%. All hits of the same gene satisfying the criteria were plotted based on –log (E-value); only hits of top –log (E-value) class were considered if clear differentiation among them was visualized, otherwise all hits were used.

3. Plant Material and Genomic Data

Sequence data of the identified C4 genes were extracted from 48 accessions of Sorghum bicolor with high mapping depth (~22× per accession, ranging from 16 to 45×) reported in previous studies [24,25,26]. These 48 accessions represent all major cultivated sorghum races and some wild progenitors (Table S1).

4. Gene-Level Population Genetic Analyses

Population genetic parameters including nucleotide diversity (θπ) [41], Tajima’s D [42], and Watterson’s Estimator (hW) [43] were directly calculated for each of the 27 genes using the Bio::PopGen::Statistics module. FST [44], which measures population differentiation, was also calculated for each of the 27 genes using the Bio::PopGen::PopStats module [26]. The Bio::PopGen::IO module was used to read input file, which was prepared using an in-house Perl script for calculation of these population genetic parameters.
The criteria used in Mace et al. (2013) were employed to identify genes under purifying selection and balancing selection, respectively. Criteria for purifying selection included: (1) θπ and hW < 5% of the empirical distribution in the cultivated group, (2) FST between the group of cultivated sorghum and the group of wild and weedy sorghum > 95% of the population pairwise distribution, (3) Tajima’s D < 0. Criteria for balancing selection included: (1) θπ and hW > 25% of the empirical distribution in the cultivated group, (2) FST between the group of cultivated sorghum and the group of wild and weedy sorghum < 90% of the population pairwise distribution, (3) Tajima’s D > 5% of the empirical distribution.

5. SNP-Level Identification of Selection Signature

Population genetics parameters including θπ, Tajima’s D, and FST between the group of cultivated sorghum and the group of wild and weedy sorghum were computed for these 27 genes using CDS sequence in PopGenome, a population genomics package implemented in the R environment (http://cran.r-project.org/) [45]. Specifically, commands diversity.stats, F_ST.stats, and neutrality.stats were called to calculate θπ, FST, and Tajima’s D for each single nucleotide polymorphism (SNP), respectively, with a slide window of 1-bp and 1-bp step size. Functional annotation of each SNP was conducted using get.codons command. Fold decrease of θπ in the cultivated sorghum group compared to the group of wild and weedy sorghum was calculated to represent reduction of diversity (RoD). The following criteria were adopted to identify sites with signature of purifying selection: (1) A RoD greater than the average of neutral genes; (2) FST > 0; (3) Tajima’s D < 0. The following criteria were adopted to identify sites with signature of balancing selection: (1) An increase in diversity (IoD) in the cultivated group and the group of wild and weedy comparison; (2) FST > 0; (3) Tajima’s D > 0.

6. Phylogenetic and Haplotype Analysis

A phylogenetic tree was constructed based on CDS of all 27 genes from C4 gene families using the neighbor-joining method with default settings (bootstrapped 100 times; support threshold, 50%) in Geneious 8.1.2 (https://www.geneious.com/, Biomatters Ltd., Auckland, New Zealand). Analysis of haplotype network was conducted using a combination of the R package ape [46] and pegas [47]. All 48 sorghum accessions were classified into four groups: Cultivated, wild and weedy, Guinea margaritiferum and S. propinquum (Table S2).

7. Results

Nucleotide Diversity of Core C4 Gene Families in Sorghum

Based on 9 genes corresponding to 8 core C4 enzymes in sorghum, 18 homologous genes were identified across the sorghum genome. In total, 5 CA genes, 2 NADP-MDH genes, 5 NADP-ME genes, 6 PEPC genes, 3 PPCK genes, 2 PPDK genes, 3 PPDK-RP genes, and 1 rbcS gene were identified (Table 1). Nucleotide diversity (θπ) of these 27 genes was investigated using sequence data of 48 genotypes covering wild and weedy, and cultivated sorghum (Mace et al., 2013). A total number of 4183 single nucleotide polymorphisms (SNPs) were identified in these 27 genes with 521 SNPs located in coding sequence (CDS) regions (Table 1). These C4 gene families displayed an average overall nucleotide diversity of θπ = 2.09 × 10−3, which is comparable to that of 130 housekeeping genes (θπ = 1.97 × 10−3, Mace et al., 2013) (t-test, p-value > 0.05). Nucleotide diversity varied dramatically among the C4 gene families, with the NADP-MDH genes displaying the lowest levels of diversity across all genotypes (average θπ = 0.25 × 10−3), followed by NADP-ME genes (θπ = 0.93 × 10−3), PPCK genes (θπ = 1.20 × 10−3), PEPC genes (θπ = 2.11 × 10−3), CA (θπ = 2.26 × 10−3), and PPDK-RP (θπ = 2.96 × 10−3), while PPDK genes showed the highest level of diversity (θπ = 5.21 × 10−3) (Table 2, Figure 2A). The only gene encoding ribulose bisphosphate carboxylase/oxygenase small-subunit (rbcS), Sobic.005G042000, had relatively high genetic diversity among C4 gene families with θπ = 4.32 × 10−3 across all 48 genotypes, 5.72 × 10−3 in the wild and weedy group, and 3.03 × 10−3 in the cultivated group.
Mixed trends were found when comparing C4 genes with non-C4 isoforms in each gene family with the average overall genetic diversity of C4 genes being comparable to that of their non-C4 counterpart (Table 2). The C4 PPDK-RP gene (Sobic.007G166300) and C4 NADP-MDH gene (Sobic.002G324400) had an overall θπ which was 161.76% and 79.85% higher than their non-C4 isoforms, respectively, whereas the θπ of the C4 PPDK gene (Sobic.009G132900) was 75.16% lower than that of the non-C4 PPDK isoform. Nucleotide diversity of C4 genes in the other gene families was within the range of variation of their non-C4 isoforms.
Genetic diversity across C4 gene families was significantly reduced during sorghum domestication (paired t-test, p-value < 0.05). Averaged across all C4 gene families genetic diversity was reduced by 22.44% in the domesticated compared with the wild and weedy group and when just the 9 core C4 genes were considered, the reduction was 22.98%. However, the reduction of genetic diversity during domestication in C4 genes was not significantly different from that in housekeeping genes (Table S2) (t-test, p-value > 0.05). Among the 27 genes, Sobic.003G292400, a non-C4 NADP-ME isoform, exhibited the most severe reduction in genetic diversity, with a reduction of 98.23%. The C4 version of that gene, the NADP-ME gene (Sobic.003G036200), showed the greatest loss of genetic diversity (51.89%) among the C4 genes, with an FST between the cultivated and wild and weedy groups of 0.06 (Figure 2B). In contrast, another non-C4 isoform of NADP-ME (Sobic.009G069600), a non-C4 isoform of PPCK (Sobic.006G148300), and a non-C4 CA isoform (Sobic.003G234600) showed a more than 2-fold increase in genetic diversity in the cultivated group.

8. Identification of Selection Signals during Domestication across the 27 Genes

The selection signature of these C4 gene families was firstly investigated at the gene level. Based on thresholds of genome-wide rankings described in Mace et al. (2013), only one gene (Sobic.001G326900, non-C4 PPDK isoform) was identified as being under balancing selection, which maintains diversity of selected genes, during sorghum domestication, while no gene was identified as being under purifying selection, which reduces diversity of selected genes (Table 1). Subsequent to this, a higher resolution detection of selection signature was conducted at the SNP level using the CDS of the 27 genes. Among 521 SNPs across 27 CDS, 176 were non-synonymous. The number of non-synonymous SNPs within genes varied from 19 in the non-C4 PPDK-RP isoform (Sobic.002G324700) to 0 in the C4 PPDK (Sobic.009G132900). The C4 PEPC gene (Sobic.010G160700) had the highest number of non-synonymous SNPs (9) among the 9 C4 genes (Table 1). In contrast to the gene-level analysis, SNP-level analysis identified 24 SNPs across 8 genes under purifying selection, including 7 non-synonymous SNPs in 6 genes (Table S3). Genes with SNPs under purifying selection included two C4 isoforms, PPDK (Sobic.009G132900) and CA (Sobic.003G234200), three of 4 non-C4 NADP-ME (Sobic.003G280900, Sobic.003G292400, Sobic.009G069600), both two non-C4 PPDK-RP (Sobic.002G324500, Sobic.002G324700), and a non-C4 PEPC gene (Sobic.007G106500). Among the 2 C4 genes with SNPs under selection, Sobic.009G132900 had 3 synonymous SNPs under purifying selection, while Sobic.003G234200 had a non-synonymous SNP under purifying selection.
A total of 60 SNPs across 8 genes were identified as being under balancing selection, 7 of which were non-synonymous SNPs distributed across 2 genes (Table S4). The non-C4 PPDK (Sobic.001G326900) had 24 SNPs under balancing selection including 5 non-synonymous SNPs, and additionally had an overall gene-level signature of balancing selection based on the previous analysis. Two C4 isoforms, PPDK-RP (Sobic.002G324400) and PEPC (Sobic.010G160700), were identified with 3 and 2 SNPs under balancing selection, respectively, although none of them were non-synonymous SNPs. Two non-C4 PEPC (Sobic.003G100600, Sobic.004G106900) were identified with SNPs under balancing selection, with Sobic.003G100600 having 21 SNPs including 2 non-synonymous SNPs exhibiting signatures of balancing selection. The other 2 genes with SNPs under balancing selection were a non-C4 CA isoform, Sobic.002G230100, and a non-C4 PPCK isoform, Sobic.004G219900.

9. Allelic Variation of Core C4 Genes under Selection in Sorghum

A phylogenetic tree was constructed using the CDS of these 27 genes to depict the genetic relationship of 48 accessions (Figure S1). The inter-and intra-species distribution of private haplotypes of each gene is detailed in Table S5, with the majority (~90%) of the genes with private inter-species haplotypes from S. propinquum, e.g., 4 unique haplotypes were observed for the C4 isoform of PEPC, with the 2 S. propinquum accessions sharing a single private haplotype. To investigate allelic variation of 4 core C4 genes with SNPs under selection in sorghum, haplotype networks were constructed using CDS SNPs. Based on 16 SNPs within the CDS of the PPDK gene (Sobic.009G132900), 8 haplotypes were identified. Five haplotypes were identified in the wild and weedy genotypes, with 3 being private haplotypes and two of them being maintained in cultivated sorghum; two new haplotypes arose in cultivated sorghum after domestication (Figure 3A). Ten haplotypes of one CA gene (Sobic.003G234200) were revealed using 33 SNPs, with 4 distinct haplotypes being characterized by the wild and weedy genotypes. Two of the wild and weedy haplotypes were maintained in cultivated sorghum during domestication, with three new haplotypes arising after domestication (Figure 3B). The loss of wild and weedy haplotypes in cultivated sorghum in these two genes was consistent with the finding that they were under purifying selection.
The PPDK-RP gene (Sobic.002G324400) had 22 SNPs in the CDS, based on which 5 haplotypes were identified. Two haplotypes were characterized by the wild and weedy genotypes, with the main wild haplotype maintained and further diversifying into two new haplotypes in the cultivated group (Figure 3C). Based on 28 SNPs in the CDS of the C4 PEPC gene (Sobic.010G160700), 4 haplotypes were identified. Wild and weedy genotypes encompassed 3 haplotypes and all of them were maintained in cultivated sorghum (Figure 3D). S. propinquum had unique haplotypes across all 4 genes, while the Sorghum bicolor race guinea margaritiferum shared haplotypes with the wild and weedy genotypes in most cases, indicating a closer relationship with the wild and weedy group.

10. Discussion

The evolution of C4 photosynthesis has been studied extensively at the cross-species level with signals of adaptive evolution identified on key genes in the C4 pathway [28,34,48,49,50]. As the evolution of C4 photosynthesis is driven by environments characterized by low CO2 availability, such as hot and dry environments in which CO2 uptake is limited by stomatal closure, it is likely that within-species adaptive variation also exists. However, to our knowledge, studies of within-species allele diversity and signatures of selection on key genes in the C4 pathway have not previously been undertaken.
Knowledge of existing natural variation and levels of genetic diversity is a pre-requisite for the optimization of C4 photosynthesis. In this study, we performed the first investigation of the genetic diversity of C4 gene families within a C4 species using a collection of 48 sorghum lines. We focused on 9 C4 genes due to their reported key roles in C4 photosynthesis. Our collection of sorghum represents all major cultivated sorghum races, landraces, and wild progenitors, and captures a good proportion of genetic diversity within sorghum. Substantial variation of nucleotide diversity was observed among these 8 C4 gene families in sorghum, with the NADP-MDH gene family showing the least diversity and the PPDK gene family showing the greatest diversity. Nine core C4 genes also exhibited varying degrees of genetic diversity, ranging from θπ values of 5.04 × 10−3 and 4.32 × 10−3 in PPDK-RP and rbcS to θπ values of 0.33 × 10−3 and 0.67 × 10−3 in NADP-MDH and NADP-ME. However, despite such low levels of diversity, non-synonymous SNPs were identified in both NADP-MDH and NADP-ME (Table 1). C4 PPDK was the only gene which did not contain a non-synonymous SNP, despite its fairly large size (gene size, 12748bp; CDS, 2847bp), indicating the function of this gene is highly conserved.
Cultivated sorghum was domesticated more than five thousand years ago in Africa [51,52,53]. This artificial selection process has morphologically and physiologically reshaped sorghum to better suit human needs, and also resulted in substantial reduction of genetic diversity genome wide in cultivated sorghum compared with wild and weedy types [26,54,55]. In this study, reduction of genetic diversity during sorghum domestication was also observed in the C4 gene families, indicating that wild sorghum, as a repository for genetic diversity, might harbor alleles useful for improving C4 photosynthesis.
However, the overall reduction in diversity of C4 gene families was not significantly different from the genome-wide average, indicating that this gene family has not been under particularly strong selection pressure. Similarly, none of the 9 core C4 genes showed a domestication signal at the gene level. The absence of large sequence variation at the gene level is also consistent with previous evolutionary studies suggesting that relatively minor changes to pre-existing regulatory networks and the use of pre-existing cis-elements were often sufficient to recruit genes into the C4 pathway [56,57]. The C4 isoform of the NADP-ME gene found in maize and sorghum is one such gene that has been found to be activated for C4 photosynthesis via subtle changes to its promoter, while the rest of the gene is highly conserved [33]. This is consistent with the low diversity in this gene family observed in our study.
A further high-resolution investigation of domestication signature at the SNP level revealed 2 C4 genes, PPDK (Sobic.009G132900) and CA (Sobic.003G234200), with SNPs under purifying selection, while the other 2 C4 genes, PPDK-RP (Sobic.002G324400) and PEPC (Sobic.010G160700), were identified with SNPs under balancing selection. Previous studies have demonstrated that SNP-level analysis using less stringent criteria is superior for capturing soft selection signals compared with genome-wide ranking [54,58]. However, the higher sensitivity may come with a cost of a greater chance of false positives, and therefore requires cautious interpretation. The contrasting selection signals on genes from the same pathway within taxa found in this study was also reported previously in signal transduction pathways [59] and the starch biosynthesis pathway [60].
The C4 isoforms of PPDK and PEPC were also found to show signals of positive selection in a previous cross-species evolutionary study using orthologous groups from closely related C3 and C4 grass species including sorghum [28]. PPDK and PPDK-RP regulate the regeneration of PEP and as such have a direct effect on CO2 assimilation rate [61], especially under cool temperatures [62,63]. However, it is thought that only minor changes to the enzyme properties of PPDK were sufficient to recruit it into the C4 pathway and its residues and regions involved in catalyzes are highly conserved in C4 species [64], possibly validating the fact that only soft selection signals via SNP-level were found for the C4 isoform of the PPDK gene in our study.
PEPC is also regarded as a potential limiting step in the assimilation of CO2, and variation of its affinity for CO2/HCO3− amongst species has been documented [65,66,67]. CA is also critical to C4 photosynthesis as it catalyzes the first step of the C4 pathway, converting CO2 to HCO3− [68]. It was reported in the C4 dicot Flaveria bidentis, where antisense plants with <10% of wild-type CA activity required high CO2 for growth and showed reduced CO2 assimilation rates [69,70]. Recent experiments showed CA and PEPC will be more limiting when stomates are partially closed, e.g., under water limitation [71].
The signal of soft purifying selection on PPDK and CA may suggest the C4 pathway was indirectly improved during sorghum domestication. Without photosynthetic rate being a direct selection target in breeding programs, a steady increase in leaf photosynthetic rate over time of cultivar release has been shown in other cereals, e.g., in Australian bread wheat [72]. The balancing selection signal on C4 PPDK-RP and PEPC may reflect adaptation to diverse environments, as both PPDK-RP and PEPC are associated with abiotic stress [73,74]. Interestingly, within the PPDK-RP and PPDK gene families, the non-C4 genes all showed selection signals contrasting with their C4 counterparts with both two non-C4 PPDK-RP (Sobic.002G324500, Sobic.002G324700) containing SNPs under purifying selection and the non-C4 PPDK (Sobic.001G326900) containing SNPs under balancing selection.
After domestication, sorghum was introduced from tropical to temperate areas, and adapted to divergent local environments. New mutations also arose during this diversification process, and played an important role in local adaptation. In the haplotype analysis, these haplotypes unique to cultivated sorghum are likely to be young alleles arising after domestication, while haplotypes unique to the wild progenitor indicate that some haplotypes were lost during domestication of sorghum. Nevertheless, the loss of wild haplotypes of C4 genes in cultivated sorghum does not mean these haplotypes are inferior in terms of photosynthetic efficiency, as photosynthesis was not specifically targeted during sorghum domestication [11]. On the contrary, bringing these wild haplotypes back to breeding programs after evaluation of their functions may enrich breeders’ toolkits to manipulate photosynthetic efficiency, ultimately contributing to yield improvements.
C4 photosynthesis has been well studied over the past 50 years and key components of this complex pathway have been identified following the advent of transgenic and sequencing technologies [9]. Understanding the genetic diversity of the key enzymes of the C4 pathway is an important step towards mining the natural allelic variation for the improvement of photosynthesis.
Further investigation of these allelic variation to link them with agronomical traits will provide new targets for sorghum improvement [75].

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/2073-4425/11/7/806/s1. Table S1: List of re-sequenced sorghum accessions and their racial and geographic origins. Table S2: List of housekeeping genes and their genetic diversity. Table S3 List of SNPs under purifying selection. Table S4: List of SNPs under balancing selection. Table S5: Inter- and intra-species distribution of private alleles across 27 genes from C4 gene families. Figure S1: Phylogenetic tree of 48 sorghum accessions based on CDS of 27 genes from C4 gene families.

Author Contributions

D.J. and E.M. conceived the original idea. Y.T., M.B.-P., S.T., A.C., and E.M. analyzed the data. Y.T. and B.G.-J. write the manuscript. All authors discussed the results and contributed to the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded partially by the Australian Government through the Australian Research Council Centre of Excellence for Translational Photosynthesis (grant number CE140100015) and State Key Laboratory of Agricultural Genomics, China (grant number 2011DQ782025).

Acknowledgments

We thank Susanne von Caemmerer and Robert Furbank for their valuable comments and suggestions towards the improvement of this manuscript.

Conflicts of Interest

The authors declare that there is no conflict of interest.

Abbreviations

CA, carbonic anhydrase; PEPC, phosphoenolpyruvate carboxylase; PPCK, phosphoenolpyruvate carboxylase kinase; NADP-MDH, NADP-malate dehydrogenase; NADP-ME, NADP-malic enzyme; PPDK, pyruvate orthophosphate dikinase; PPDK-RP, PPDK regulatory protein; RbcS, ribulose bisphosphate carboxylase/oxygenase small-subunit.

References

  1. Sage, R.F.; Sage, T.L.; Kocacinar, F. Photorespiration and the evolution of C4 photosynthesis. Annu. Rev. Plant Biol. 2012, 63, 19–47. [Google Scholar] [CrossRef] [PubMed]
  2. Sage, R.F. The evolution of C4 photosynthesis. New Phytol. 2004, 161, 341–370. [Google Scholar] [CrossRef]
  3. Zelitch, I.; Schultes, N.P.; Peterson, R.B.; Brown, P.; Brutnell, T.P. High glycolate oxidase activity is required for survival of maize in normal air. Plant Physiol. 2009, 149, 195–204. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Edwards, E.J.; Smith, S.A. Phylogenetic analyses reveal the shady history of C4 grasses. Proc. Natl. Acad. Sci. USA 2010, 107, 2532–2537. [Google Scholar] [CrossRef] [Green Version]
  5. Hibberd, J.M.; Covshoff, S. The regulation of gene expression required for C4 photosynthesis. Annu. Rev. Plant Biol. 2010, 61, 181–207. [Google Scholar] [CrossRef]
  6. von Caemmerer, S.; Furbank, R.T. The C4 pathway: An efficient CO2 pump. Photosynth. Res. 2003, 77, 191. [Google Scholar] [CrossRef] [PubMed]
  7. Hibberd, J.M.; Sheehy, J.E.; Langdale, J.A. Using C4 photosynthesis to increase the yield of rice—Rationale and feasibility. Curr. Opin. Plant Biol. 2008, 11, 228–231. [Google Scholar] [CrossRef] [PubMed]
  8. Langdale, J.A. C4 cycles: Past, present, and future research on C4 photosynthesis. Plant Cell 2011, 23, 3879–3892. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. von Caemmerer, S.; Furbank, R.T. Strategies for improving C4 photosynthesis. Curr. Opin. Plant Biol. 2016, 31, 125–134. [Google Scholar] [CrossRef] [Green Version]
  10. Still, C.J.; Berry, J.A.; Collatz, G.J.; DeFries, R.S. Global distribution of C3 and C4 vegetation: Carbon cycle implications. Glob. Biogeochem. Cy. 2003, 17, 1006. [Google Scholar] [CrossRef]
  11. Long, S.P.; Zhu, X.G.; Naidu, S.L.; Ort, D.R. Can improvement in photosynthesis increase crop yields? Plant Cell Environ. 2006, 29, 315–330. [Google Scholar] [CrossRef] [PubMed]
  12. Zhu, X.G.; Shan, L.L.; Wang, Y.; Quick, W.P. C4 Rice—An ideal arena for systems biology research. J. Integr. Plant Biol. 2010, 52, 762–770. [Google Scholar] [CrossRef] [PubMed]
  13. von Caemmerer, S.; Quick, W.P.; Furbank, R.T. The development of C4 rice: Current progress and future challenges. Science 2012, 336, 1671–1672. [Google Scholar] [CrossRef] [PubMed]
  14. Covshoff, S.; Szecowka, M.; Hughes, T.E.; Smith-Unna, R.; Kelly, S.; Bailey, K.J.; Sage, T.L.; Pachebat, J.A.; Leegood, R.; Hibberd, J.M. C4 Photosynthesis in the Rice Paddy: Insights from the Noxious Weed Echinochloa glabrescens. Plant Physiol. 2016, 170, 57–73. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Hatch, M.D.; Kagawa, T.; Craig, S. Subdivision of C4-pathway species based on differing C4 acid decarboxylating systems and ultrastructural features. Funct. Plant Biol. 1975, 2, 111–128. [Google Scholar] [CrossRef]
  16. Bräutigam, A.; Schliesky, S.; Külahoglu, C.; Osborne, C.P.; Weber, A.P.M. Towards an integrative model of C4 photosynthetic subtypes: Insights from comparative transcriptome analysis of NAD-ME, NADP-ME, and PEP-CK C4 species. J. Exp. Bot. 2014, 65, 3579–3593. [Google Scholar] [CrossRef] [Green Version]
  17. Sonawane, B.V.; Sharwood, R.E.; Whitney, S.; Ghannoum, O. Shade compromises the photosynthetic efficiency of NADP-ME less than PEP-CK and NAD-ME C4 Grasses. J. Exp. Bot. 2018, 69, 3053–3068. [Google Scholar] [CrossRef] [PubMed]
  18. Grass Phylogeny Working Group II. New grass phylogeny resolves deep evolutionary relationships and discovers C4 origins. New Phytol. 2012, 193, 304–312. [Google Scholar] [CrossRef] [Green Version]
  19. Kidambi, S.P.; Krieg, D.R.; Rosenow, D.T. Genetic variation for gas-exchange rates in grain sorghum. Plant Physiol. 1990, 92, 1211–1214. [Google Scholar] [CrossRef] [Green Version]
  20. Peng, S.B.; Krieg, D.R. Gas-exchange traits and their relationship to water-use efficiency of grain sorghum. Crop Sci. 1992, 32, 386–391. [Google Scholar] [CrossRef]
  21. Henderson, S.; von Caemmerer, S.; Farquhar, G.D.; Wade, L.J.; Hammer, G. Correlation between carbon isotope discrimination and transpiration efficiency in lines of the C4 species Sorghum bicolor in the glasshouse and the field. Aust. J. Plant Physiol. 1998, 25, 111–123. [Google Scholar] [CrossRef]
  22. Balota, M.; Payne, W.A.; Rooney, W.; Rosenow, D. Gas exchange and transpiration ratio in sorghum. Crop Sci. 2008, 48, 2361–2371. [Google Scholar] [CrossRef]
  23. Fernandez, M.G.S.; Strand, K.; Hamblin, M.T.; Westgate, M.; Heaton, E.; Kresovich, S. Genetic analysis and phenotypic characterization of leaf photosynthetic capacity in a sorghum (Sorghum spp.) diversity panel. Genet. Resour. Crop Ev. 2015, 62, 939–950. [Google Scholar] [CrossRef]
  24. Zheng, L.Y.; Guo, X.S.; He, B.; Sun, L.J.; Peng, Y.; Dong, S.S.; Liu, T.F.; Jiang, S.Y.; Ramachandran, S.; Liu, C.M.; et al. Genome-wide patterns of genetic variation in sweet and grain sorghum (Sorghum bicolor). Genome Biol. 2011, 12, R114. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Paterson, A.H.; Bowers, J.E.; Bruggmann, R.; Dubchak, I.; Grimwood, J.; Gundlach, H.; Haberer, G.; Hellsten, U.; Mitros, T.; Poliakov, A.; et al. The Sorghum bicolor genome and the diversification of grasses. Nature 2009, 457, 551–556. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Mace, E.S.; Tai, S.S.; Gilding, E.K.; Li, Y.H.; Prentis, P.J.; Bian, L.L.; Campbell, B.C.; Hu, W.S.; Innes, D.J.; Han, X.L.; et al. Whole-genome sequencing reveals untapped genetic potential in Africa’s indigenous cereal crop sorghum. Nat. Commun. 2013, 4, 2320. [Google Scholar] [CrossRef] [Green Version]
  27. Fankhauser, N.; Aubry, S. Post-transcriptional regulation of photosynthetic genes is a key driver of C4 leaf ontogeny. J. Exp. Bot. 2017, 68, 137–146. [Google Scholar] [CrossRef] [Green Version]
  28. Huang, P.; Studer, A.J.; Schnable, J.C.; Kellogg, E.A.; Brutnell, T.P. Cross species selection scans identify components of C4 photosynthesis in the grasses. J. Exp. Bot. 2017, 68, 127–135. [Google Scholar] [CrossRef] [Green Version]
  29. Burgess, S.J.; Hibberd, J.M. Insights into C4 metabolism from comparative deep sequencing. Curr. Opin. Plant Biol. 2015, 25, 138–144. [Google Scholar] [CrossRef]
  30. Reeves, G.; Grangé-Guermente, M.J.; Hibberd, J.M. Regulatory gateways for cell-specific gene expression in C4 leaves with Kranz anatomy. J. Exp. Bot. 2017, 68, 107–116. [Google Scholar] [CrossRef]
  31. Christin, P.-A.; Osborne, C.P.; Chatelet, D.S.; Columbus, J.T.; Besnard, G.; Hodkinson, T.R.; Garrison, L.M.; Vorontsova, M.S.; Edwards, E.J. Anatomical enablers and the evolution of C4 photosynthesis in grasses. Proc. Natl. Acad. Sci. USA 2013, 110, 1381–1386. [Google Scholar] [CrossRef] [Green Version]
  32. Moreno-Villena, J.J.; Dunning, L.T.; Osborne, C.P.; Christin, P.-A. Highly expressed genes are preferentially co-opted for C4 photosynthesis. Mol. Biol. Evol. 2018, 35, 94–106. [Google Scholar] [CrossRef] [Green Version]
  33. Borba, A.R.; Serra, T.S.; Gorska, A.; Gouveia, P.; Cordeiro, A.M.; Reyna-Llorens, I.; Knerova, J.; Barros, P.M.; Abreu, I.A.; Oliveira, M.M.O.; et al. Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state. Mol. Biol. Evol. 2018, 35, 1690–1705. [Google Scholar] [CrossRef] [Green Version]
  34. Christin, P.-A.; Petitpierre, B.; Salamin, N.; Büchi, L.; Besnard, G. Evolution of C4 phosphoenolpyruvate carboxykinase in grasses, from genotype to phenotype. Mol. Biol. Evol. 2008, 26, 357–365. [Google Scholar] [CrossRef] [Green Version]
  35. Christin, P.-A.; Salamin, N.; Muasya, A.M.; Roalson, E.H.; Russier, F.; Besnard, G. Evolutionary switch and genetic convergence on rbcL following the evolution of C4 Photosynthesis. Mol. Biol. Evol. 2008, 25, 2361–2368. [Google Scholar] [CrossRef] [Green Version]
  36. Gowik, U.; Bräutigam, A.; Weber, K.L.; Weber, A.P.; Westhoff, P. Evolution of C4 photosynthesis in the genus Flaveria: How many and which genes does it take to make C4? Plant Cell 2011, 23, 2087–2105. [Google Scholar] [CrossRef] [Green Version]
  37. Mallmann, J.; Heckmann, D.; Bräutigam, A.; Lercher, M.J.; Weber, A.P.; Westhoff, P.; Gowik, U. The role of photorespiration during the evolution of C4 photosynthesis in the genus Flaveria. eLife 2014, 3, e02478. [Google Scholar] [CrossRef]
  38. Williams, B.P.; Aubry, S.; Hibberd, J.M. Molecular evolution of genes recruited into C4 photosynthesis. Trends Plant Sci. 2012, 17, 213–220. [Google Scholar] [CrossRef]
  39. Wang, X.; Gowik, U.; Tang, H.; Bowers, J.E.; Westhoff, P.; Paterson, A.H. Comparative genomic analysis of C4 photosynthetic pathway evolution in grasses. Genome Biol. 2009, 10, R68. [Google Scholar] [CrossRef] [Green Version]
  40. Ermakova, M.; Danila, F.R.; Furbank, R.T.; von Caemmerer, S. On the road to C4 rice: Advances and perspectives. Plant J. 2020, 101, 940–950. [Google Scholar] [CrossRef] [Green Version]
  41. Nei, M.; Li, W.-H. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 1979, 76, 5269–5273. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Tajima, F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 1989, 123, 585–595. [Google Scholar] [PubMed]
  43. Watterson, G.A. On the number of segregating sites in genetical models without recombination. Theor. Popul. Biol. 1975, 7, 256–276. [Google Scholar] [CrossRef]
  44. Hudson, R.; Boos, D.D.; Kaplan, N. A statistical test for detecting geographic subdivision. Mol. Biol. Evol. 1992, 9, 138–151. [Google Scholar] [PubMed] [Green Version]
  45. Pfeifer, B.; Wittelsbürger, U.; Onsins, S.E.R.; Lercher, M.J. PopGenome: An efficient Swiss army knife for population genomic analyses in R. Mol. Biol. Evol. 2014, 31, 1929–1936. [Google Scholar] [CrossRef] [Green Version]
  46. Paradis, E.; Claude, J.; Strimmer, K.J.B. APE: Analyses of phylogenetics and evolution in R language. Bioinformatics 2004, 20, 289–290. [Google Scholar] [CrossRef] [Green Version]
  47. Paradis, E. Pegas: An R package for population genetics with an integrated–modular approach. Bioinformatics 2010, 26, 419–420. [Google Scholar] [CrossRef] [Green Version]
  48. Ehleringer, J.R.; Sage, R.F.; Flanagan, L.B.; Pearcy, R.W. Climate change and the evolution of C4 photosynthesis. Trends Ecol. Evol. 1991, 6, 95–99. [Google Scholar] [CrossRef]
  49. Christin, P.A.; Salamin, N.; Savolainen, V.; Duvall, M.R.; Besnard, G. C4 Photosynthesis evolved in grasses via parallel adaptive genetic changes. Curr. Biol. CB 2007, 17, 1241–1247. [Google Scholar] [CrossRef] [Green Version]
  50. Christin, P.A.; Samaritani, E.; Petitpierre, B.; Salamin, N.; Besnard, G. Evolutionary insights on C4 photosynthetic subtypes in grasses from genomics and phylogenetics. Genome Biol. Evol. 2009, 1, 221–230. [Google Scholar] [CrossRef] [Green Version]
  51. Clark, J.D.; Stemler, A. Early domesticated sorghum from central Sudan. Nature 1975, 254, 588–591. [Google Scholar] [CrossRef]
  52. Mann, J.A.; Kimber, C.T.; Miller, F.R. The Origin and Early Cultivation of Sorghums in Africa; Bulletin 1454; Texas Agricultural Experiment Station: College Station, TX, USA, 1983. [Google Scholar]
  53. Wendorf, F.; Close, A.E.; Schild, R.; Wasylikowa, K.; Housley, R.A.; Harlan, J.R.; Królik, H. Saharan exploitation of plants 8,000 years BP. Nature 1992, 359, 721–724. [Google Scholar] [CrossRef]
  54. Tao, Y.; Mace, E.S.; Tai, S.; Cruickshank, A.; Campbell, B.C.; Zhao, X.; Van Oosterom, E.J.; Godwin, I.D.; Botella, J.R.; Jordan, D.R. Whole-genome analysis of candidate genes associated with seed size and weight in Sorghum bicolor reveals signatures of artificial selection and insights into parallel domestication in cereal crops. Front. Plant Sci. 2017, 8, 1237. [Google Scholar] [CrossRef] [Green Version]
  55. Tao, Y.; Zhao, X.; Mace, E.; Henry, R.; Jordan, D. Exploring and exploiting pan-genomics for crop improvement. Mol. Plant 2019, 12, 156–169. [Google Scholar] [CrossRef] [Green Version]
  56. Kümpers, B.M.C.; Burgess, S.J.; Reyna-Llorens, I.; Smith-Unna, R.; Boursnell, C.; Hibberd, J.M. Shared characteristics underpinning C4 leaf maturation derived from analysis of multiple C3 and C4 species of Flaveria. J. Exp. Bot. 2017, 68, 177–189. [Google Scholar] [CrossRef] [Green Version]
  57. Külahoglu, C.; Denton, A.K.; Sommer, M.; Maß, J.; Schliesky, S.; Wrobel, T.J.; Berckmans, B.; Gongora-Castillo, E.; Buell, C.R.; Simon, R.; et al. Comparative transcriptome atlases reveal altered gene expression modules between two Cleomaceae C3 and C4 plant species. Plant Cell 2014, 26, 3243–3260. [Google Scholar] [CrossRef] [Green Version]
  58. Massel, K.; Campbell, B.C.; Mace, E.S.; Tai, S.; Tao, Y.; Worland, B.G.; Jordan, D.R.; Botella, J.R.; Godwin, I.D. Whole genome sequencing reveals potential new targets for improving nitrogen uptake and utilization in Sorghum bicolor. Front. Plant Sci. 2016, 7, 1544. [Google Scholar] [CrossRef] [Green Version]
  59. Riley, R.M.; Jin, W.; Gibson, G. Contrasting selection pressures on components of the Ras-mediated signal transduction pathway in Drosophila. Mol. Ecol. 2003, 12, 1315–1323. [Google Scholar] [CrossRef]
  60. Campbell, B.C.; Gilding, E.K.; Mace, E.S.; Tai, S.; Tao, Y.; Prentis, P.J.; Thomelin, P.; Jordan, D.R.; Godwin, I.D. Domestication and the storage starch biosynthesis pathway: Signatures of selection from a whole sorghum genome sequencing strategy. Plant Biotechnol. J. 2016, 14, 2240–2253. [Google Scholar] [CrossRef]
  61. Wang, Y.-M.; Xu, W.-G.; Hu, L.; Zhang, L.; Li, Y.; Du, X.-H. Expression of maize gene encoding C4-pyruvate orthophosphate dikinase (PPDK) and C4-phosphoenolpyruvate carboxylase (PEPC) in transgenic Arabidopsis. Plant Mol. Biol. Rep. 2012, 30, 1367–1374. [Google Scholar] [CrossRef]
  62. Wang, D.; Portis, A.R.; Moose, S.P.; Long, S.P. Cool C4 photosynthesis: Pyruvate Pi dikinase expression and activity corresponds to the exceptional cold tolerance of carbon assimilation in Miscanthus × giganteus. Plant Physiol. 2008, 148, 557–567. [Google Scholar] [CrossRef] [Green Version]
  63. Naidu, S.L.; Moose, S.P.; AL-Shoaibi, A.K.; Raines, C.A.; Long, S.P. Cold Tolerance of C4 photosynthesis in Miscanthus × giganteus: Adaptation in amounts and sequence of C4 photosynthetic enzymes. Plant Physiol. 2003, 132, 1688–1697. [Google Scholar] [CrossRef] [Green Version]
  64. Chastain, C.J.; Failing, C.J.; Manandhar, L.; Zimmerman, M.A.; Lakner, M.M.; Nguyen, T.H.T. Functional evolution of C4 pyruvate, orthophosphate dikinase. J. Exp. Bot. 2011, 62, 3083–3091. [Google Scholar] [CrossRef] [Green Version]
  65. Bauwe, H.; Chollet, R. Kinetic properties of phosphoenolpyruvate carboxylase from C3, C4, and C3-C4 intermediate species of Flaveria (Asteraceae). Plant Physiol. 1986, 82, 695–699. [Google Scholar] [CrossRef] [Green Version]
  66. von Caemmerer, S.; Edwards, G.E.; Koteyeva, N.; Cousins, A.B. Single cell C4 photosynthesis in aquatic and terrestrial plants: A gas exchange perspective. Aquat. Bot. 2014, 118, 71–80. [Google Scholar] [CrossRef] [Green Version]
  67. Boyd, R.A.; Gandin, A.; Cousins, A.B. Temperature responses of C4 photosynthesis: Biochemical analysis of rubisco, phosphoenolpyruvate carboxylase, and carbonic anhydrase in Setaria viridis. Plant Physiol. 2015, 169, 1850–1861. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  68. Hatch, M.D.; Burnell, J.N. Carbonic anhydrase activity in leaves and its role in the first step of C4 photosynthesis. Plant Physiol. 1990, 93, 825–828. [Google Scholar] [CrossRef] [Green Version]
  69. von Caemmerer, S.; Quinn, V.; Hancock, N.; Price, G.D.; Furbank, R.T.; Ludwig, M. Carbonic anhydrase and C4 photosynthesis: A transgenic analysis. Plant Cell Environ. 2004, 27, 697–703. [Google Scholar] [CrossRef]
  70. Cousins, A.B.; Badger, M.R.; von Caemmerer, S. Carbonic anhydrase and its influence on carbon isotope discrimination during C4 photosynthesis. Insights from antisense RNA in Flaveria bidentis. Plant Physiol. 2006, 141, 232–242. [Google Scholar] [CrossRef] [Green Version]
  71. Osborn, H.L.; Alonso-Cantabrana, H.; Sharwood, R.E.; Covshoff, S.; Evans, J.R.; Furbank, R.T.; von Caemmerer, S. Effects of reduced carbonic anhydrase activity on CO2 assimilation rates in Setaria viridis: A transgenic analysis. J. Exp. Bot. 2016, 68, 299–310. [Google Scholar] [CrossRef] [Green Version]
  72. Watanabe, N.; Evans, J.R.; Chow, W.S. Changes in the Photosynthetic Properties of Australian wheat cultivars over the last century. Aust. J. Plant Physiol. 1994, 21, 169–183. [Google Scholar] [CrossRef]
  73. Liu, X.; Li, X.; Zhang, C.; Dai, C.; Zhou, J.; Ren, C.; Zhang, J. Phosphoenolpyruvate carboxylase regulation in C4-PEPC-expressing transgenic rice during early responses to drought stress. Physiol. Plant. 2017, 159, 178–200. [Google Scholar] [CrossRef] [PubMed]
  74. Jeanneau, M.; Gerentes, D.; Foueillassar, X.; Zivy, M.; Vidal, J.; Toppan, A.; Perez, P. Improvement of drought tolerance in maize: Towards the functional validation of the Zm-Asr1 gene and increase of water use efficiency by over-expressing C4–PEPC. Biochimie 2002, 84, 1127–1135. [Google Scholar] [CrossRef]
  75. Tao, Y.; Zhao, X.; Wang, X.; Hathorn, A.; Hunt, C.; Cruickshank, A.W.; van Oosterom, E.J.; Godwin, I.D.; Mace, E.S.; Jordan, D.R. Large-scale GWAS in sorghum reveals common genetic control of grain size among cereals. Plant Biotechnol. J. 2020, 18, 1093–1105. [Google Scholar] [CrossRef] [Green Version]
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Figure 1. Diagram of the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) biosynthetic pathway of C4 photosynthesis (adapted from [40]). In the mesophyll cells, CO2 is converted to HCO3− catalyzed by carbonic anhydrase (CA) and fixed into the four-carbon acid, oxaloacetate (OAA), by phosphoenolpyruvate carboxylase (PEPC). Phosphorylation of PEPC is carried out by PEPC kinase (PPCK). The OAA generated by PEPC is then reduced to malate by the NADP-malate dehydrogenase (NADP-MDH) or trans-aminated to aspartate. The resultant C4 acids, malate and aspartate, are transported to the bundle sheath and then decarboxylated in the vicinity of Rubisco to release CO2 and pyruvate. Pyruvate is transported back to mesophyll cells to regenerate PEP by pyruvate orthophosphate dikinase (PPDK), while CO2 enters the Calvin–Benson–Bassham cycle and is fixed by ribulose-1,5-bisphosphate carboxylase (Rubisco). Activation and inactivation of PPDK is catalyzed by PPDK regulatory protein (PPDK-RP).
Figure 1. Diagram of the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) biosynthetic pathway of C4 photosynthesis (adapted from [40]). In the mesophyll cells, CO2 is converted to HCO3− catalyzed by carbonic anhydrase (CA) and fixed into the four-carbon acid, oxaloacetate (OAA), by phosphoenolpyruvate carboxylase (PEPC). Phosphorylation of PEPC is carried out by PEPC kinase (PPCK). The OAA generated by PEPC is then reduced to malate by the NADP-malate dehydrogenase (NADP-MDH) or trans-aminated to aspartate. The resultant C4 acids, malate and aspartate, are transported to the bundle sheath and then decarboxylated in the vicinity of Rubisco to release CO2 and pyruvate. Pyruvate is transported back to mesophyll cells to regenerate PEP by pyruvate orthophosphate dikinase (PPDK), while CO2 enters the Calvin–Benson–Bassham cycle and is fixed by ribulose-1,5-bisphosphate carboxylase (Rubisco). Activation and inactivation of PPDK is catalyzed by PPDK regulatory protein (PPDK-RP).
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Figure 2. Genetic diversity and fixation index (FST) of C4 gene families between cultivated sorghum and the wild and weedy group. (A) Genetic diversity (pi) for each of the C4 gene families. Gene IDs in red indicate core C4 genes. Red bars represent the pi of cultivated sorghum, while dark blue bars represent the pi of wild and weedy. (B) FST between cultivated and wild and weedy of each of C4 gene families. Gene IDs in red indicate core C4 genes.
Figure 2. Genetic diversity and fixation index (FST) of C4 gene families between cultivated sorghum and the wild and weedy group. (A) Genetic diversity (pi) for each of the C4 gene families. Gene IDs in red indicate core C4 genes. Red bars represent the pi of cultivated sorghum, while dark blue bars represent the pi of wild and weedy. (B) FST between cultivated and wild and weedy of each of C4 gene families. Gene IDs in red indicate core C4 genes.
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Figure 3. Haplotype network of 4 core C4 gene with selection signal based on individual SNP analysis. (A) The PPDK gene (Sobic.009G132900) with signal of purifying selection; (B) one of the CA genes (Sobic.003G234200) with signal of purifying selection; (C) the PPDK-RP gene (Sobic.002G324400) with signal of balancing selection; (D) the PEPC gene (Sobic.010G160700) with signal of balancing selection. Group classification of sorghum accessions used as detailed in Table S1. Color-coding as follows; cultivated sorghum (red), wild and weedy genotypes (purple), Sorghum propinquum (blue), and Sorghum guinea margaritiferum (green). The size of the circles in the haplotype networks is proportionate to the number of accessions with that haplotype. The branch length represents the genetic distance between two haplotypes.
Figure 3. Haplotype network of 4 core C4 gene with selection signal based on individual SNP analysis. (A) The PPDK gene (Sobic.009G132900) with signal of purifying selection; (B) one of the CA genes (Sobic.003G234200) with signal of purifying selection; (C) the PPDK-RP gene (Sobic.002G324400) with signal of balancing selection; (D) the PEPC gene (Sobic.010G160700) with signal of balancing selection. Group classification of sorghum accessions used as detailed in Table S1. Color-coding as follows; cultivated sorghum (red), wild and weedy genotypes (purple), Sorghum propinquum (blue), and Sorghum guinea margaritiferum (green). The size of the circles in the haplotype networks is proportionate to the number of accessions with that haplotype. The branch length represents the genetic distance between two haplotypes.
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Table
Table 1. Single nucleotide polymorphism (SNP) information and selection signals across 27 genes from C4 gene families.
Table 1. Single nucleotide polymorphism (SNP) information and selection signals across 27 genes from C4 gene families.
Gene IDEnzymeGLCDSLNoSNoSiCNoNSNoSSUPSGLUBSGLNoSUPSNoNSUPSNoSUBSNoNSUBS
Sobic.002G230100CA4823101411514410NoNo0010
Sobic.003G234200CA10440137147533726NoNo1100
Sobic.003G234400CA474961513813310NoNo0000
Sobic.003G234500CA29866091731156NoNo0000
Sobic.003G234600CA475077121018108NoNo0000
Sobic.007G166200NADP-MDH33541308531165NoNo0000
Sobic.007G166300NADP-MDH381612901081239NoNo0000
Sobic.003G036000NADP-ME610719411111147NoNo0000
Sobic.003G036200NADP-ME544719111411239NoNo0000
Sobic.003G280900NADP-ME5691178217522139NoNo1100
Sobic.003G292400NADP-ME452717829522814NoNo10200
Sobic.009G069600NADP-ME36241713118341024NoNo3100
Sobic.002G167000PEPC56322904411165NoNo0000
Sobic.003G100600PEPC8881311737143934NoNo00212
Sobic.003G301800PEPC7610290113819317NoNo0000
Sobic.004G106900PEPC6977288314634529NoNo0070
Sobic.007G106500PEPC56162895641284NoNo1100
Sobic.010G160700PEPC6647308719328919NoNo0020
Sobic.004G219900PPCK161292440918NoNo0020
Sobic.004G338000PPCK174985537944NoNo0000
Sobic.006G148300PPCK199790064413NoNo0000
Sobic.001G326900PPDK84942730321461828NoYes00245
Sobic.009G132900PPDK12748284744116016NoNo3000
Sobic.002G324400PPDK-RP250712907922814NoNo0030
Sobic.002G324500PPDK-RP307212606920515NoNo4000
Sobic.002G324700PPDK-RP4662158722228199NoNo1122
Sobic.005G042000RbcS155651045743NoNo0000
Gene ID is according to sorghum reference genome V3.1. Gene IDs in bold indicate their C4 genes. Enzyme: Encoded enzyme. GL: Gene length. CDSL: Length of coding sequence (CDS). NoS: Total number of SNPs identified across the gene. NoSiC: Number of SNPs identified in CDS. NoNS: Number of non-synonymous SNPs. NoSS: Number of synonymous SNPs. UPSGL: Under purifying selection based on gene level analysis. UBSGL: Under balancing selection based on gene level analysis. NoSUPS: Number of SNPs under purifying selection. NoNSUPS: Number of non-synonymous SNPs under purifying selection. NoSUBS: Number of SNPs under balancing selection. NoNSUBS: Number of non-synonymous SNPs under balancing selection.
Table
Table 2. Genetic diversity (θπ) and fixation index (FST) of 27 genes from C4 gene families.
Table 2. Genetic diversity (θπ) and fixation index (FST) of 27 genes from C4 gene families.
GeneIDEnzymeθπ–Allθπ-Cultivatedθπ-W&WFST
Sobic.002G230100CA0.800.740.900.19
Sobic.003G234200CA2.652.462.660.16
Sobic.003G234400CA1.010.910.880.37
Sobic.003G234500CA5.555.514.560.07
Sobic.003G234600CA1.271.350.650.06
Sobic.007G166200NADP-MDH0.180.210.130.07
Sobic.007G166300NADP-MDH0.330.330.420.08
Sobic.003G036000NADP-ME0.880.651.590.15
Sobic.003G036200NADP-ME0.890.671.390.06
Sobic.003G280900NADP-ME0.930.851.110.09
Sobic.003G292400NADP-ME1.430.084.440.32
Sobic.009G069600NADP-ME0.520.490.100.45
Sobic.002G167000PEPC0.580.510.850.04
Sobic.003G100600PEPC5.365.183.560.05
Sobic.003G301800PEPC0.640.222.370.22
Sobic.004G106900PEPC3.183.022.140.07
Sobic.007G106500PEPC0.440.220.470.21
Sobic.010G160700PEPC2.492.252.860.04
Sobic.004G219900PPCK2.081.942.120.12
Sobic.004G338000PPCK1.030.960.910.03
Sobic.006G148300PPCK0.480.390.130.41
Sobic.001G326900PPDK8.345.645.640.40
Sobic.009G132900PPDK2.071.792.190.13
Sobic.002G324400PPDK-RP5.043.824.550.41
Sobic.002G324500PPDK-RP1.270.103.750.24
Sobic.002G324700PPDK-RP2.582.503.510.05
Sobic.005G042000rbcS4.323.415.720.12
Gene ID is according to sorghum reference genome V3.1. Gene IDs in bold indicate the C4 gene versions. Enzyme: Encoded enzyme. θπ-All: Nucleotide diversity across all 48 genotypes. θπ-Cultivated: Nucleotide diversity across cultivated genotypes. θπ-W&W: Nucleotide diversity across wild and weedy genotypes. All θπ values are in unites of per kb. FST: Fixation index between cultivated genotypes and wild and weedy genotypes.

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Tao, Y.; George-Jaeggli, B.; Bouteillé-Pallas, M.; Tai, S.; Cruickshank, A.; Jordan, D.; Mace, E. Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.). Genes 2020, 11, 806. https://0-doi-org.brum.beds.ac.uk/10.3390/genes11070806

AMA Style

Tao Y, George-Jaeggli B, Bouteillé-Pallas M, Tai S, Cruickshank A, Jordan D, Mace E. Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.). Genes. 2020; 11(7):806. https://0-doi-org.brum.beds.ac.uk/10.3390/genes11070806

Chicago/Turabian Style

Tao, Yongfu, Barbara George-Jaeggli, Marie Bouteillé-Pallas, Shuaishuai Tai, Alan Cruickshank, David Jordan, and Emma Mace. 2020. "Genetic Diversity of C4 Photosynthesis Pathway Genes in Sorghum bicolor (L.)" Genes 11, no. 7: 806. https://0-doi-org.brum.beds.ac.uk/10.3390/genes11070806

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