Plastome Data of Red Currant and Gooseberry Reveal Potential Taxonomical Issues within the Ribes Genus (Grossulariaceae)

Abstract

The complete chloroplast genomes of red currant cultivar ‘Belaya Potapenko’ and gooseberry cultivar ‘Nekrasovskij’ were sequenced and assembled for the first time. The plastomes are 157,802 bp and 157,559 bp in length for Ribes rubrum and R. uva-crispa, respectively. The R. rubrum cp genome is 243 b.p. longer. It has one more protein-coding gene ycf1, which is pseudogenized in the R. uva-crispa cp genome. In total, 56 and 54 simple sequence repeats (SSRs) were identified within the assembled plastid genomes. The SSR content of plastid genomes was assessed for the 18 Saxifragales species. Phylogeny inference based on plastome data of 18 Saxifragales revealed that all Ribes species are clustered together on the phylogenetic tree, though R. fasciculatum seems to be the most distant from the other analyzed Ribes species. The position of taxa inside the Ribes genus clade does not support the concept of its division into five subgenera. All Ribes species share approximately the same set of protein-coding genes in their plastome sequences. There was multiple independent pseudogenization of the ycf1 gene within the Ribes genus as well as other Saxifragales taxa. Negative selection was observed for most of the genes in both the Ribes group and Saxifragales. A positive selection ratio was observed only inside the Ribes group for the ycf4 and clpP genes. Together with positive selection signatures, pseudogenization events of ycfs genes perhaps reflect that these genes’ evolution was important for Ribes’ adaptation. Thus, our study provides genomic resources and valuable reference for marker development, and makes some clarifications of the phylogenomics of the Ribes genus.

1. Introduction

Ribes L. is the only genus in the family Grossulariaceae of the order Saxifragales. Ribes includes about 200 shrub species, which mainly grow in the temperate zone of the Northern Hemisphere. Some species have economic value due to ornamental or berry features. Ribes species have been used for detoxification, glaucoma, cardiovascular disease, stomachache, hepatitis, hyperlipidemia, hypertension, and other ailments [1]. Black and red currant and gooseberry are the most economically important Ribes species growing as berry crops [2]. The leading black currant producers are Poland, the United Kingdom, and Germany [3]. The main red currant producers are Poland, Germany, Holland, Belgium, France, and Hungary [4]. Germany and the Slovak Republic are the leading producers of white currants. The leading gooseberry producers are Russia, Ukraine, the United Kingdom, Switzerland, and Kyrgyzstan [5]. The center of biodiversity of Ribes rubrum L. and Ribes nigrum L. is located in northern and north-western Europe and both species have been cultivated as garden shrubs in Germany, Holland, Denmark, and the eastern shore of the Baltic Sea for 500–600 years [6]. One of the oldest French texts that mentions the cultivation of red currant is dated to the beginning of the 14th century [6,7]. Gooseberry species are native to Europe, Asia, northern Africa, and North America. According to U. P. Hedrick, gooseberry domestication probably took place almost at the same time in several countries of northern Europe. However, the first records on gooseberry cultivation in Europe are known from England and are dated to the middle of the 16th century [8]. The beginning of the gooseberry culture in Russia dates even earlier, where it was cultivated in the gardens of the monasteries in the 11th century. It seems that the gooseberry germplasm in Russia was genetically isolated from those in Europe until the 19th century. But in the 19th century, the main part of Russian cultivars was replaced by Western European ones [9]. Early settlers from England and Holland tried to cultivate European gooseberries in America but it was not successful due to climate and disease issues. Native American wild gooseberries were grown by American pioneers in gardens, but true domestication of the species seems not to have been attempted until less than a hundred years ago. The first gooseberry cultivar derived from a Native American species was the ‘Houghton’, which was recorded for the first time in 1847 [8]. The gooseberry was very popular in North America until it was discovered that it is a vector of blister rust, deadly to certain pines, resulting in its removal from forest areas [10].

The varietal diversity of cultivated Ribes species is very large. About 1200 black currant cultivars, about 200 red currant cultivars, and over 1000 gooseberry cultivars are known [8,9,11]. According to Harmat et al. (1990), at least 18 species have contributed to the domestication of edible fruited currant [12]. Other research states that only 10 to 12 of the worldwide known Ribes species comprise the primary gene pool from which domesticated currants and gooseberries were developed [6,13]. The observed discrepancy in the number of species is probably the result of different numbers of species being distinguished within the genus Ribes by different researchers.

In earlier research, seven different taxa were discriminated in the pedigrees of red currant modern cultivars: garden currant (R. vulgare Lam.), large-fruited variety (R. vulgare var. macrocarpum), red currant (R. rubrum L.), rock red currant (R. petraeum Wulfen), multi-flowered currant (R. multiflorum Kit. ex Schult.), Varshevich currant (R. warszewiczii Jancz.), and Palchevsky currant (R. palczewskii (Janch.) Pojark) [9]. However, according to modern classifications, all of them can be reduced to only four species (in addition to Ribes warszewiczii, whose status is currently uncertain): Ribes rubrum L., Ribes petraeum Wulfen, R. multiflorum Kit. ex Schult., and Ribes spicatum subsp. lapponicum Hyl [14].

Modern gooseberry cultivars are based on both European (R.uva-crispa) and American (R. × robustum, R. hirtellum) species. Interestingly, about seven gooseberry species represent spontaneous interspecific hybrids. For example, Ribes × robustum (formerly Grossularia robusta) is a hybrid between R. niveum and R. inerme [9].

Previous, botanical references defined separate genera for currants and gooseberries [15,16,17]. Nowadays, most taxonomists recognize a single genus Ribes [18,19]. The fact that gooseberry and currant species are able to hybridize supports the single-genus concept [20].

According to the most recent concept, the genus Ribes is subdivided into five subgenera: (1) Berisia, European dioecious plants; (2) Grossularia, gooseberries; (3) Grossularioides, thorny currants; (4) Parilla, South American natives; and (5) Ribes, currants [21,22]. Infrageneric classification of the genus Ribes has undergone many revisions, and even in the era of molecular phylogenetics, its status is still debatable. Clarification of taxonomic subdivisions within the Ribes genus based on a chloroplast genome sequence comparison could be an interesting alternative to the classical taxonomy approach.

For a number of reasons, the plastid genome has long been the primary source of data for studies of plant phylogeny and evolution. The first chloroplast genomes were sequenced in the 1980s [23]. Currently, the Chloroplast Genome Database (CpGDB) consists of complete chloroplast genome sequences of 3823 plant species belonging to 1527 genera from 256 families [24]. It is worth mentioning that the artificial integration of foreign genes (>300) into chloroplast genomes results in their highest levels of expression, which makes the chloroplast genome an interesting target for genetic transformation. Species-specific chloroplast vectors with endogenous genes and regulatory sequences are required for efficient foreign gene expression. Thus, an understanding of the diversity of chloroplast genomes, in terms of both structure and sequence, is important for developing efficient systems for genetic engineering and phylogenomics research [23].

There has been an enormous reduction in gene content from the ancestral cyanobacteria (over 3200 genes of cyanobacterium Synechocystis PCC 6803) to the plastid genomes found in photosynthetic eukaryotes [25]. Most land plant plastid genomes contain 110–130 genes, with ~80 genes coding for proteins involved in photosynthesis and other processes [26]. Thanks to advances in next-generation sequencing technologies, more and more chloroplast genomic sequences are being analyzed, and phylogenetics has entered a new era at the same time [27].

As it mentioned above, black and red currant and gooseberry are the most economically important Ribes species. In the current study, plastomes of two of the three most economically important Ribes species, red currant and gooseberry, were sequenced and compared with plastomes of other Ribes species. It should be noted as well, the different clades of Ribes that those species are representatives of. Red currant Ribes rubrum L. is a type species of the genera and belongs to subg. Ribes sect. Ribes. Gooseberry (Ribes uva-crispa) for a long period was treated as the separate genera Grossularia and currently belongs to the sect. Grossularia of subg. Grossularia. Data obtained in this study can raise our awareness of this species, and the results will enrich the genetic information of the genus Ribes that we already have, providing a theoretical basis for study on the evolution of Ribes and species identification.

2. Materials and Methods

2.1. Plant Material, DNA Extraction, and Sequencing

We sampled young leaves of red currant cultivar ‘Belaya Potapenko’ (Ribes rubrum) and gooseberry cultivar ‘Nekrasovskij’ (Ribes uva-crispa) from VNIISPK bioresource collection (VNIISPK BRC) [28], Orel Province, Russia. Total genomic DNA was extracted from sampled leaves using the DNeasy^® Plant Pro Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The isolated genomic DNA was used to prepare genome libraries, which were sequenced on Illumina genome analyzer (Illumina, San Diego, CA, USA, NovaSeq 6000) with a 150 bp paired-end read.

2.2. Chloroplast Genome Assembly and Annotation

To evaluate the quality of raw sequencing data, FastQC software (version 0.11.9) was applied [29]. Ten million pairs of raw sequencing reads of each sample were preprocessed with the Trimmomatic tool (version 0.39) to clip Nextera adapter sequences and reads with length below 75 bp [30]. Assembly of plastid genomes was performed by the NOVOPlasty program (version 4.3.1) with K-mer size equal to 33 [31]. The assembled plastomes had 703x (Ribes rubrum) and 810x (Ribes uva-crispa) coverage depth. Genome annotation based on sequence similarity with the complete chloroplast genome sequence Ribes odoratum (MT081309) was performed in Geneious software version 2023.0.2 [32]. CPGAVAS2 with its own pipeline using tRNAscan-SE, ARAGORN, and tRNAdb was applied to detect tRNA genes with default settings [33,34,35,36]. Annotated protein-coding sequences (CDS) were extracted and then translated into a set of amino acid sequences. Then, multiple alignments of protein sequences were accomplished in the MAFFT plugin of Geneious tool to manually verify the genome annotation. Annotated plastid genome sequences were submitted to GenBank under the following accession numbers: OR227935 for R. rubrum and OR227937 for R. uva-crispa. A circular genome maps were drawn using CHLOROPLOT web server [37].

2.3. Genome Comparison and Adaptive Evolution Analysis

To visualize the structural variations among the cp genomes of 8 Ribes species, the R. rubrum genome was compared with the plastomes of Ribes glaciale, Ribes fasciculatum, Ribes roezlii, Ribes nigrum, Ribes odoratum, and Ribes nevadense by using the mVISTA program in LAGAN model [38]. The annotation of R. rubrum was used as a reference.

To estimate the evolutionary rate variation within the Saxifragales species, 67 protein-coding regions of 18 Saxifragales species (including 8 Ribes species) were used. The non-synonymous (Ka) and synonymous (Ks) substitution rates of each gene were estimated using the software Mega 11 [39] and calculating overall mean distance in group of 8 Ribes species and group of 18 Saxifragales species (including 8 Ribes species). Ka/Ks ratio was also calculated.

2.4. Simple Sequence Repeats Analysis

Simple sequence repeats were detected and located in the chloroplast (cp) genomes by GMATA tool version 2.3 [40]. For the cp SSR identification, the following minimal number of repeated units were applied: 10 for mononucleotide repeats; 5 for dinucleotide repeats; 4 for trinucleotide repeats; and 3 for tetra-, penta-, and hexanucleotide repeats.

2.5. Phylogenetic Analysis

Phylogenetic reconstruction was performed by two methods. First method was maximum likelihood (ML) analyses with RAxML version 8.2.11, using the GTR CAT model [41]. The number of bootstrap replications was equal to 10,000. Second method was Bayesian inference with MrBayes [42]. All Ribes plastomes available in the GenBank were downloaded on 15 May 2023 (Table S1).

3. Results

3.1. General Features of the R. rubrum and R. uva-crispa Chloroplast Genomes

In this study, the cp genome of two commercially important crops (red currant and gooseberry) were sequenced, which belong to different subgenera of the Ribes genus: Ribes (section Ribes) and Grossularia (section Grossularia), respectively.

Assembled plastomes are 157,802 bp and 157,559 bp in length, with a GC content of 38.1 and 38.2% for Ribes rubrum and R. uva-crispa, respectively (Figure 1). Both genomes have a typical quadripartite structure, consisting of a pair of inverted repeat regions (26,005 bp and 26,001 bp) separated by a large single copy region (87,369 bp and 87,391), and a small single copy region (18,423 bp and 18,166 bp). Values in brackets are given for R. rubrum and R. uva-crispa plastomes, respectively. The length of R. rubrum and R. uva-crispa plastomes is in the range of the previously sequenced chloroplast genomes of R. odoratum (MT081309) and R. glaciale (NC_062156), which are 157,152 bp and 157,848 bp, respectively.

Within the R. rubrum plastome, 133 genes were annotated, including 86 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 2 pseudogenes (Table S3). Overall, the number of genes in the R. uva-crispa chloroplast genome is 133, including 85 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 3 pseudogenes. Hence, R. uva-crispa has one protein-coding gene less than R. rubrum; i.e., a normal copy of ycf1 is absent because it was pseudogenized. The shortest genes in the sequenced plastomes are the same-trnG (GCC) and trnC (GCA) with equal length of 71 bp. The shortest among protein-coding genes are petN and petL, having the same length equal to 96 bp in the sequenced chloroplast genomes. The longest gene in both sequenced cp genomes is ycf2 (6879 bp) in R. rubrum and 6873 bp in R. uva-crispa, occurring in two copies in each of the presented genomes.

3.2. SSR Analysis

In total, 56 and 54 microsatellite loci were found in the chloroplast genomes of R. rubrum and R. uva-crispa, respectively.

In both Ribes plastomes, most microsatellites refer to mono- and di-nucleotide classes (39 and 10 SSR loci in R. rubrum; 39 and 7 in R. uva-crispa). Tri- and tetra-nucleotides are the least frequent SSR groups in both genomes (1 and 6 SSR loci in R. rubrum; 2 and 6 in R. uva-crispa). Penta- and hexa-nucleotides were not identified within the investigated plastomes. The total length of the SSR loci in the R. rubrum cp genome is 606 bp, which comprises approximately 0.38% of the plastome length. The total length of microsatellite loci in the R. uva-crispa genome is 594 bp, i.e., approximately 0.38% of its length. There are five SSR loci located within protein-coding sequences (CDS) in each of the chloroplast genomes.

3.3. Genome Comparison and Adaptive Evolution Analysis

In addition to the R. rubrum and R. uva-crispa plastomes, sequences of chloroplast genomes for six other Ribes species and ten additional representatives of Saxifragales were retrieved from the GenBank (Table S1). A comparison of 8 Ribes plastomes using the mVISTA program revealed that the cp genome of R. rubrum exhibited a high degree of sequence similarity with all other Ribes species, especially with R. glaciale (Figure S1). The cp genomes of R. fasciculatum followed by R. odoratum showed the most variations compared to R. rubrum. In total, non-coding regions were found to be more divergent than the protein-coding regions, though the sequences of the longer copy of ycf1 gene were found to be quite divergent.

In our evaluation, none of the genes reported a Ka value above one, of which clpP reported the highest value (Ka = 0.5361) in a group of 8 Ribes species and ycf4 reported the highest value (Ka = 0.1914) in a group of 18 Saxifragales species. The highest Ks value was recorded also for clpP (Ks = 0.4626) in a group of 8 Ribes species and ycf4 (Ks = 0.2904) in a group of 18 Saxifragales species.

Negative selection (conservation) and a Ka\Ks ratio < 1 were observed for most of the genes in both the Ribes group and the Saxifragales group (Table S3, Figure S2). Positive selection (adaptive evolution) and a Ka\Ks ratio > 1 were observed only inside the Ribes group for two genes (ycf4 and clpP).

3.4. Phylogenetic Analysis

In a phylogenetic analysis, 11 out of 15 Saxifragales families were represented. There are no complete chloroplast genome sequences available in the GenBank for the plant families Peridiscaceae, Aphanopetalaceae, and Tetracarpaeaceae. Representatives of Cynomoriaceae were excluded from the analysis due to extreme plastome reduction [43]. Vitis vinifera L. was used as the outgroup, which is the Rosides representative, and Saxifragales was considered as the sister group.

Phylogenetic reconstruction was performed by two methods. The phylogenies performed by the maximum likelihood (ML) analyses were constructed using both whole sequences of plastomes and concatenated CDSs. In both cases, the topological structure was similar, though the whole genome tree showed better resolution of the Daphniphyllaceae-Cercidifhyllaceae-Hamamelidaceae-Altingiaceae clade and higher bootstrap values at certain nodes. A phylogenetic tree based on whole sequencing data and performed by maximum likelihood (ML) analyses is represented (Figure 2). The second method of phylogenetic reconstruction was Bayesian inference based on whole sequencing data. The Bayesian phylogenetic tree (Figure S3) was identical to the ML phylogenetic tree. Hence, it supports the robustness of the tree topology.

As demonstrated on the tree, most nodes showed maximal bootstrap values (BV) of 100%. All Ribes species were clustered together (BV 100%). It was evident that pairs of species consisting of R. roezlii and R. uva-crispa, R. nigrum, and R. nevadense formed two monophyletic clades with BV 100%. They were further clustered with R. odoratum (low BV 66%), a monophyletic clade of R. rubrum and R. glaciale (BV100%), and completed by R. fasciculatum.

The clade of Ribes species is followed by Saxifragaceae (Astilbe koreana), then Iteaceae (Itea Chinensis), and the monophyletic clade of Crassulaceae, Haloragaceae, and Penthoraceae. Representatives of five other Saxifragales families (Paeoniaceae, Hamamelidaceae, Altingiaceae, Daphniphyllaceae, and Cercidifhyllaceae) were clustered quite separately from the rest.

4. Discussion

‘Belaya Potapenko’ [44] is a variety of red currant with white berries originating from Novosibirsk, Russia (Figure 3a). On the maternal line, ‘Belaya Potapenko’ represents the third generation from a large-fruited variety of garden currant (R. vulgare var. macrocarpum). Remarkably, databases of modern taxonomy—Plants of the World Online (POWO) [45] and World Flora Online (WFO) [46] refer to Ribes vulgare Lam. As a synonym of Ribes rubrum L. Though, according to Pikunova et al., an admixture analysis based on 7.5 K SNPs of 75 individuals of red currant placed descendants of R. rubrum and R. vulgare into different groups [14]. Ribes rubrum is representative of sect. Ribes of the subgenus Ribes.

‘Nekrasovskij’ [47] is a modern gooseberry cultivar that originated from VNIISPK (Russian Research Institute of Fruit Crop Breeding, Orel, Russia). The cultivar is an interspecific hybrid of Ribes uva-crispa, Ribes hirtellum, and Ribes × robustum with a chloroplast genome inherited from Ribes × robustum (former Grossularia robusta) (Figure 3b). Ribes × robustum is a hybrid species that originated from the crossing of two species of section Grossularia of subgenus Grossularia-R. niveum and R. inerme.

A comparative analysis of cp SSRs for 8 Ribes species and 10 Saxifragales species was performed (Table S2,). Except for R. rubrum and R. uva-crispa, whose cp genomes are reported in the current study, the cp SSRs analysis included R. glaciale, R. fasciculatum, R. roezlii, R. nigrum, R. odoratum, and R. nevadense, whose plastome sequences were retrieved from the GenBank, along with plastome sequences of 10 Saxifragales species. Thus, the analysis included species from 3 out of 5 subgenera and 7 out of 16 sections of the genus Ribes (Figure 4).

The total number of SSRs identified within the plastomes of selected Ribes species ranged from 53 (R. glaciale) to 72 (R.nigrum) loci. Mononucleotide repeats were the predominant types in all Ribes species, with their frequency ranging from 68% (for Ribes nevadense and Ribes glaciale) to 80% (Ribes odoratum) of the total number of SSRs identified. Pentanucleotide repeats were observed only in Ribes roezlii, Ribes nevadense, and Ribes fasciculatum var. chinense cp-genomes. There were no hexanucleotides within the analyzed Ribes plastomes, though hexanucleotides were observed in two Saxifragales species (Itea chinensis and Penthorum chinense). Within the analyzed Ribes plastomes, the number of SSR loci located within protein-coding sequences was equal to either five (in the R. rubrum, R. uva-crispa, R. odoratum, and R. nevadense chloroplast genomes) or six (R. nigrum, R. glaciale, R. fasciculatum, and R. roezlii). A wider range was observed within analyzed Saxifragales plastomes: up to 15 SSR loci were found within protein-coding sequences in Crassula perforata. The total length of microsatellite loci within the Ribes cp genomes varied from 579 bp (R. glaciale) to 809 bp (R. nigrum), i.e., from 0.37% to 0.51% of their length. On average, the total length of SSR loci within the Ribes cp genomes (691 bp) was shorter than that of the other 10 Saxifragales species (896 bp). Hence, significant variation in the SSR content among Ribes species was identified, though variation in the SSR content among the Saxifragales species was even more diverse. Information about SSR loci in the chloroplast genome will serve as the basis for the development of new DNA markers for the investigation of Ribes germplasm diversity and breeding.

In this study, Ka and Ks values were estimated in 67 genes computed as overall mean distance in a group of 8 Ribes species and a group of 18 Saxifragales species (including 8 Ribes species). Surprisingly, no genes with positive selection were detected for the Saxifrageles group. For comparison, 19 genes with positive selection sites were determined in the plastomes of Dipsacales [48]. This can probably be explained by the divergence of evolution in different lineages of Saxifrageles. This is an extremely diverse group of plants, including trees, shrubs, perennial herbs, succulents, aquatic, and parasitic plants. The diversity of vegetative and floristic characters makes it difficult to identify common features of this taxon. The fossil record and molecular data show an earlier origin in the early Cretaceous (102–108 Myr) with rapid early diversification to more modern forms [49,50]. This assumption is consistent with the fact that although no genes with positive selection signatures were identified in Saxifrageles, in general, two genes (ycf4 and clpP) under positive selection were identified by the analysis of the Ribes species group. The chloroplast clpP gene encodes the proteolytic subunit of an ATP-dependent protease. This group of enzymes plays an important role in the removal of damaged proteins by performing intracellular proteolysis. Also, by exhibiting peptidase and chaperone activities, they are involved in the fine control of some key cellular components [51]. Interestingly, clpP was one of the 19 genes with positive selection sites detected in Dipsacales chloroplast genomes [48]. The function of ycfs (hypothetical chloroplast open reading frames) genes is still not fully understood. The ycf4 gene has been described as a highly conserved protein in plants, green algae, and cyanobacteria. Recently, a study on Nicotiana tabacum showed that the chloroplast ycf4 gene is essential for transcriptional gene regulation and plant photoautotrophic growth [52]. Positive selection signatures in ycf4 and clpP show that these genes may have played important roles in the speciation of Ribes species and their adaptation to diverse environments.

The size and structure showed high conservation across the plastomes of the 8 Ribes species, implying a close phylogenetic relationship between them. The non-coding regions were more divergent than the protein-coding regions, and this is consistent with comparative analyses in other angiosperms [53,54].

All the Ribes species along with Astilbe from the sister family Saxifragaceae, share approximately the same set of protein-coding genes in their plastome sequences (Table S4). Like in other Ribes species, there are two copies of the rps12 gene in the R. nevadense and R. roezlii plastomes, but one of them is not annotated in each of the mentioned genomes. Each of the R. nevadense and R. roezlii plastomes contain two functional copies of the rpl2 gene, but the gene is completely missing according to the genome annotation. A short rps19 pseudogene remnant was annotated in some Ribes genomes. The chloroplast genomes of R. nugrum, R. uva-crispa, R. nevadense, R. roezlii, and R. glaciale lack the second functional copy of the ycf1 gene, which was retained only as a pseudogene in the plastomes of the mentioned species. At the same time, there are two functional copies of the gene within the R. rubrum, R. fasciculatum, and R. odoratum chloroplast genomes. It is interesting that in all cases, the same copy of the gene was pseudogenized, which could be due to its position in the genome. Pseudogenization of one copy of the ycf1 gene was found in the plastomes of some other Saxifragales representatives. In particular, pseudogenization of one copy of the ycf1 gene occurred in all the analyzed genomes of species from the Crassulaceae-Haloragaceae-Penthoraceae clade as well as Cercidiphyllum and Paeonia within the Paeoniaceae-Daphniphyllaceae-Cercidiphyllaceae-Hamamelidaceae-Altingiaceae clade. There is only one annotated copy of the gene within the Disanthus (Hamamelidaceae) plastome. The genome of Vitis within the Rosids clade, which is considered sister to the Saxifragales group, lacks one functional copy of the ycf1 gene. Thus, taking into account all the above-mentioned findings regarding ycf1 occurrence within the investigated genomes, it may be concluded that there was multiple independent pseudogenization of the gene within the Ribes genus as well as other Saxifragales taxa. One pseudogenized copy of the ycf2 gene in the R. nevadense chloroplast genome was found. Both copies of the ycf15 gene were pseudogenized in the plastomes of all Ribes genus representatives. It is worth mentioning that the genomes of R. nevadense, R. roezlii, and Astilbe contained two non-functional copies of the ycf15 gene, which were not annotated. All ycf15 gene copies are either non-functional or even not annotated in the genomes of Crassulaceae-Haloragaceae-Penthoraceae clade representatives on the constructed phylogenetic tree. However, every species of the Paeoniaceae-Daphniphyllaceae-Cercidiphyllaceae-Hamamelidaceae-Altingiaceae clade has two functional copies of the ycf15 gene in their plastome according to the genome annotation. There are no annotated ycf15 genes within the Vitis vinifera chloroplast genome. The genome of Astilbe (Saxifragaceae), which is the closest taxon to Ribes (Grossulariaceae) on the dendrogram, contains the ycf1 pseudogene and two non-functional copies of the ycf15 gene. Remarkably, in Itea from the same Grossulariaceae-Saxifragaceae-Iteaceae clade of the tree, there are two functional copies of both the ycf1 and ycf15 genes in the chloroplast genome. The pseudogenization of genes is a part of chloroplast evolution, accompanying the selection of a “better” copy of the gene. Together with positive selection signatures, pseudogenization events of ycfs genes perhaps reflect that the evolution of these genes was important for Ribes adaptation.

Phylogenetic reconstruction reveals that the eight analyzed Ribes species form a monophyletic clade inside the Saxifragales. Ribes is the single genus of the Grossulariaceae family clustered together with representatives of Iteaceae and Saxifragaceae, with the latter being the closest one. The obtained results of phylogeny inference within the order Saxifragales confirm previous phylogenetic studies [54,55,56].

Remarkably, the tree topology does not support known taxonomic subdivisions within the Ribes genus. Analyzed Ribes species belong to three different subgenera. R. nigrum and R. rubrum, R. nevadense, and R. odoratum belong to subgenus Ribes. R. roezlii and Ribes uva-crispa belong to subgenus Grossularia. R. glaciale and R. fasciculatum belong to subgenus Berisia. Only those from different sections of the same subgenus Grossularia (R. roezlii and Ribes uva-crispa) clustered together.

As for subgenus Ribes, only two of the four representatives, R. nigrum (sect. Ribes) and R. nevadense (sect. Calobotrya) clustered together. R. odoratum (subg. Ribes sect. Symphocalyx) formed a separate branch. Ribes rubrum from the same section Ribes as R. nigrum was placed together with R. glaciale (subg. Berisia) on the phylogenetic tree. The second species of subg. Berisia, R. fasciculatum seems to be most distant from the other analyzed Ribes species. Remarkably, previously R. fasciculatum was placed in another subgenus—Parilla [15]. Data on the basal position of R. fasciculatum are in agreement with the results of ITS analysis conducted by Senters and Soltis (2003) [57]. In their study, R. fasciculatum was the most distant sister to the other 65 Ribes species and was not clustered either with other species of subgenus Parilla or with the subgenus Berisia.

The separate position of R. odoratum supports the point of view of Weigend et al. (2002) [58]. Based on the analysis of 5S-NTS sequences, the authors suggest that Symphocalyx (golden currants) ought to be recognized at the subgenus level.

In our study, Ribes rubrum was clustered together with R. glaciale (subg. Berisia). Remarkably, according to the ITS rDNA polymorphism, R. rubrum also clustered with samples mainly represented by species of the Berisia subgenus [57].

Our data support the gooseberry (R. uva-crispa) position inside the Ribes genus. In earlier studies that were based on the analysis of nuclear and chloroplast DNA polymorphisms, it was determined that all currant and gooseberry species were clustered together [57,58,59,60].

Interestingly, the clade of the subgenus Grossularia species had a terminal position on the tree close to the R. nigrum and R. nevadense clades. This observation probably points to the recent origin of gooseberry, despite the fact that it was for a long time treated as a separate genus. A relatively closer relationship between gooseberry and black currant was earlier also detected based on RAPD analysis [61].

The results obtained show that the current delimitation of sections and subgenera of the genus Ribes could be artificial and needs to be extensively revised. Broader sampling and involvement of nuclear genome data are required for a deeper understanding of the evolution history and systematics of Ribes.

5. Conclusions

The complete chloroplast genomes of red currant cultivar ‘Belaya Potapenko’ and gooseberry cultivar ‘Nekrasovskij’ were sequenced and assembled for the first time. The plastomes are 157,802 bp and 157,559 bp in length, with a GC content of 38.1 and 38.2% for Ribes rubrum and R. uva-crispa, respectively. The R. rubrum cp genome is 243 b.p. longer. It has one more protein-coding gene ycf1, though, in the R. uva-crispa cp genome, this gene is a pseudogene.

The size and structure showed high level of conservation across the plastomes of the eight Ribes species, implying a close phylogenetic relationship between them. Positive selection was observed only inside the Ribes group for ycf4 and clpP genes. Together with positive selection signatures, pseudogenization events of ycfs genes perhaps reflect that the evolution of these genes was important for Ribes adaptation.

In total, 56 and 54 simple sequence repeats (SSRs) were identified within the assembled R. rubrum and R. uva-crispa plastid genomes. The SSR content of plastid genomes was assessed for the eight Ribes species.

The chloroplast genome sequences of 18 Saxifragales species (including eight Ribes species representing three subgenera) were used to reconstruct their phylogenetic relationship. Phylogenetic analysis revealed that Ribes species form a monophyletic clade inside the Saxifragales group, with R. fasciculatum being the most distant Ribes species. R. odoratum formed a separate branch within the Ribes genus clade but its position in the tree is not well supported by bootstrap. Our data support the gooseberry (R. uva-crispa) position inside the Ribes genus and also its similarity to black currant (R. nigrum). The tree topology does not support the known taxonomic subdivision within the Ribes genus. Analysis has revealed the high stability of protein-coding gene content among Ribes plastomes. Thus, our study provides genomic resources and valuable reference for marker development, and makes some clarifications regarding the phylogeny of the genus Ribes. Investigating Ribes phylogeny based on a broader taxonomic range are possible future research directions.