Complete Chloroplast Genome of Krascheninnikovia ewersmanniana: Comparative and Phylogenetic Analysis

Krascheninnikovia ewersmanniana is a dominant desert shrub in Xinjiang, China, with high economic and ecological value. However, molecular systematics research on K. ewersmanniana is lacking. To resolve the genetic composition of K. ewersmanniana within Amaranthaceae and its systematic relationship with related genera, we used a second-generation Illumina sequencing system to detect the chloroplast genome of K. ewersmanniana and analyze its assembly, annotation, and phylogenetics. Total length of the chloroplast genome of K. ewersmanniana reached 152,287 bp, with 84 protein-coding genes, 36 tRNAs, and eight rRNAs. Codon usage analysis showed the majority of codons ending with base A/U. Mononucleotide repeats were the most common (85.42%) of the four identified simple sequence repeats. A comparison with chloroplast genomes of six other Amaranthaceae species indicated contraction and expansion of the inverted repeat boundary region in K. ewersmanniana, with some genes (rps19, ndhF, ycf1) differing in length and distribution. Among the seven species, the variation in non-coding regions was greater. Phylogenetic analysis revealed Krascheninnikovia ceratoides, Dysphania ambrosioides, Dysphania pumilio, and Dysphania botrys to have a close monophyletic relationship. By sequencing the K. ewersmanniana chloroplast genome, this research resolves the relatedness among 35 Amaranthaceae species, providing molecular insights for germplasm utilization, and theoretical support for studying evolutionary relationships.


Introduction
K. ewersmanniana is a perennial, strongly drought-tolerant shrub belonging to the genus Krascheninnikovia in the family Amaranthaceae [1].In addition, the shrub has tolerance to salt and alkali, cold resistance, and other beneficial characteristics [2][3][4].This species is widely distributed in China, Kazakhstan, Russia, and Mongolia; in China, K. ewersmanniana grows exclusively in the arid desert grasslands of the Xinjiang Altay and Tianshan Mountains [5][6][7].K. ewersmanniana is highly effective as a windbreak and aids in sand fixation and water and soil conservation.It is thereby considered the main species in the arid desert area of Xinjiang and plays an indispensable role in restoration and improvement of grassland vegetation [8].As well as its significance for ecological construction, K. ewersmanniana is a good forage shrub for livestock, with high crude protein content and nutritional value [9].Since K. ewersmanniana plants grow tall, they can be used as a life-saving grass in winter and spring in Xinjiang and play an important role in disaster relief [9].Therefore, K. ewersmanniana has high economic and ecological value and is a valuable germplasm resource.Extensive research on the development and utilization of K. ewersmanniana has elucidated its morphology, anatomy, physiological and biochemical characteristics, ecology, and cultivation management technology [10][11][12][13][14].However, few certified specimen was collected along with the leaves (collector: Youzheng Li).The specimen was identified as K. ewersmanniana, a Krascheninnikovia plant of family Amaranthaceae, by Mei Ke, a researcher at the Grassland Research Institute of the Xinjiang Academy of Animal Sciences.Specimens were stored in the plant specimen library of the Grassland Research Institute, Xinjiang Academy of Animal Sciences.Seedlings were raised in the Xinjiang Academy of Animal Sciences AI climate chamber.Fresh and young leaves from healthy plants were collected for sequencing.

Genomic DNA Extraction and Sequencing
Genomic DNA was extracted from fresh leaves of plants using cetyltrimethyl ammonium bromide, and the DNA concentration was measured using a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and a Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA).The Illumina Miseq platform was used to sequence the chloroplast genome of the sample (Personalbio, Shanghai, China).
The data quality control was performed by fastp (v0.19.4) and high-quality sequences were produced.Junction contamination at the 3 ′ end was removed by Adapter Removal (version 2) [28], and the sliding window method was used to filter the quality.The average Q value of the bases in the window was calculated.If the Q value was <20, the bases in the window were deleted, and if Q was ≥20, sliding was stopped.If the length of any reads in the double-end is ≤50 bp and the number of bases in the double-end is ≥5, the double-end sequence is removed to ensure that the sequences contained in the dataset have sufficient length and quality.

Analysis of Chloroplast Genome Repeats and Codon Usage Preferences
Misa software (version 2.1) was used to predict simple sequence repeats (SSRs) of K. ewersmanniana chloroplast genomes, with the minimum number of repetitions for single nucleotide, double nucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide sets as 10, 6, 4, 3, 3, and 3, respectively [33].The online tool REPuter [34] was used to analyze dispersed repeat, with the Hamming distance and minimum repeat fragment size parameters set to 3 and 30, respectively.The relative synonymous codon usage (RSCU) value was calculated using CodonW software v.1.4.2 [35].RSCU is the ratio of the actual codon frequency to expected frequency.A RSCU value of 1 depicts codon usage without bias, while RSCU value < 1 indicates its relative rarity and >1 indicates that codon usage is greater than expected [36].

Comparison of Chloroplast Genomes
The IRscope online tool was used to analyze the infrared boundary information of the chloroplast genomes of seven Amaranthaceae plants [37].mVISTA software [38] was used to perform collinearity analysis of the chloroplast genomes of different species, with Chenopodium quinoa as a reference.

Phylogenetic Analysis
A phylogenetic tree of 35 species in the Amaranthaceae family was established, in which the annotated K. ewersmanniana chloroplast genome was assembled, and the genome sequences of 34 other Amaranthaceae species were downloaded from NCBI (Table S1), with Oryza sativa and Arabidopsis thaliana as outgroups.Nucleotide sequences corresponding to PCG in the selected genome were connected, and multiple sequence alignments were performed using the MAFFT program [39].A phylogenetic tree was constructed using the maximum likelihood (ML) algorithm and Bayesian analysis, and the best model was selected using ModelFinder [40].The ML method uses the software RAxML and selects the nucleotide substitution model as GTR + F + I + G4 [41].The best-fit GTR + F + I + G4 model was selected using Bayesian imputation in MrBayes v3.2.6 [42].

Genomic Features of K. ewersmanniana
Second-generation Illumina sequencing on the K. ewersmanniana chloroplast genome generated 36,875,778 raw reads, of which 712,889 were used for subsequent genome assembly.In this study, we assembled and annotated the complete chloroplast genome of K. ewersmanniana for the first time.The chloroplasts of K. ewersmanniana comprised four parts (SSC, IRa, LSC, and IRb) and exhibited a unique double-stranded circular structure with a genome size of 152,287 bp (Figure 1).The lengths of the IR, SSC, and LSC sequences were 49,182, 19,007, and 84,098 bp, respectively.The GC levels of the IR, SSC, and LSC regions were 41.99%, 30.58%, and 34.74%, respectively (Table S2).Table 1.Gene functional classification and composition of the K. ewersmanniana chloroplast genome.
Among the 128 annotated genes, a total of 19 genes contained exons and introns.Among these, only two genes (ycf3 and clpP) had three exons and two introns.The remaining 17 genes, namely ndhB(2), trnI-GAU(2), petD, ndhA, atpF, petB, trnV-UAC, rpl16, trnA-UGC, rpoC1, trnR-UCU, rps12, trnL-UAA, trnK-UU, and rps16 had two exons and one intron.The two introns of clpP were 811 bp and 603 bp long, whereas those of ycf3 were 779 bp and 805 bp long.Among all genes containing introns, trnK-UUU has the longest length of 2493 bp and rps12 has the shortest length of 543 bp.Among all genes containing exons, ndhB had the largest number of exons (1533 bp); 777 bp for exon I and 756 bp for exon II.The three genes with the smallest number of exons were trnA-UGC, trnA-UGC, and trnK-UUU, all with 72 exons.Among them, the length of exon I of the three genes was 37 bp, whereas that of exon II was 35 bp (Table 2).Note: + positive strand; − negative strand.

Analysis of the Codon Usage Profiles
According to codon analysis, we detected 22,696 codons in K. ewersmanniana, encoding 20 amino acids (Figure 2, Table 3).Of these, 2395 codons encoded leucine (Leu), accounting for 10.55% of the total codons, exhibiting the highest coding rate.However, only 253 codons encoded cysteines, accounting for the smallest proportion (1.11%).According to the RSCU analysis, 30 codons had RSCU > 1, implying overrepresentation of these codons, whereas 32 codons had RSCU < 1, implying that these codons were used less frequently.In Leu, the UUA had the largest RSCU value, at 2.07, and the GUG encoding Leu had the lowest at 0.32.Moreover, 29 codons had bases ending in A/U, whereas the one remaining codon ended in G.The proportion of codons ending in A/U was 96.67%.Consequently, K. ewersmanniana favors codons ending in A/U.

Repeat Analysis
Repeat sequence analysis performed on the genomes of two plants of Krascheninnikovia (K.ewersmanniana and K. ceratoides) detected two different types of scattered repetitive sequences: palindromic (P) and forward (F) (Figure 3A).The number of scattered repetitive sequences, arranged according to length, differed significantly between the two Krascheninnikovia species (Figure 3B).A total of 48 SSRs sequences were identified in the chloroplast genome of K. ewersmanniana, encompassing seven sequence types.Among them, mononucleotide repeats were the dominant sequence (all A/T), with 41 sequences, accounting for 85.42%.Similar SSRs were found in chloroplast genomes of two Krascheninnikovia plants.The total number of SSRs in the K. ceratoides chloroplast genome was 45, encompassing eight sequence types.Among them, 37 were single nucleotide repeats (A/T).ACTAT/AGTAT was the only different repeat sequence type identified in K. ceratoides (Figure 4A; Table S3).Most of the SSRs in both species were distributed in the intergenic spacer region (26 in K. ewersmanniana and 22 in K. ceratoides), accounting for more than 50% of all SSRs (Figure 4B).The number of SSRs distributed in the intergenic spacer, PCG, and intron regions was 22, 10, and 10, respectively (Figure 4B).There are 48 SSRs located in the intergenic spacer, and 19 SSRs had introns (Table S4).
nucleotide repeats (A/T).ACTAT/AGTAT was the only different repeat sequence type identified in K. ceratoides (Figure 4A; Table S3).Most of the SSRs in both species were distributed in the intergenic spacer region (26 in K. ewersmanniana and 22 in K. ceratoides), accounting for more than 50% of all SSRs (Figure 4B).The number of SSRs distributed in the intergenic spacer, PCG, and intron regions was 22, 10, and 10, respectively (Figure 4B).There are 48 SSRs located in the intergenic spacer, and 19 SSRs had introns (Table S4).

Comparison of the Chloroplast Genomes of Seven Amaranthaceae Plants
We compared the IR boundary regions and locations of the adjacent genes of K. ewersmanniana and six other species of Amaranthaceae (Figure 5).The chloroplast genome structure of K. ewersmanniana was highly conserved; however, structural changes were observed in the IR boundary regions.The rps19 gene exhibited variable expansion at the LSC/IRb regional junction in seven species, including K. ewersmanniana, K. ceratoides, Atriplex canescens, and Dysphania ambrosioidis.Among these species, expansion ranged from 139-149 bp in four species and 64-79 bp in three species (Atriplex gmelinii, C. quinoa, and Dysphania botris).The ndhF gene exhibited variable contraction or expansion at the identified in K. ceratoides (Figure 4A; Table S3).Most of the SSRs in both species were distributed in the intergenic spacer region (26 in K. ewersmanniana and 22 in K. ceratoides), accounting for more than 50% of all SSRs (Figure 4B).The number of SSRs distributed in the intergenic spacer, PCG, and intron regions was 22, 10, and 10, respectively (Figure 4B).There are 48 SSRs located in the intergenic spacer, and 19 SSRs had introns (Table S4).

Comparison of the Chloroplast Genomes of Seven Amaranthaceae Plants
We compared the IR boundary regions and locations of the adjacent genes of K. ewersmanniana and six other species of Amaranthaceae (Figure 5).The chloroplast genome structure of K. ewersmanniana was highly conserved; however, structural changes were observed in the IR boundary regions.The rps19 gene exhibited variable expansion at the LSC/IRb regional junction in seven species, including K. ewersmanniana, K. ceratoides, Atriplex canescens, and Dysphania ambrosioidis.Among these species, expansion ranged from 139-149 bp in four species and 64-79 bp in three species (Atriplex gmelinii, C. quinoa, and Dysphania botris).The ndhF gene exhibited variable contraction or expansion at the

Comparison of the Chloroplast Genomes of Seven Amaranthaceae Plants
We compared the IR boundary regions and locations of the adjacent genes of K. ewersmanniana and six other species of Amaranthaceae (Figure 5).The chloroplast genome structure of K. ewersmanniana was highly conserved; however, structural changes were observed in the IR boundary regions.The rps19 gene exhibited variable expansion at the LSC/IRb regional junction in seven species, including K. ewersmanniana, K. ceratoides, Atriplex canescens, and Dysphania ambrosioidis.Among these species, expansion ranged from 139-149 bp in four species and 64-79 bp in three species (Atriplex gmelinii, C. quinoa, and Dysphania botris).The ndhF gene exhibited variable contraction or expansion at the IRb/SSC region junction in five species: K. ewersmanniana, A. canescens, A. gmelinii, D. botrys, and C. quinoa.Except for K. ewersmanniana, which exhibited contraction of 3 bp, expansion of 31-57 bp was observed in the other species.Except for K. ewersmanniana and C. quinoa, the ycf1 gene was expanded at the IRb/SSC region junction, with a range of 1-55 bp.In all seven species, ycf1 showed similar expansion at the SSC/IRa region junction.The psbA-trnH gene exhibited variable expansion at the IRA/LSC region junction in all species except K. ewersmanniana, K. ceratoides, and C. quinoa.Contraction of 82 bp and 1 bp was observed in A. gmelinii and D. ambrosioides, respectively, and rpl2-rps19 contraction of 1 bp was only observed at the IRa/LSC region junction in A. canescens.
The comparison showed similar chloroplast genome sequences for the seven species; however, the variation of non-coding region was significantly higher than that of coding region.PCGs such as ycf3 and rpl16 showed significant variation.The regions between genes with a high degree of variation included psbA-trnK-UUU, rps2-rpoC2, atpI-atpF, rpl32-ccsn, and dhB-tmL-CAA (Figure 6).
1 bp was only observed at the IRa/LSC region junction in A. canescens.
The comparison showed similar chloroplast genome sequences for the seven species; however, the variation of non-coding region was significantly higher than that of coding region.PCGs such as ycf3 and rpl16 showed significant variation.The regions between genes with a high degree of variation included psbA-trnK-UUU, rps2-rpoC2, atpI-atpF, rpl32-ccsn, and dhB-tmL-CAA (Figure 6).
Analysis of the nucleotide diversity of the two Krascheninnikovia species revealed average Pi values of 0-0.00758 (Figure 7).Three highly variable regions were identified with Pi > 0.005, including psaJ, psbK, and psbK; these sites may contain more rapidly evolving site information and show potential as molecular markers.Analysis of the nucleotide diversity of the two Krascheninnikovia species revealed average Pi values of 0-0.00758 (Figure 7).Three highly variable regions were identified with Pi > 0.005, including psaJ, psbK, and psbK; these sites may contain more rapidly evolving site information and show potential as molecular markers.

Phylogenetic Analysis
To determine the phylogenetic position of K. ewersmanniana, we extracted and analyzed the shared PCGs of 35 representative Amaranthaceae species (Figure 8) with O. sativa and A. thaliana as outgroups.A total of 16 species of Chenopodium, Atriplex, and Beta were grouped into a single cluster.The 10 species of Salicornis, Suaeda, Bassia, and Salsola were incorporated into one group.Nine species of Ostosia, Celosia, and Amaranth were incorporated into another group.Each genus of plants, all of which are monophyletic, was clustered together.Alternanthera philoxeroides was located in the bottom of the phylogenetic tree and is the earliest isolated species among the 35 species in the Amaranthaceae family.Krascheninnikovia was located in the middle of the phylogenetic tree.K. ewersmanniana and K. ceratoides form a separate clade, which was most closely related to two other clades, comprising, respectively, D. ambrosioides, D. botrys, D. pumilio, and Beta vulgaris, Beta intermedia, Beta lomatogona, Atriplex (A. gmelinii and A. canescens), and Chenopodium (C.

Phylogenetic Analysis
To determine the phylogenetic position of K. ewersmanniana, we extracted and analyzed the shared PCGs of 35 representative Amaranthaceae species (Figure 8) with O. sativa and A. thaliana as outgroups.A total of 16 species of Chenopodium, Atriplex, and Beta were grouped into a single cluster.The 10 species of Salicornis, Suaeda, Bassia, and Salsola were incorporated into one group.Nine species of Ostosia, Celosia, and Amaranth were incorporated into another group.Each genus of plants, all of which are monophyletic, was clustered together.Alternanthera philoxeroides was located in the bottom of the phylogenetic tree and is the earliest isolated species among the 35 species in the Amaranthaceae family.Krascheninnikovia was located in the middle of the phylogenetic tree.K. ewersmanniana and K. ceratoides form a separate clade, which was most closely related to two other clades, comprising, respectively, D. ambrosioides, D. botrys, D. pumilio, and Beta vulgaris, Beta intermedia, Beta lomatogona, Atriplex (A. gmelinii and A. canescens), and Chenopodium (C.petiolare and C. quinoa).These clades were located at the top of the phylogenetic tree and are the most recently isolated of the 35 species of the Amaranthaceae family.

Discussion
The Amaranthaceae family, formerly also named Chenopodiaceae, harbors 80 genera and approximately 2500 species [27], including Krascheninnikovia, earlier referred to as Ceratoides or Eurotia, whose taxonomic position remains unclear.Prior studies have focused on the botanical, biological, and ecological characteristics of this plant [43][44][45].With the development of sequencing technology, more complete cp genomes have been reported.According to incomplete statistics, approximately 86 complete genomes of the Amaranthaceae family were deposited in the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/(accessed on 22 April 2024) [27,[46][47][48][49][50].However, merely one Krascheninnikovia species, K. ceratoides, was reported.Genetic and phylogenetic characteristics, therefore, have been significantly lacking for the Krascheninnikovia species.K. ewersmanniana is a major species of high-quality wild forage grass, providing wind-resistance and sand-fixation for the vegetation in northern Xinjiang.It plays a vital role in vegetation restoration and ecological construction of the desert steppe in Xinjiang.To construct the phylogeny and lay a solid foundation for the evolution of Krascheninnikovia and the Amaranthaceae family, the K. ewersmanniana complete cp genome was assembled and annotated.The results depict that the chloroplast genome length of K. ewersmanniana is 152,287 bp and 128 genes, similar to that of Chenopodium acuminatum Willd.(152,200 bp, 113 genes) [51], Chenopodium album (152,200 bp, 110 genes) [52], and Amaranthaceae (149,726-153,474 bp, according to NCBI).Their complete cp genomes were all typical tetrad structures and composed of SSC, LSC, and two IRs.Overall, the basic features of the K. ewersmanniana chloroplast genome were consistent with those of most Amaranthaceous species, especially for congeners such as K. ceratoides, indicating that chloroplast genome size and structures of Krascheninnikovia species are highly conserved.The GC content in the IR region was higher than that in the LSC and SSC regions, which may be attributed to the presence of GC-rich genes in the IR region.
Repetitive sequences were the main force of chloroplast genome repeat, deletion, and rearrangement which resulted in the evolution of chloroplast [53].The two tandem types identified in the K. ewersmanniana chloroplast genome were forward (F) and palindromic (P), which is consistent with those of K. ceratoides, C. album, and B. vulgaris [46,54].Only one tandem type was detected in the chloroplast genomes of spinach and quinoa, both of which are F-types [55].These results also differ from those of Carthamus (Asteraceae) species [27].Thus, a correlation exists between the genetic relationships among species and the type and number of repeats.DNA molecular markers are useful tools widely applied in genetic structure analysis, identification of generational relations, and evolution of species.Being highly polymorphic and codominant, SSR markers were given more prominence than RAPD, ISSR, and other markers [56].In another study, 48 SSRs were detected in K. ewersmanniana cp genomes, and mononucleotide A/T as a repeat unit was dominant.Compared with K. ceratoides, there was no pentanucleotide [25].Chloroplast SSR markers can greatly facilitate species detection since they are maternally inherited.For example, a difference only in SSRs (ACTAT/AGTAT) found in the cp genome of two plants allowed for the differentiation and identification of species.Whether this hypothesis is true requires further validation via publication of more cp genomes along with development of SSR molecular markers.
DNA barcoding has been an effective tool to carry out species identification [57], and greatly promoted the development of species classification and phylogeny [58].DNA barcoding research in higher plants mainly focuses on the chloroplast and nuclear genomes.According to the Consortium for the Barcode of Life, Chloroplast DNA fragments such as psbA-trnH and ribosomal DNA fragments such as ITS are widely used DNA barcodes [59].In the literature, several variations in hotspots, such as psbA-trnK-UUU, rps2-rpoC2, atpI-atpF, rpl32-ccsn, and dhB-tmL-CAA and genes, psaJ, psbK, and psbK have been elucidated.Nevertheless, accD-psaI, ndhF-trnL, petA-psbJ, psbF-petL, trnC-psbM, trnS-trnG, and ycf2-trnL and PCGs such as accD, matK, ndhF, ndhK, ycf1, and ycf2 are those with significant variation [25].Hence, further developing and screening structural variations in the chloroplast genome of K. ewersmanniana could be applied to species identification and phylogenetic development for K. ewersmanniana with its related species.
The main reason for the structural variation of the chloroplast genome is the variation of IR/LSC boundaries, and its contraction and expansion play key roles in the evolution of plant chloroplast DNA.Compared with that in other species, genetic structural changes in K. ewersmanniana occurred in all IR boundary regions.Genes and regions with high degrees of variation were identified and validated as potential molecular markers.
The phylogenetic tree based on chloroplast genome construction showed that 35 species of Amaranthaceae plants were clustered into one group.K. ewersmanniana and K. ceratoides were grouped together with 100% bootstrap values, and were most closely related to the clade of Atriplex, Chenopodium, and Dysphania, species with 100% bootstrap values; this is consistent with previous research results on the comparative analysis of K. ceratoides chloroplast genomes [25].Atriplex and Dysphania were inserted in the Chenopodium, along with Krascheninnikovia, together divided into Tribe Chenopodieae [60].Among Amaranthaceae, Liu et al. [25] found that Suaeda had the closest phylogeny to Atriplex.Atriplex and Suaeda formed different topological structures with other genera in this study.Determining the taxonomy based on morphology of Krascheninnikovia species has been difficult due to their characteristic transition from entomophily to anemophily and minor flowers.A comprehensive analysis of the morphology has not yet been attained due to lack of research at the molecular level, and the identification and taxonomy of Krascheninnikovia and even Amarathaceae are therefore yet to be elucidated.

Conclusions
To sum up, our study for the first time assembled the chloroplast genome of K. ewersmanniana, a plant that is widely distributed in the Xinjiang desert and has important ecological and economic value.This research not only enriches existing molecular biology data for this species, but also expands available genomic resources.In addition, we reported the evolutionary relationship between K. ewersmanniana and other species of Amaranthaceae.The results of the phylogenetic tree analysis suggested that K. ewersmanniana and K. ceratoides, along with D. ambrosioids, D. pumilio, and D. botrys, form a closely related monophyletic group.The phylogenetic insights gained from this study can help address future taxonomic and nomenclature challenges associated with K. ewersmanniana.In addition to expanding the available genetic resources of the Krascheninnikovia species, this work provides a scientific basis for future research on population genetic diversity and the sustainable utilization of K. ewersmanniana resources.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes15050546/s1,Table S1: Accession number and sampled chloroplast genomes obtained from GenBank; Table S2: Characteristics of the chloroplast genome of K. ewersmanniana; Table S3: Identified SSRs motifs in the chloroplast genomes of two Krascheninnikovia species; Table S4: SSR locations in the chloroplast genomes of two Krascheninnikovia species.

Genes 2024 , 16 Figure 1 .
Figure 1.Map of the chloroplast genome structure of K. ewersmanniana.Genes drawn in circles are transcribed clockwise, whereas genes outside the circle are transcribed counterclockwise.Different colors are annotated according to the different functions of the genes.

Figure 1 .
Figure 1.Map of the chloroplast genome structure of K. ewersmanniana.Genes drawn in circles are transcribed clockwise, whereas genes outside the circle are transcribed counterclockwise.Different colors are annotated according to the different functions of the genes.

Figure 2 .
Figure 2. Relative synonymous codon usage of amino acids of K. ewersmanniana.The blocks underneath stand for different codon encoding amino acids.The columns on the top depict the sums of RSCU values of the 20 amino acids.

Figure 2 .
Figure 2. Relative synonymous codon usage of amino acids of K. ewersmanniana.The blocks underneath stand for different codon encoding amino acids.The columns on the top depict the sums of RSCU values of the 20 amino acids.

Figure 3 .
Figure 3. Analysis of tandem repeat sequences in the plastomes of two Krascheninnikovia plants.(A) type of scattered repeats; (B) length of scattered repeats.

Figure 4 .
Figure 4. Analysis of SSRs in the chloroplast genome of two Krascheninnikovia plants.(A) Frequency of identified SSR motifs; (B) location distribution of all SSR motifs.

Figure 3 .
Figure 3. Analysis of tandem repeat sequences in the plastomes of two Krascheninnikovia plants.(A) type of scattered repeats; (B) length of scattered repeats.

Figure 3 .
Figure 3. Analysis of tandem repeat sequences in the plastomes of two Krascheninnikovia plants.(A) type of scattered repeats; (B) length of scattered repeats.

Figure 4 .
Figure 4. Analysis of SSRs in the chloroplast genome of two Krascheninnikovia plants.(A) Frequency of identified SSR motifs; (B) location distribution of all SSR motifs.

Figure 4 .
Figure 4. Analysis of SSRs in the chloroplast genome of two Krascheninnikovia plants.(A) Frequency of identified SSR motifs; (B) location distribution of all SSR motifs.

Figure 5 .
Figure 5. Comparative analysis of four boundary regions in the chloroplast genomes of seven Amaranthaceae plants.

Figure 5 .
Figure 5. Comparative analysis of four boundary regions in the chloroplast genomes of seven Amaranthaceae plants.Genes 2024, 15, x FOR PEER REVIEW 10 of 16

Figure 6 .
Figure 6.Global alignment of seven chloroplast genomes using C. quinoa as a reference.Horizontal axis represents coordinates in the chloroplast genome; vertical axis indicates the average percentage sequence similarity in the aligned regions from 50% to 100%.

Figure 7 .
Figure 7.Nucleotide polymorphisms in chloroplast genome of two Krascheninnikovia plants.Horizontal and vertical axes show the name of the gene and Pi value, respectively.

Figure 6 .
Figure 6.Global alignment of seven chloroplast genomes using C. quinoa as a reference.Horizontal axis represents coordinates in the chloroplast genome; vertical axis indicates the average percentage sequence similarity in the aligned regions from 50% to 100%.

Figure 6 .
Figure 6.Global alignment of seven chloroplast genomes using C. quinoa as a reference.Horizontal axis represents coordinates in the chloroplast genome; vertical axis indicates the average percentage sequence similarity in the aligned regions from 50% to 100%.

Figure 7 .
Figure 7.Nucleotide polymorphisms in chloroplast genome of two Krascheninnikovia plants.Horizontal and vertical axes show the name of the gene and Pi value, respectively.

Genes 2024 ,
15, x FOR PEER REVIEW 11 of 16 petiolare and C. quinoa).These clades were located at the top of the phylogenetic tree and are the most recently isolated of the 35 species of the Amaranthaceae family.

Figure 8 .
Figure 8. Phylogenetic tree of K. ewersmanniana with 34 other representative Amaranthaceae species.A. thaliana and O. sativa were selected as outgroups.The maximum likelihood and Bayesian tree were determined based on shared protein-coding genes.Maximum likelihood bootstrap support values/Bayesian posterior probabilities are shown for each node.

Figure 8 .
Figure 8. Phylogenetic tree of K. ewersmanniana with 34 other representative Amaranthaceae species.A. thaliana and O. sativa were selected as outgroups.The maximum likelihood and Bayesian tree were determined based on shared protein-coding genes.Maximum likelihood bootstrap support values/Bayesian posterior probabilities are shown for each node.

Author
Contributions: P.W. conducted experiments, collected and analyzed data, and drafted the manuscript.Z.W. designed the experiment, and revised the manuscript.Y.L. collected plant materials.M.K., A.A. and Y.H. contributed to the interpretation of results.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Xinjiang Tianshan Talents Training Program "Agriculture, Rural Backbone" Talent Training Program (2022SNGGNT096), National Key Research and Development Program of China (2023YFD1200305), Xinjiang Tianshan Talents "Program-Science and Technology Innovation Leading Talents (team) (20221100619), Key Projects in Science and Technology of Inner Mongolia (2021ZD0031), and the Hohhot Science and Technology Plan (2022-she-zhong-1-2). Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.

Table 1 .
Gene functional classification and composition of the K. ewersmanniana chloroplast genome.

Table 2 .
Length of exons and introns of split genes in the chloroplast genome of K. ewersmanniana.
remaining codon ended in G.The proportion of codons ending in A/U was 96.67%.Consequently, K. ewersmanniana favors codons ending in A/U.Note: * Terrestrial amino acid (Ter).
Nucleotide polymorphisms in chloroplast genome of two Krascheninnikovia plants.Horizontal and vertical axes show the name of the gene and Pi value, respectively.