Hystrix Chakr Research Articles

Transcriptomic Analysis of Cucumis hystrix and the Functional Identification of ChTrxh under NaCl Stress

Salinity is a prominent environmental stressor that significantly impacts plant growth and development. Here, we conducted research on the physiological and transcriptomic mechanism of a wild cucumber, Cucumis hystrix Chakr, under NaCl stress. Physiological data showed that contents of malondialdehyde, peroxide (H2O2), proline, soluble sugar, and activities of antioxidant enzymes of superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione reductase in wild cucumber plants were increased significantly after NaCl treatment. Transcriptomic analysis revealed that 3509 transcripts were differentially expressed in leaves and 5516 transcripts in roots after NaCl treatment. Numerous genes were related to the signal transduction, transcription factor, ion transport, osmotic metabolism, and reactive oxygen species scavenging. Moreover, the thioredoxin H type gene of Cucumis hystrix Chakr (ChTrxh) was isolated and characterized. Our study demonstrated that the transgenic tobacco plants overexpressing ChTrxh exhibited enhanced tolerance to NaCl stress compared to wild-type plants. These findings contribute valuable insights into the functional characteristics of important genes in wild cucumber under NaCl stress.

Chromosome-scale genome assembly of Cucumis hystrix\u2014a wild species interspecifically cross-compatible with cultivated cucumber

Cucumis hystrix Chakr. (2n = 2x = 24) is a wild species that can hybridize with cultivated cucumber (C. sativus L., 2n = 2x = 14), a globally important vegetable crop. However, cucumber breeding is hindered by its narrow genetic base. Therefore, introgression from C. hystrix has been anticipated to bring a breakthrough in cucumber improvement. Here, we report the chromosome-scale assembly of C. hystrix genome (289 Mb). Scaffold N50 reached 14.1 Mb. Over 90% of the sequences were anchored onto 12 chromosomes. A total of 23,864 genes were annotated using a hybrid method. Further, we conducted a comprehensive comparative genomic analysis of cucumber, C. hystrix, and melon (C. melo L., 2n = 2x = 24). Whole-genome comparisons revealed that C. hystrix is phylogenetically closer to cucumber than to melon, providing a molecular basis for the success of its hybridization with cucumber. Moreover, expanded gene families of C. hystrix were significantly enriched in “defense response,” and C. hystrix harbored 104 nucleotide-binding site–encoding disease resistance gene analogs. Furthermore, 121 genes were positively selected, and 12 (9.9%) of these were involved in responses to biotic stimuli, which might explain the high disease resistance of C. hystrix. The alignment of whole C. hystrix genome with cucumber genome and self-alignment revealed 45,417 chromosome-specific sequences evenly distributed on C. hystrix chromosomes. Finally, we developed four cucumber–C. hystrix alien addition lines and identified the exact introgressed chromosome using molecular and cytological methods. The assembled C. hystrix genome can serve as a valuable resource for studies on Cucumis evolution and interspecific introgression breeding of cucumber.

The complete chloroplast genome sequence of the wild cucumber Cucumis hystrix Chakr. (Cucumis, cucurbitaceae)

The complete nucleotide sequence of the wild cucumber (C. hystrix Chakr.) chloroplast genome has been determined in this study. The genome was composed of 155,031 bp containing a pair of inverted repeats (IRs) of 25,150 bp, which was separated by a large single-copy region of 86,564 bp and a small single-copy region of 18,166 bp. The chloroplast genome contained 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species) and 37 tRNA genes (30 tRNA species), with 18 of them located in the IR region. In these genes, 16 contained 1 intron, and 2 genes and one ycf contained 2 introns.

Retrotransposon- and microsatellite sequence-associated genomic changes in early generations of a newly synthesized allotetraploid Cucumis × hytivus Chen & Kirkbride

Allopolyploidization is considered an essential evolutionary process in plants that could trigger genomic shock in allopolyploid genome through activation of transcription of retrotransposons, which may be important in plant evolution. Two retrotransposon-based markers, inter-retrotransposon amplified polymorphism and retrotransposon-microsatellite amplified polymorphism and a microsatellite-based marker, inter simple sequence repeat were employed to investigate genomic changes in early generations of a newly synthesized allotetraploid Cucumis×hytivus Chen & Kirkbride (2n=4x=38) which was derived from crossing between cultivated cucumber C. sativus L. (2n=2x=14) and its wild relative C. hystrix Chakr. (2n=2x=24). Extensive genomic changes were observed, most of which involved the loss of parental DNA fragments and gain of novel fragments in the allotetraploid. Among the 28 fragments examined, 24 were lost while four were novel, suggesting that DNA sequence elimination is a relatively frequent event during polyploidization in Cucumis. Interestingly, of the 24 lost fragments, 18 were of C. hystrix origin, four were C. sativus-specific, and the remaining two were shared by both species, implying that fragment loss may be correlated with haploid DNA content (genome size) of diploid parents. Most changes were observed in the first generation after polyploidization (S(1)) and stably inherited in the subsequent three generations (S(2)-S(4)), indicating that genomic changes might be a rapid driving force for the stabilization of allotetraploids. Sequence analysis of 11 of the 28 altered DNA fragments showed that genomic changes in the allotetraploid occurred in both coding and non-coding regions, which might suggest that retrotransposons inserted into genome randomly and had a genome-wide effect on the allotetraploid evolution. Fluorescence in situ hybridization (FISH) analysis revealed a unique distribution of retrotransposon and/or microsatellite flanking sequences in mitotic and meiotic chromosomes, where the preferential FISH signals occurred in the centromeric and telomeric regions, implying that these regions were the possible hotspots for genomic changes.

Marker-assisted backcross selection in an interspecific Cucumis population broadens the genetic base of cucumber (Cucumis sativus L.)

Cucumber (Cucumis sativus L.) is a major cucurbit vegetable species whose genetic base has been drastically reduced during its domestication. The crop’s narrow genetic base (3–12% DNA polymorphism) has resulted from the use of limited genetic material and intense selection during plant improvement. Recently, however, interspecific hybridization has been successful in Cucumis via mating of C. hystrix Chakr. and C. sativus, which resulted in the amphidiploid C. hytivus. We report herein a marker-assisted strategy for increasing genetic diversity in cucumber through introgression backcrossing employing C. hytivus. The comparatively late-flowering but high-yielding, indeterminate, monoecious line WI 7012A (P1; donor parent) derived from a C. hytivus × C. sativus-derived line (long-fruited Chinese C. sativus cv. Beijingjietou) was initially crossed to the determinate, gynoecious C. sativus line WI 7023A (P2; recurrent parent 1), and then advanced backcross generation progeny (BC2) were crossed with the gynoecious indeterminate line WI 9-6A (P3; recurrent parent 2). More specifically, a single F1 individual (P1 × P2) was backcrossed to P2, and then BC progeny were crossed to P2 and P3, where marker-assisted selection (MAS) for genetic diversity (8 mapped and 16 unmapped markers; designated Sel) or no selection (designated NSel) was applied to produce BC3P2 (Sel) and BC3P3 (Sel), and BC2P2 (NSel) and BC2P2S1 (NSel) progeny. Relative vegetative growth, number of lateral branches (LB), days to flowering (DF), yield (fruit number), and fruit quality [as measured by length:diameter (L:D) and endocarp:total diameter (E:T) ratios] were assessed in parents and cross-progeny. DF varied from ~20 (BC3P2Sel) to ~25 days (BC2P3Sel) among the populations examined, where progeny derived from P2 possessed the shortest DF. Differences in cumulative yield among the populations over six harvests were detected, varying from ~8 fruits per plant in BC3P2 (Sel) to ~39 fruits per plant in BC2P3 (Sel). Although the vigorous vegetative growth of line P1 was observed in its backcross progeny, highly heterozygous and polymorphic backcross progeny derived from P3 were comparatively more vigorous and bore many high-quality fruit. Response to selection was detected for LB, DF, L:D, and E:T, but the effectiveness of MAS depended upon the parental lines used. Data indicate that the genetic diversity of commercial cucumber can be increased by introgression of the C. hystrix genome through backcrossing.

Identification and characterization of potential NBS-encoding resistance genes and induction kinetics of a putative candidate gene associated with downy mildew resistance in Cucumis.

BackgroundDue to the variation and mutation of the races of Pseudoperonospora cubensis, downy mildew has in recent years become the most devastating leaf disease of cucumber worldwide. Novel resistance to downy mildew has been identified in the wild Cucumis species, C. hystrix Chakr. After the successful hybridization between C. hystrix and cultivated cucumber (C. sativus L.), an introgression line (IL5211S) was identified as highly resistant to downy mildew. Nucleotide-binding site and leucine-rich repeat (NBS-LRR) genes are the largest class of disease resistance genes cloned from plant with highly conserved domains, which can be used to facilitate the isolation of candidate genes associated with downy mildew resistance in IL5211S.ResultsDegenerate primers that were designed based on the conserved motifs in the NBS domain of resistance (R) proteins were used to isolate NBS-type sequences from IL5211S. A total of 28 sequences were identified and named as cucumber (C. sativus = CS) resistance gene analogs as CSRGAs. Polygenetic analyses separated these sequences into four different classes. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that these CSRGAs expressed at different levels in leaves, roots, and stems. In addition, introgression from C. hystrix induced expression of the partial CSRGAs in cultivated cucumber, especially CSRGA23, increased four-fold when compared to the backcross parent CC3. Furthermore, the expression of CSRGA23 under P. cubensis infection and abiotic stresses was also analyzed at different time points. Results showed that the P. cubensis treatment and four tested abiotic stimuli, MeJA, SA, ABA, and H2O2, triggered a significant induction of CSRGA23 within 72 h of inoculation. The results indicate that CSRGA23 may play a critical role in protecting cucumber against P. cubensis through a signaling the pathway triggered by these molecules.ConclusionsFour classes of NBS-type RGAs were successfully isolated from IL5211S, and the possible involvement of CSRGA23 in the active defense response to P. cubensis was demonstrated. These results will contribute to develop analog-based markers related to downy mildew resistance gene and elucidate the molecular mechanisms causing resistance in IL5211S in the future.

Backcross Introgression of the Cucumis hystrix Genome Increases Genetic Diversity in U.S. Processing Cucumber

The genetic base of commercial cucumber (Cucumis sativus L.) is extremely narrow (about 3%–8% polymorphism). Wide-based crosses within C. sativus [i.e., C. sativus var. sativus × C. sativus var. hardwickii (R.) Alef.] and interspecific hybridization attempts before 1995 have not substantially increased genetic diversity for plant improvement. However, in 1995, an amphidiploid (Cucumis hytivus Chen and Kirkbride) was derived from a C. sativus × Cucumis hystrix Chakr. mating. A derivative of this amphidiploid was used herein to broaden the genetic base of cucumber through backcross introgression [(C. sativus × C. hytivus) × C. sativus]. Initially, the combining ability of eight genetically diverse lines was investigated for days to anthesis (DA), sex expression (SEX), lateral branch number (LBN), fruit per plant (FP), fruit length:diameter ratio (L:D), and salt-processing ability [i.e., processed fruit color (exterior and interior), shape, and seed cavity characteristics]. Based on the combining ability, inbred backcross lines [IBL (BC2S3)] were developed from an original gynoecious determinate line WI 7023A [C. sativus (recurrent parent)] × monoecious indeterminate line WI 7012A (C. sativus × C. hytivus derived) mating, where 30 of 392 (8%) BC1 progeny were selected based on their diversity at 16 mapped marker loci. These progeny were used to develop BC2 progeny, which were then self-pollinated without further selection to produce 94 IBL. These IBL were genotyped and evaluated in the open field in two plantings in 2008 for DA, SEX, LBN, leaf size, FP, and L:D. The genetic distance (GD) between parental lines was 0.85, and the GD among IBL ranged between 0.16 and 0.75. Multivariate analyses indicated that IBL differed from parental lines and possessed considerable morphological and genotypic diversity that could be used to broaden the genetic base of commercial U.S. processing cucumber.

Analysis of cytoplasmic variation in a cucumber germplasm collection using chloroplast microsatellite markers

Nine PCR-based markers were developed from the microsatellites in non-coding regions of chloroplast genome of Cucumis sativus and used to detect chloroplast DNA variation. These markers successfully detected intraspecific polymorphism among 37 cucumber accessions containing Chinese native germplasms (CNGs) and non-Chinese germplasms (NCGs). Each marker detected between two and four alleles and the diversity value of the makers ranged from 0.105 to 0.528. Based on the data from allele size variation, a total of 17 distinct haplotypes were identified from the 35 accessions (excluding the two accessions possessing null genes). Three haplotypes were prevalent among CNGs but most NCGs had unique haplotype. No identical haplotype was found between CNGs and NCGs, reflecting lack of exchange of CNGs with others in the 60–80s of last century. A wild species (C. hystrix Chakr.) tested herein shared a haplotype with some CNGs, suggesting that it could be the ancestry of C. sativus or at least had a common ancestral lineage. The genetic relationship among the 37 cucumber accessions was further analyzed through construction of dendrogram based on Jaccard coefficient of similarity obtained from the allele sizes. All the CNGs were clustered into a group (containing the wild accession) that distinctly differed from the other four groups containing NCGs. This result agreed with the findings above obtained from haplotype analysis. Our research documented here will offer useful information for cucumber breeding.

Molecular analysis of introgression lines from Cucumis hystrix Chakr. to C. sativus L

Two stable introgression lines (ILs), IL56 and IL73 with fourteen chromosomes, derived from a cross between the wild relative Cucumis hystrix Chakr. (2 n = 24) and the cultivated cucumber “beijingjietou” ( Cucumis sativus L., 2 n = 14) were characterized using SSR and AFLP markers. Twenty-three SSR primers were used to detect the introgression from C. hystrix to C. sativus, and one locus at 210 bp was revealed and assigned to introgressive fragment of C. hystrix genome in IL56. Substantial genomic changes in these two lines were detected by AFLP analysis with thirty different primer combinations. C. hystrix-specific bands, novel bands for the two parents and deleted bands in C. sativus were observed. One hundred and forty-seven polymorphic bands were observed, but only four and five introgressed bands were found in IL56 and IL73, respectively. Although representing only a small proportion of the C. hystrix genome, these two introgression lines with downy mildew and fusarium wilt resistance will be valuable to resistant genetics and breeding.

Inheritance of aspartate aminotransferase (AAT) inCucumisspecies as revealed by interspecific hybridization

The inheritance of aspartate aminotransferase (AAT) isozymes was investigated in Cucumis sativus L. (CC; 2n = 2x = 14), Cucumis hystrix Chakr. (HH; 2n = 2x = 24), the synthetic amphidiploid species C. hytivus Chen & Kirkbride (HHCC; 2n = 4x = 38), and the allotriploid (HCC; 2n = 3x = 26) from backcrossing C. hytivus to C. sativus. Two polymorphic loci, Aat-1 and Aat-2, and one monomorphic locus, Aat-3, were detected among these parents and their progenies by using polyacrylamide gel electrophoresis. Cucumis sativus displayed the fast-migrating anodic band for Aat-1 (2), while C. hystrix contained a slow-migrating cathodic band (1). For Aat-2, the slow-migrating cathodic band was observed with C. sativus (1), whereas C. hystrix contained the fast-migrating anodic band (2). Cucumis hytivus, a synthetic species derived from doubling the chromosome number of a F1from a C. sativus × C. hystrix mating, exhibited the typical hybrid dimeric banding pattern resulting from the combination of two parental homomeric products with equal staining intensity and a heteromeric product with intermediate mobility and greater staining intensity than either homomeric product. The difference in band intensity between C. hytivus and its backcross progenies, when C. sativus was the recurrent parent, were due to the dosage effects of alleles at Aat-1 and Aat-2. These banding morphotypes can be used for typing of C. hytivus and BC1progenies that are similar in morphology. Aat-3 was monomorphic in this mating, encoding a single allele Aat-3 (1).

Taxonomic Relationships of A Rare Cucumis Species (C. hystrix Chakr.) and Its Interspecific Hybrid with Cucumber

The current Cucumis taxonomic classification places C. hystrix Chakr. in subgen. Cucumis based on its morphological similarities to cucumber (C. sativus L., 2n = 14). However, the chromosome number of C. hystrix was identified as 2n = 24, the same number as in subgen. Melo. Cucumis hystrix is therefore considered the first wild Cucumis species of Asiatic origin possessing 12 basic chromosomes. Thus, any research regarding its biosystematics would challenge the basic chromosome number and geographic location theories that govern the current taxonomic system. The production of the amphidiploid species (Cucumis ×hytivus Chen and Kirkbride, 2n = 38) obtained from the cross between C. hystrix and C. sativus and subsequent chromosome doubling would provide an effective means of investigating the relationship between Cucumis species with two different basic chromosome numbers. Thus, RAPD markers were used to study the taxonomic placement of C. hystrix and its interspecific hybrid with cucumber. Of the 220 arbitrary primers screened, 31 were used for analysis where 402 (96.3%) fragments were polymorphic among the germplasm examined. A UPGMA-based cluster analysis partitioned 31 accessions into two main groups [C. sativus (CS) and C. melo (CM)]. Under the similarity coefficient threshold of 0.23, these two groups can be further divided into five clusters with C. hystrix, C. ×hytivus, and C. sativus as separate clusters in the CS group. A modified taxonomic system is proposed based on these results and findings of a previous chloroplast DNA analysis with the genus Cucumis containing subgen. Cucumis with three species and subgen. Melo with six series.

Molecular phylogeny ofCucumisspecies as revealed by consensus chloroplast SSR marker length and sequence variation

To investigate phylogenetic relationships in the genus Cucumis, 9 consensus chloroplast simple sequence repeat (ccSSR) primer pairs (ccSSR3, 9, 11, 13, 14, 17, 20, 21, and 23) were employed for DNA fragment length variation and 5 amplified fragments, ccSSR4, 12, 13, 19, and 20, were sequenced using total DNA from 13 accessions representing 7 African Cucumis species (x = 12), 3 Cucumis melo L. (x = 12) accessions, 2 Cucumis sativus L. (x = 7) accessions, and 1 Cucumis hystrix Chakr. (x = 12) accession. A Citrullus lanatus (Thunb.) Matsum. & Nakai (x = 11) accession was used as an outgroup. While fragment length analysis revealed the existence of 3 major species clusters (i.e., a group of African Cucumis species, a group composed of C. melo accessions, and a group containing C. sativus and C. hystrix species), sequence variation analysis identified 2 major species clusters (i.e., a group of African Cucumis species and a group composed of C. melo, C. sativus, and C. hystrix species). Comparative analysis using nuclear DNA (previous studies) and cpDNA sequence substitution data resulted in the placement of C. melo and C. sativus in different cluster groupings. Thus, both nuclear and cytoplasmic DNA should be employed and compared when a putative progenitor or specimens of an ancestral Cucumis species lineage is investigated. In addition, C. ficifolius (2x) and C. aculeatus (4x) of the African Cucumis species clustered together in this study. This result does not agree with reported isozyme analyses, but does agree with previously characterized chromosome homologies between these 2 species. Although African Cucumis species and C. hystrix do not share a close relationship, genetic affinities between C. sativus and C. hystrix are considerable. Combined evidence from previously published studies and data presented herein lend support to the hypothesis that C. hystrix is either a progenitor species of C. sativus or that they at least share a common ancestral lineage.

Synthesis and preliminary characterization of a new species (amphidiploid) in Cucumis

A successful interspecific hybridization between cucumber (Cucumis sativus L., 2n = 14) and a wild it Cucumis species, C. hystrix Chakr. (2n = 24) was made via embryo rescue. Hybrid plants (2n = 19; 7 from cucumber and 12 from C. hystrix) were sterile, but morphologically uniform. Self-pollination and backcrossing of F1 hybrid plants to either parent confirmed presence of both male- and female-sterility that were likely caused by lack of homology and improper pairing during meiosis. While the multiple-branching habit, densely brown hairs (on corolla and pistil), orange-yellow corolla, and ovate fruit of F1 hybrid plants were similar to that of the C. hystrix parent, the appearance of the first pistillate flower was more similar to that of C. sativus parent. The diameter and internode length of the stem, and the shape and size of leaves and flowers were intermediate when compared to the parents. The chromosome number in the hybrid was doubled through somaclonal variation during embryo culture and regeneration process to restore the fertility. Pollen grains were released and fruits with viable seeds matured on fertile, synthetic amphidiploid plants. The results from flow cytometry indicated that, on average, 7.3% of the morphologically unique regenerants had the 4C DNA content of 2.35 pg relative to the 2C DNA content of the original F1 hybrid at 1.17 pg and, therefore, were likely chromosome-doubled F1 hybrids (2n = 38). Nutrition alanalysis indicated that the synthetic species had higher protein (0.78%)and mineral (0.35%) content compared to the normal pickling cucumber(0.62% and 0.27%, respectively), and could be considered a new Cucumis crop having a special place in the future agriculture. Preliminarily evaluation indicates that C. hystrix possesses a high level of root-knot nematode resistance, and that this resistance is partially expressed in the interspecific F1 and chromosome-doubled F1. This and the fact that the fruit morphology of the fertile amphidiploid differs during the growing season (e.g., short and long fruit) suggest that it could be useful in broadening the germplasm base of cucumber.

A New Synthetic Species of Cucumis (Cucurbitaceae) from Interspecific Hybridization and Chromosome Doubling

A new synthetic species,Cucumis hytivus J.-F. Chen & J. H. Kirkbr. (2n=38), obtained by doubling the number of chromosomes in the sterile interspecific F1 hybrid (2n=19) betweenC. sativus L. (2n=14) andC. hystrix Chakr. (2n=24), is described.

115 Micropropagation of a Rare Cucumis Species

Cucumis hystrix Chakr. is a rare cucurbit species native to Asia. The species is valued by breeders because of its multiple branching habit and has been used in interspecific crosses with Cucumis sativus. However, individual C. hystrix plants have not been identified in the wild since 1990. Therefore, it was our objective to develop a micropropagation protocol that would allow us to clonally propagate plants in cultivation. Shoots tips (2 cm) were excised from a single C. hystrix plant grown in the greenhouse. All tendrils and leaves were removed before surface-sterilization in 1.25% NaOCl for 5 or 10 min and rinsed six times with sterile distilled water. Shoot tips were trimmed to 1 cm (meristem with two to three young leaf primordia) and placed into 25 × 125-mm test tubes containing 25 ml of initiation medium [MS plus (per liter) 100 mg inositol, 30 g sucrose and 5 g Agargel; pH 5.7-5.8]. PGR combinations tested were initiation medium with 1 μM BA, and initiation medium with 1.7 μM IBA, 0.5 μM kinetin and 0.3 μM GA3 (IKG). Explant survival was greater when shoot tips were surface-sterilized for 5 min (75%) compared to 10 min (33%). More axillary shoots formed when shoot tips were cultured in IKG medium (10.8) than in medium with BA (5.5). Shoots were considerably longer (10 mm) when cultured in medium with IKG compared to BA (1.5 mm). About 64% of shoots place in medium containing 8 μM NAA formed roots and were acclimatized to greenhouse conditions.

Successful interspecific hybridization between Cucumis sativus L. and C. hystrix Chakr

Interspecific F1 hybrids were obtained from a cross between Cucumis sativus L. (2n = 2x = 14) and C. hystrix Chakr. (2n = 2x = 24). Controlled crossing resulted in fruit containing embryos which were excised and rescued on a Murashige and Skoog solid medium. A total of 59 vigorous plants were obtained from a fruit containing 159 embryos (37.3% regeneration rate). Hybrid plants were morphologically uniform. The multiple branching habit, densely brown hairs (especially on corolla and pistil), orange-yellow collora, and ovate fruit of F1 hybrid plants were similar to that of the C. hystrix paternal parent. While appearance of the first pistillate flower was more similar to that of C. sativus maternal parent than to C. hystrix, staminate flower appearance was mid-parent in occurence. The diameter and internode length of stem, shape and size of leaves and flowers were intermediate when compared to the parents. An elongated green, trilobate style/stigma which was not apparent in either parent was observed in staminate flowers of F1 plants. Similarly, the style/stigma of pistillate flower of F1 plants were longger when compared to their parents. The brown pubescence observed on pistillate flowers of the F1 and C. hystrix was not observed on the C. sativus parent. The somatic chromosome number of F1 plants was 19. Two morphologically distinct groups of chromosomes were observed in the F1 hybrid; 7 relatively large chromosomes characteristic of C. sativus, and 12 smaller chromosomes characteristic of C. hystrix. Analysis of malate dehydrogenase isozyme banding patterns provided additional comfirmation of hybridity. Reciprocal crossing of F1 plants to either parent and self-crossing indicated that the hybrids were male and female sterile.