Rapid and visual monitoring of alien sequences using crop wild relatives specific oligo-painting: The case of cucumber chromosome engineering.

Wild species related to domesticated crops (crop wild relatives, or CWRs) represent a high level of genetic diversity that provides a practical gene pool for crop pre-breeding employed to address climate change and food demand challenges globally. Nevertheless, rapid identifying and visual tracking of alien chromosomes and sequences derived from CWRs have been a technical challenge for crop chromosome engineering. Here, a species-specific oligonucleotide (oligo) pool was developed by using the reference genome of Cucumis hystrix (HH, 2n = 2x = 24), a wild species carrying many favorable traits and interspecific compatibility with cultivated cucumber (C. sativus, CC, 2n = 2x = 14). These synthetic double-stranded oligo probes were applied to validate the assembly and characterize the chromosome architectures of C. hystrix, as well as to rapidly identify C. hystrix-chromosomes in diverse C. sativus-hystrix chromosome-engineered germplasms, including interspecific hybrid F1 (HC), synthetic allopolyploids (HHCC, CHC, and HCH) and alien additional lines (CC-H). Moreover, a ∼2Mb of C. hystrix-specific sequences, introduced into cultivated cucumber, were visualized by CWR-specific oligo-painting. These results demonstrate that the CWR-specific oligo-painting technique holds broad applicability for chromosome engineering of numerous crops, as it allows rapid identification of alien chromosomes, reliable detection of homoeologous recombination, and visual tracking of the introgression process. It is promising to achieve directed and high-precision crop pre-breeding combined with other breeding techniques, such as CRISPR/Cas9-mediated chromosome engineering.

Identification and Expression Analysis of the CsMYB Gene Family in Root Knot Nematode-Resistant and Susceptible Cucumbers.

MYB (myeloblastosis) transcription factors (TFs) play important roles in controlling various physiological processes in plants, such as responses to biotic and abiotic stress, metabolism, and defense. A previous study identified a gene, Csa6G410090, encoding a plant lipid transfer protein (LTP), as a possible regulator in cucumber (Cucumis sativus L.) of the resistance response to root-knot nematode (RKN) [Meloidogyne incognita Kofoid and White (Chitwood)]. Myb-type DNA-binding TFs were presumed to regulate downstream genes expression, including LTPs, however, the regulation mechanism remained unclear. To elucidate whether and which MYB TFs may be involved in regulation of the resistance response, this study identified 112 genes as candidate members of the CsMYB gene family by combining CDD and SMART databases, using the Hidden Markov Model (HMM) and manual calibration. Within this group, ten phylogenetic subgroups were resolved according to sequence-based classification, consistent with results from comprehensive investigation of gene structure, conserved motifs, chromosome locations, and cis-element analysis. Distribution and collinearity analysis indicated that amplification of the CsMYB gene family in cucumber has occurred mainly through tandem repeat events. Spatial gene expression analysis showed that 8 CsMYB genes were highly expressed at differing levels in ten different tissues or organs. The roots of RKN-resistant and susceptible cucumbers were inoculated with M. incognita, finding that CsMYB (Csa6G538700, Csa1G021940, and Csa5G641610) genes showed up-regulation coincident with upregulation of the "hub" gene LTP (Csa6G410090) previously implicated as a major gene in the resistance response to RKN in cucumber. Results of this study suggest hypotheses regarding the elements and regulation of the resistant response as well as possible RKN resistance-enhancing strategies in cucumber and perhaps more broadly in plants.

Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis.

Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double-stranded labelled oligos, which produced much stronger signals than single-stranded labelled oligos, by amplification using fluorophore-conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross-species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo-painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome.

Candidate genes underlying the quantitative trait loci for root-knot nematode resistance in a Cucumis hystrix introgression line of cucumber based on population sequencing.

The southern root-knot nematode (RKN), Meloidogyne incognita (Kofoid & White) Chitwood, is one of most destructive species of plant parasitic nematodes, causing significant economic losses to numerous crops including cucumber (Cucumis sativus L. 2n = 14). No commercial cultivar is currently available with resistance to RKN, severely hindering the genetic improvement of RKN resistance in cucumber. An introgression line, IL10-1, derived from the interspecific hybridization between the wild species Cucumis hystrix Chakr. (2n = 24, HH) and cucumber, was identified with resistance to RKN. In this study, an ultrahigh-density genetic linkage bin-map, composed of high-quality single-nucleotide polymorphisms (SNPs), was constructed based on low-coverage sequences of the F2:6 recombinant inbred lines derived from the cross between inbred line IL10-1 and cultivar 'Beijingjietou' CC3 (hereinafter referred to as CC3). Three QTLs were identified accounting for 13.36% (qRKN1-1), 9.07% and 9.58% (qRKN5-1 and qRKN5-2) of the resistance variation, respectively. Finally, four genes with nonsynonymous SNPs from chromosome 5 were speculated to be the candidate RKN-resistant related genes, with annotation involved in disease resistance. Though several gaps still exist on the bin-map, our results could potentially be used in breeding programs and establish an understanding of the associated mechanisms underlying RKN resistance in 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.

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

BACKGROUND: Due 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. RESULTS: Degenerate 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. CONCLUSIONS: Four 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.

Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber.

Quantitative real-time polymerase chain reaction (QRT-PCR) has become one of the most widely used methods for gene expression analysis. However, the expression profile of a target gene may be misinterpreted due to unstable expression of the reference genes under different experimental conditions. Thus, a systematic evaluation of these reference genes is necessary before experiments are performed. In this study, 10 putative reference genes were chosen for identifying expression stability using geNorm, NormFinder, and BestKeeper statistical algorithms in 12 different cucumber sample pools, including those from different plant tissues and from plants treated with hormones and abiotic stresses. EF1alpha and UBI-ep exhibited the most stable expression across all of the tested cucumber samples. In different tissues, in addition to expression of EF1alpha and UBI-ep, the expression of TUA was also stable and was considered as an appropriate reference gene. Evaluation of samples treated with different hormones revealed that TUA and UBI-ep were the most stably expressed genes. However, for abiotic stress treatments, only EF1alpha showed a relatively stable expression level. In conclusion, TUA, UBI-ep, and EF1alpha will be particularly helpful for reliable QRT-PCR data normalization in these types of samples. This study also provides guidelines for selecting different reference genes under different conditions.

Cucumis monosomic alien addition lines: morphological, cytological, and genotypic analyses.

Cucumis hystrix Chakr. (HH, 2n=24), a wild relative of the cultivated cucumber, possesses several potentially valuable disease-resistance and abiotic stress-tolerance traits for cucumber ( C. sativus L., CC, 2n=14) improvement. Numerous attempts have been made to transfer desirable traits since the successful interspecific hybridization between C. hystrix and C. sativus, one of which resulted in the production of an allotriploid (HCC, 2n=26: one genome of C. hystrix and two of C. sativus). When this genotype was treated with colchicine to induce polyploidy, two monosomic alien addition lines (MAALs) (plant nos. 87 and 517: 14 CC+1 H, 2n=15) were recovered among 252 viable plants. Each of these plants was morphologically distinct from allotriploids and cultivated cucumbers. Cytogenetic and molecular marker analyses were performed to confirm the genetic constitution and further characterize these two MAALs. Chromosome counts made from at least 30 meristematic cells from each plant confirmed 15 nuclear chromosomes. In pollen mother cells of plant nos. 87 and 517, seven bivalents and one univalent were observed at diakinesis and metaphase I; the frequency of trivalent formation was low (about 4-5%). At anaphase I and II, stochastic and asymmetric division led to the formation of two gamete classes: n=7 and n=8; however, pollen fertility was relatively high. Pollen stainability in plant no. 87 was 86.7% and in plant no. 517 was 93.2%. Random amplified polymorphic DNA analysis was performed using 100 random 10-base primers. Genotypes obtained with eight primers (A-9, A-11, AH-13, AI-19, AJ-18, AJ-20, E-19, and N-20) showed a band common to the two MAAL plants and C. hystrix that was absent in C. sativus, confirming that the alien chromosomes present in the MAALs were derived from C. hystrix. Morphological differences and differences in banding patterns were also observed between plant nos. 87 and 517 after amplification with primers AI-5, AJ-13, N-12, and N-20, suggesting that these plants may contain different C. hystrix chromosomes.