TCP transcription factor StAST1 represses potato tuberization by regulating tuberigen complex activity.

Potato (Solanum tuberosum L.) is cultivated worldwide for its underground tubers, which provide an important part of human nutrition and serve as a model system for belowground storage organ formation. Similar to flowering, stolon-expressed FLOWERING LOCUS T-like (FT-like) protein SELF-PRUNING 6A (StSP6A) plays an instrumental role in tuberization by binding to the bZIP transcription factors StABI5-like 1 (StABL1) and StFD-like 1 (StFDL1), causing transcriptional reprogramming at the stolon subapical apices. However, the molecular mechanism regulating the widely conserved FT-bZIP interactions remains largely unexplored. Here, we identified a TCP transcription factor StAST1 (StABL1 and StSP6A-associated TCP protein 1) binding to both StSP6A and StABL1. StAST1 is specifically expressed in the vascular tissue of leaves and developing stolons. Silencing of StAST1 leads to accelerated tuberization and a shortened life cycle. Molecular dissection reveals that the interaction of StAST1 with StSP6A and StABL1 attenuates the formation of the alternative tuberigen activation complex (aTAC). We also observed StAST1 directly activates the expression of potato GA 20-oxidase gene (StGA20ox1) to regulate GA responses. These results demonstrate StAST1 functions as a tuberization repressor by regulating plant hormone levels; our findings also suggest a mechanism by which the widely conserved FT-FD genetic module is fine-tuned.

Suppression of the tonoplast sugar transporter, StTST3.1, affects transitory starch turnover and plant growth in potato.

Transitory starch and vacuolar sugars function as highly dynamic pools of instantly accessible metabolites in plant leaf cells. Their metabolic regulation is critical for plant survival. The tonoplast sugar transporters (TSTs), responsible for sugar uptake into vacuoles, regulate cellular sugar partitioning and vacuolar sugar accumulation. However, whether TSTs are involved in leaf transient starch turnover and plant growth is unclear. Here, we found that suppressing StTST3.1 resulted in growth retardation and pale green leaves in potato plants. StTST3.1-silenced plants displayed abnormal chloroplasts and impaired photosynthetic performance. The subcellular localization assay and the oscillation expression patterns revealed that StTST3.1 encoded a tonoplast-localized protein and responded to photoperiod. Moreover, RNA-seq analyses identified that starch synthase (SS2 and SS6) and glucan water, dikinase (GWD), were downregulated in StTST3.1-silenced lines. Correspondingly, the capacity for starch synthesis and degradation was decreased in StTST3.1-silenced lines. Surprisingly, StTST3.1-silenced leaves accumulated exceptionally high levels of maltose but low levels of sucrose and hexose. Additionally, chlorophyll content was reduced in StTST3.1-silenced leaves. Analysis of chlorophyll metabolic pathways found that Non-Yellow Coloring 1 (NYC1)-like (NOL), encoding a chloroplast-localized key enzyme that catalyzes the initial step of chlorophyll b degradation, was upregulated in StTST3.1-silenced leaves. Transient overexpression of StNOL accelerated chlorophyll b degradation in tobacco leaves. Our results indicated that StTST3.1 is involved in transitory starch turnover and chlorophyll metabolism, thereby playing a critical role in normal potato plant growth.

Karyotyping of aneuploid and polyploid plants from low coverage whole-genome resequencing.

BACKGROUND: Karyotype, as a basic characteristic of species, provides valuable information for fundamental theoretical research and germplasm resource innovation. However, traditional karyotyping techniques, including fluorescence in situ hybridization (FISH), are challenging and low in efficiency, especially when karyotyping aneuploid and polyploid plants. The use of low coverage whole-genome resequencing (lcWGR) data for karyotyping was explored, but existing methods are complicated and require control samples. RESULTS: In this study, a new protocol for molecular karyotype analysis was provided, which proved to be a simpler, faster, and more accurate method, requiring no control. Notably, our method not only provided the copy number of each chromosome of an individual but also an accurate evaluation of the genomic contribution from its parents. Moreover, we verified the method through FISH and published resequencing data. CONCLUSIONS: This method is of great significance for species evolution analysis, chromosome engineering, crop improvement, and breeding.

Genome sequence analysis provides insights into the mode of 2n egg formation in Solanum malmeanum.

KEY MESSAGE: Our genomic investigation confirms the mechanism of 2n eggs formation in S. malmeanum and aid in optimizing the use of wild germplasm. Wild potatoes are a valuable source of agronomic traits. However, substantial reproductive barriers limit gene flow into cultivated species. 2n gametes are instrumental in preventing endosperm abortion caused by genetic imbalances in the endosperm. However, little is known about the molecular mechanisms underlying the formation of 2n gametes. Here, the wild species Solanum malmeanum Bitter (2x, 1EBN, endosperm balance number) was used in inter- and intrapoloid crosses with other Solanum species, with viable seeds being produced only when S. malmeanum was used as the female parent to cross the 2EBN Solanum genus and with the likely involvement of 2n gametes. Subsequently, we substantiated the formation of 2n eggs in S. malmeanum using fluorescence in situ hybridization (FISH) and genomic sequencing technology. Additionally, the transmission rate of maternal heterozygous polymorphism sites was assessed from a genomic perspective to analyze the mode of 2n egg formation in S. malmeanum × S. tuberosum and S. malmeanum × S. chacoense crosses; each cross acquired an average of 31.12% and 22.79% maternal sites, respectively. This confirmed that 2n egg formation in S. malmeanum attributed to second-division restitution (SDR) coupled with the occurrence of exchange events. The high-throughput sequencing technology used in this study has strong advantages over traditional cytological analyses. Furthermore, S. malmeanum, which has a variety of excellent traits not available from present cultivated potato genepool, has received little research attention and has successfully achieved gene flow in cultivated species in the current study. These findings will facilitate the understanding and optimization of wild germplasm utilization in potatoes.

Combining genome composition and differential gene expression analyses reveals that SmPGH1 contributes to bacterial wilt resistance in somatic hybrids.

KEY MESSAGE: Clarification of the genome composition of the potato + eggplant somatic hybrids cooperated with transcriptome analysis efficiently identified the eggplant gene SmPGH1 that contributes to bacterial wilt resistance. The cultivated potato is susceptible and lacks resistance to bacterial wilt (BW), a soil-borne disease caused by Ralstonia solanacearum. It also has interspecies incompatibility within Solanaceae plants. Previously, we have successfully conducted the protoplast fusion of potato and eggplant and regenerated somatic hybrids that showing resistance to eggplant BW. For efficient use of these novel germplasm and improve BW resistance of cultivated potato, it is essential to dissect the genetic basis of the resistance to BW obtained from eggplant. The strategy of combining genome composition and transcriptome analysis was established to explore the gene that confers BW resistance to the hybrids. Genome composition of the 90 somatic hybrids was studied using genomic in situ hybridization coupled with 44 selected eggplant-specific SSRs (smSSRs). The analysis revealed a diverse set of genome combinations among the hybrids and showed a possibility of integration of alien genes along with the detection of 7 smSSRs linked to BW resistance (BW-linked SSRs) in the hybrids. Transcriptome comparison between the resistant and susceptible gene pools identified a BW resistance associated gene, smPGH1, which was significantly induced by R. solanacearum in the resistant pool. Remarkably, smPGH1 was co-localized with the BW-linked SSR emh01E15 on eggplant chromosome 9, which was further confirmed that smPGH1 was activated by R. solanacearum only in the resistant hybrids. Taken together, the identified gene smPGH1 and BW-linked SSRs have provided novel genetic resources that will aid in potato breeding for BW resistance.

Examining the association between delay discounting, delay aversion and physical activity in Chinese adults with type-2 diabetes mellitus.

BACKGROUND: The role of physical activity in diabetes is critical, influencing this disease's development, man-agement, and overall outcomes. In China, 22.3% of adults do not meet the minimum level of physical activity recommended by the World Health Organization. Therefore, it is imperative to identify the factors that contributing to lack of physical activity must be identified. AIM: To investigate the relationship among delay discounting, delay aversion, glycated hemoglobin (HbA1c), and various levels of physical activity in Chinese adults diagnosed with type 2 diabetes mellitus (T2DM). METHODS: In 2023, 400 adults with T2DM were recruited from the People's Hospital of Linxia Hui Autonomous Prefecture of Gansu Province. A face-to-face questionnaire was used to gather demographic data and details on physical activity, delay discounting, and delay aversion. In addition, HbA1c levels were measured in all 400 participants. The primary independent variables considered were delay discounting and delay aversion. The outcome variables included HbA1c levels and different intensity levels of physical activity, including walking, moderate physical activity, and vigorous physical activity. Multiple linear regression models were utilized to assess the relationship between delay discounting, delay aversion, and HbA1c levels, along with the intensity of different physical activity measured in met-hours per week. RESULTS: After controlling for the sample characteristics, delay discounting was negatively associated with moderate physical activity (ß = -2.386, 95%CI: -4.370 to -0.401). Meanwhile, delay aversion was negatively associated with the level of moderate physical activity (ß = -3.527, 95% CI: -5.578 to -1.476) in the multiple linear regression model, with statistically significant differences. CONCLUSION: Elevated delay discounting and increased delay aversion correlated with reduced levels of moderate physical activity. Result suggests that delay discounting and aversion may influence engagement in moderate physical activity. This study recommends that health administration and government consider delay discounting and delay aversion when formulating behavioral intervention strategies and treatment guidelines involving physical activity for patients with T2DM, which may increase participation in physical activity. This study contributes a novel perspective to the research on physical activity in adults with T2DM by examining the significance of future health considerations and the role of emotional responses to delays.

Nuclear and cytoplasmic genome components of Solanum tuberosum + S. chacoense somatic hybrids and three SSR alleles related to bacterial wilt resistance.

The somatic hybrids were derived previously from protoplast fusion between Solanum tuberosum and S. chacoense to gain the bacterial wilt resistance from the wild species. The genome components analysis in the present research was to clarify the nuclear and cytoplasmic composition of the hybrids, to explore the molecular markers associated with the resistance, and provide information for better use of these hybrids in potato breeding. One hundred and eight nuclear SSR markers and five cytoplasmic specific primers polymorphic between the fusion parents were used to detect the genome components of 44 somatic hybrids. The bacterial wilt resistance was assessed thrice by inoculating the in vitro plants with a bacterial suspension of race 1. The disease index, relative disease index, and resistance level were assigned to each hybrid, which were further analyzed in relation to the molecular markers for elucidating the potential genetic base of the resistance. All of the 317 parental unique nuclear SSR alleles appeared in the somatic hybrids with some variations in the number of bands detected. Nearly 80 % of the hybrids randomly showed the chloroplast pattern of one parent, and most of the hybrids exhibited a fused mitochondrial DNA pattern. One hundred and nine specific SSR alleles of S. chacoense were analyzed for their relationship with the disease index of the hybrids, and three alleles were identified to be significantly associated with the resistance. Selection for the resistant SSR alleles of S. chacoense may increase the possibility of producing resistant pedigrees.

Introgression of bacterial wilt resistance from eggplant to potato via protoplast fusion and genome components of the hybrids.

KEY MESSAGE: Bacterial wilt resistant somatic hybrids were obtained via protoplast fusion between potato and eggplant and three types of nuclear genomes were identified in the hybrids through GISH and SSR analysis. ABSTRACT: Cultivated potato (Solanum tuberosum L.) lacks resistance to bacterial wilt caused by Ralstonia solanacearum. Interspecific symmetric protoplast fusion was conducted to transfer bacterial wilt resistance from eggplant (S. melongena, 2n = 2x = 24) into dihaploid potato (2n = 2x = 24). In total, 34 somatic hybrids were obtained, and of these, 11 rooted and were tested for genome components and resistance to race 1 of R. solanacearum. The hybrids exhibited multiple ploidy levels and contained the dominant nuclear genome from the potato parent. Three types of nuclear genomes were identified in the hybrids through genomic in situ hybridization (GISH) and simple sequence repeat (SSR) analysis, including (1) the potato type of the tetraploids in which eggplant chromosomes could not be detected by GISH but their nuclear DNA was confirmed by SSR, (2) the biased type of the hexaploids in which the chromosome dosage was 2 potato:1 eggplant, and (3) the chromosome translocation type of the mixoploids and aneuploids that was characterized by various rates of translocations of nonhomologous chromosomes. Cytoplasmic genome analysis revealed that mitochondrial DNA of both parents coexisted and/or recombined in most of the hybrids. However, only potato chloroplast DNA was retained in the hybrids speculating a compatibility between cpDNA and nuclear genome of the cell. The pathogen inoculation assay suggested a successful transfer of bacterial wilt resistance from eggplant to the hybrids that provides potential resistance for potato breeding against bacterial wilt. The genome components characterized in present research may explain partially the inheritance behavior of the hybrids which is informative for potato improvement.

Molecular Marker-Assisted Selection for Frost Tolerance in a Diallel Population of Potato.

A multi-parental population is an innovative tool for mapping large numbers of loci and genetic modifications, particularly where they have been used for breeding and pre-breeding in crops. Frost injury is an environmental stress factor that greatly affects the growth, development, production efficiency, and geographical distribution of crops. No reported study has focused on genetic mapping and molecular marker development using diallel populations of potatoes. In this study, 23 successful cross combinations, obtained by a half diallel cross among 16 parents, including eight frost-tolerant advanced breeding lines and eight cultivars, were used to map the genetic loci for frost tolerance and to create a molecular marker-assisted selection (MAS) system. Three candidate regions related to frost tolerance on chromosomes II, V, and IX were mapped by bulked segregant analysis (BSA). Furthermore, six SNP markers associated with frost tolerance from candidate regions were developed and validated. Above all, a MAS system for the frost tolerance screening of early breeding offspring was established. This study highlights the practical advantages of applying diallel populations to broaden and improve frost-tolerant germplasm resources.

Simple Webserver-Facilitated Method to Design and Synthesize Artificial miRNA Gene and Its Application in Engineering Viral Resistance.

Plant viruses impose serious threats on crop production. Artificial miRNAs can mediate specific and effective gene silencing in plants and are widely used in plant gene function studies and to engineer plant viral resistance. To facilitate the design of artificial miRNA genes, we developed a webserver, AMIRdesigner, which can be used to design oligos for artificial miRNA synthesis using wild-type and permutated MIR171 and MIR164 backbones. The artificial miRNA genes designed by AMIRdesigner can be easily assembled into miRNA clusters for multiple target sites. To validate the server functionality, we designed four artificial miRNA genes targeting four conserved regions in the potato leafroll virus genome using AMIRdesigner. These genes were synthesized with the server-designed oligos and further assembled into a quadruple miRNA cluster, which was cloned into an overexpression vector and transformed into potato plants. Small RNA Northern blot and virus inoculation analyses showed that a high level of artificial miRNA expression and good viral resistance were achieved in some of the transgenic lines. These results demonstrate the utility of our webserver AMIRdesigner for engineering crop viral resistance.

Rychc Confers Extreme Resistance to Potato virus Y in Potato.

The Potato virus Y (PVY) is responsible for huge economic losses for the potato industry worldwide and is the fifth most consequential plant virus globally. The main strategies for virus control are to limit aphid vectors, produce virus-free seed potatoes, and breed virus-resistant varieties. The breeding of PVY-resistant varieties is the safest and most effective method in terms of cost and environmental protection. Rychc, a gene that confers extreme resistance to PVY, is from S. chacoense, which is a wild diploid potato species that is widely used in many PVY-resistant breeding projects. In this study, Rychc was fine mapped and successfully cloned from S. chacoense accession 40-3. We demonstrated that Rychc encodes a TIR-NLR protein by stably transforming a diploid susceptible cultivar named AC142 and a tetraploid potato variety named E3. The Rychc conferred extreme resistance to PVYO, PVYN:O and PVYNTN in both of the genotypes. To investigate the genetic events occurring during the evolution of the Rychc locus, we sequenced 160 Rychc homologs from 13 S. chacoense genotypes. Based on the pattern of sequence identities, 160 Rychc homologs were divided into 11 families. In Family 11 including Rychc, we found evidence for Type I evolutionary patterns with frequent sequence exchanges, obscured orthologous relationships and high non-synonymous to synonymous substitutions (Ka/Ks), which is consistent with rapid diversification and positive selection in response to rapid changes in the PVY genomes. Furthermore, a functional marker named MG64-17 was developed in this study that indicates the phenotype with 100% accuracy and, therefore, can be used for marker-assisted selection in breeding programs that use S. chacoense as a breeding resource.

Meiotic behavior of pollen mother cells in relation to ploidy level of somatic hybrids between Solanum tuberosum and S. chacoense.

Potato somatic hybrids obtained by protoplast fusion between Solanum tuberosum (4x) and Solanum chacoense (2x) were investigated for genome stability and meiotic behavior associated with the pollen viability in order to elucidate the mechanism influencing the fertility of the somatic hybrids. The ploidy level detections conducted in 2004 and 2007 demonstrated that 68 out of 108 somatic hybrids had their ploidy level changed to be uniform and euploidy after successive in vitro subcultures, which mainly occurred in octaploids, aneuploids, and mixoploids, while 74% hexaploids were still stable in their genome dosage in 2007. Different types of abnormal meiotic behavior were observed during the development of pollen mother cells (PMCs) including the formation of univalents, multivalents, laggard chromosomes, and chromosomal bridges, as well as triads and polyads. A higher proportion of abnormal meiosis seemed to be accompanied with a genome dosage higher than the hexaploids expected in this study. A significant positive correlation between defective PMCs and the number of small pollen grains and negative correlation between number of small pollen grains and pollen viability strongly suggested that abnormal meiosis could be a causal factor influencing the fertility of the somatic hybrids. The hexaploids with stable genome dosage and a certain level of fertility will have great potential in a potato breeding program.

QTL mapping for microtuber dormancy and GA3 content in a diploid potato population.

The genetic control of dormancy is poorly understood in most plant species, but dormancy is a prominent feature for the potato industry. We used the microtuber system, in which tubers were produced in vitro and stored at 20°C, to perform quantitative trait locus (QTL) analysis for dormancy and gibberellic acid (GA3) content in an F1 population consisting of 178 genotypes derived from an interspecific cross between Solanum chacoense acc. PI 320285 (long dormancy) and Solanum phureja acc. DM1-3 516 R44 (short dormancy). In this analysis, 163 markers were used to construct a genetic map with a total length of 591.8 cM. Through QTL analysis, we identified 22 markers closely linked to the timing of dormancy release and GA3 content. The male parent alleles were closely related with long dormancy, with the most significant effect on chromosome I, which accounted for 9.4% of phenotypic variation. The dormancy and GA3 QTLs localized to the same position in the genome, confirming that same genomic region controls GA3 content at different developmental stages or in dormant and sprouting tubers. The identified QTLs may be useful for future breeding strategies and studies of dormancy in potato.