Genetic fingerprint construction and genetic diversity analysis of sweet potato (Ipomoea batatas) germplasm resources.

BACKGROUND: China is the largest producer of sweet potato in the world, accounting for 57.0% of the global output. Germplasm resources are the basis for promoting innovations in the seed industry and ensuring food security. Individual and accurate identification of sweet potato germplasm is an important part of conservation and efficient utilization. RESULTS: In this study, nine pairs of simple sequence repeat molecular markers and 16 morphological markers were used to construct genetic fingerprints for sweet potato individual identification. Combined with basic information, typical phenotypic photographs, genotype peak graphs, and a two-dimensional code for detection and identification were generated. Finally, a genetic fingerprint database containing 1021 sweet potato germplasm resources in the "National Germplasm Guangzhou Sweet Potato Nursery Genebank in China" was constructed. Genetic diversity analysis of the 1021 sweet potato genotypes using the nine pairs of simple sequence repeat markers revealed a narrow genetic variation range of Chinese native sweet potato germplasm resources, and Chinese germplasm was close to that from Japan and the United States, far from that from the Philippines and Thailand, and the furthest from that from Peru. Sweet potato germplasm resources from Peru had the richest genetic diversity, supporting the view that Peru is the center of origin and domestication of sweet potato varieties. CONCLUSIONS: Overall, this study provides scientific guidance for the conservation, identification, and utilization of sweet potato germplasm resources and offers a reference to facilitate the discovery of important genes to boost sweet potato breeding.

Exploration of molecular mechanism of intraspecific cross-incompatibility in sweetpotato by transcriptome and metabolome analysis.

Cross-incompatibility, frequently happening in intraspecific varieties, has seriously restricted sweetpotato breeding. However, the mechanism of sweetpotato intraspecific cross-incompatibility (ICI) remains largely unexplored, especially for molecular mechanism. Treatment by inducible reagent developed by our lab provides a method to generate material for mechanism study, which could promote incompatible pollen germination and tube growth in the ICI group. Based on the differential phenotypes between treated and untreated samples, transcriptome and metabolome were employed to explore the molecular mechanism of sweetpotato ICI in this study, taking varieties 'Guangshu 146' and 'Shangshu 19', a typical incompatible combination, as materials. The results from transcriptome analysis showed oxidation-reduction, cell wall metabolism, plant-pathogen interaction, and plant hormone signal transduction were the essential pathways for sweetpotato ICI regulation. The differentially expressed genes (DEGs) enriched in these pathways were the important candidate genes to response ICI. Metabolome analysis showed that multiple differential metabolites (DMs) involved oxidation-reduction were identified. The most significant DM identified in comparison between compatible and incompatible samples was vitexin-2-O-glucoside, a flavonoid metabolite. Corresponding to it, cytochrome P450s were the most DEGs identified in oxidation-reduction, which were implicated in flavonoid biosynthesis. It further suggested oxidation-reduction play an important role in sweetpotato ICI regulation. To validate function of oxidation-reduction, reactive oxygen species (ROS) was detected in compatible and incompatible samples. The green fluorescence was observed in incompatible but not in compatible samples. It indicated ROS regulated by oxidation-reduction is important pathway to response sweetpotato ICI. The results in this study would provide valuable insights into molecular mechanisms for sweetpotato ICI.

Genome Resource for Elsinoë batatas, the Causal Agent of Stem and Foliage Scab Disease of Sweet Potato.

Elsinoë batatas is a phytopathogenic fungus causing stem and foliage scab disease of sweet potato. At present, there is no reference genome available for E. batatas, limiting basic research for the pathogen. The present study applied the Nanopore single-molecule sequencing technology to sequence the E. batatas genome. This study reports the first high-quality genome sequence of E. batatas, with a total contig size of 26.49 Mb, 50.8% GC content, and an N50 of 2,546,814 bp. The sequences obtained serve as a reference for analysis of E. batatas isolates and provide a resource to better understand the biology of stem and foliage scab disease of sweet potato.

First Report of White Rust on Sweetpotato Caused by Albugo ipomoeae-panduratae in Guangdong, China.

Sweetpotato (Ipomoea batatas) is the eighth major food crop cultivated worldwide with annual production of 89.5 million tons (FAO 2020). China is the world's biggest producer of sweetpotato, and Guangdong Province has the fourth-largest sweetpotato growing area and the biggest sweetpotato market in China (Huang et al. 2020a). Sweetpotato leaves are a key organ providing nutrients for humans and animals, and are popular with customers in Guangdong. On October 14, 2021, a white rust affecting sweetpotato leaves was observed in the fields of Yunfu, Guangdong (22°54'55''N, 112°02'40''E) when conditions were humid, rainy and relatively mild. The adaxial surface of the infected leaves initially exhibited irregular light-yellow or yellow spots, which gradually turned to brown and necrotic. Meanwhile, tiny, powdery, chalky-white pustules, typical of white rust, dispersed individually or in clusters were observed on the corresponding underside of lesions, resulting in wrinkled leaves or abscission. For further analysis in the laboratory, affected leaf pieces (5 mm × 5 mm) containing raised pustules were examined using a scanning electron microscope (S-3400N-Ⅱ, Hitachi, Japan) at 5kv. The micrographs revealed numerous cylindrical-shaped sporangia released from broken pustules. The surfaces of globose oospores were covered with tiny papillae in a reticular pattern. Based on the morphological analyses, the pathogen was preliminarily identified as an Albugo sp. Crude genomic DNA of a few pustules from the diseased leaves were extracted and subjected to PCR amplification using a 2×T5 Direct PCR kit (TSE011, Tsingke, China) with the primers, ITS1/ITS4 (White et al. 1990). PCR products were detected using agarose gel electrophoresis and sequenced by Tsingke company (Guangzhou, China). The sequences were compared against the NCBI database using the BLASTn search tool. The two best-matched alignments with over 90% query coverage showed that ITS sequence amplified from the sample, which was deposited in GenBank (OM182104), was ≥97% identical to those from two isolates of A. ipomoeae-panduratae from China (AY742741) and Korea (DQ643920). A. ipomoeae-panduratae primarily causes white rust on sweetpotato (Moyer and Clark 2013) and is an obligate parasite not culturable in vitro. To demonstrate pathogenicity, spores collected from symptomatic sweetpotato leaves were mixed with sterile water and sprayed onto leaves of three healthy sweetpotato plants. Inoculated plants were then placed at 21℃ and 93% relative humidity. Three other healthy plants treated with sterile water without spores served as the control group. After 12 days, chlorosis and necrosis were observed on the upper leaf surface; and raised white pustules appeared on the lower leaf surface. No symptoms were observed in the noninoculated control plants. To the best of our knowledge, this is the first report of white rust, caused by A. ipomoeae-panduratae, affecting sweetpotato in Guangdong, China. White rust on sweetpotato has also been reported in other provinces in China (Huang et al. 2020b), and the detrimental effects and control measures of this disease should be studied further.

Draft Genome Sequence of Diaporthe batatatis Causing Dry Rot Disease in Sweetpotato.

Dry rot caused by Diaporthe batatatis leads to the serious decay of sweetpotato storage roots during postharvest storage, which can result in considerable economic loss. Genomic research of the pathogen could provide a basis for study and prevention of sweetpotato dry rot. Herein, we report a high-quality draft genome sequence of D. batatatis CRI 302-4 isolated from infected sweetpotato storage roots in Taizhou City, Zhejiang Province, China. The size of the genome was 54.38 Mb and consisted of 36 scaffolds with a G+C content of 50.56% and an N50 of 2,950,914 bp. The information provided in this genome sequence will be an invaluable resource for molecular genetic research and disease control in sweetpotato production.

High-Quality Genome Resource of Diaporthe destruens Causing Foot Rot Disease of Sweet Potato.

Foot rot of sweet potato caused by Diaporthe destruens severely affects yield and quality worldwide. Research on this pathogen is limited due to nonavailability of genome resources. Here, we report a high-quality genome sequence of D. destruens isolate CRI 305-2, which was originally isolated from infected stem of sweet potato in Taizhou City, Zhejiang Province, China. The genome comprised a total length of 56,108,228 bp, consisted of 47 scaffolds with an overall G+C content of 48.7% and an N50 of 2,479,481 bp. This resource can be used as a reference for evolution mechanisms and comparative genomic research.

Isolation and Identification of Trichoderma asperellum, the Novel Causal Agent of Green Mold Disease in Sweetpotato.

Postharvest disease is an important limiting factor for sweetpotato production. Recently, a new green mold disease was found in sweetpotato storage roots. To investigate the mechanism underlying the pathogenesis of the disease, the pathogen was isolated and identified based on morphological and molecular features, and its characteristics were further analyzed by pathogenic and antagonistic evaluations. The results showed that the isolated pathogen (CRI-Ta1) was identified as Trichoderma asperellum based on the similar growth and morphological features with Trichoderma spp., 99% homology of internal transcribed spacer (ITS) sequence, and membership to the same phylogenetic group with the model strain of T. asperellum (CBS 433.97). The pathogenic analysis revealed that CRI-Ta1 could cause green mold disease through wound infection on the storage roots and the strains reisolated from infected storage roots could cause disease in different sweetpotato varieties, which was fulfilled in Koch's postulate. Moreover, CRI-Ta1 could also infect other common crop species, including chestnut, carrot, apple, pear, and others. It indicated that CRI-Ta1 was the pathogen to the storage roots of sweetpotato and had a wide host range. Additionally, in vitro antagonistic evaluation showed that CRI-Ta1 effectively inhibited the growth of common sweetpotato pathogens, including Fusarium solani and Rhizopus nigricans. However, further research is needed on the potential of CRI-Ta1 to control sweetpotato diseases in vivo. Collectively, our findings provided valuable insights into the characteristics of the T. asperellum CRI-Ta1 in sweetpotato and would be helpful to the prevention and control of sweetpotato green mold disease.

Temporal patterns of gene expression associated with tuberous root formation and development in sweetpotato (Ipomoea batatas).

BACKGROUND: The tuberous root of sweetpotato is undisputedly an important organ from agronomic and biological perspectives. Little is known regarding the regulatory networks programming tuberous root formation and development. RESULTS: Here, as a first step toward understanding these networks, we analyzed and characterized the genome-wide transcriptional profiling and dynamics of sweetpotato root in seven distinct developmental stages using a customized microarray containing 39,724 genes. Analysis of these genes identified temporal programs of gene expression, including hundreds of transcription factor (TF) genes. We found that most genes active in roots were shared across all developmental stages, although significant quantitative changes in gene abundance were observed for 5,368 (including 435 TFs) genes. Clustering analysis of these differentially expressed genes pointed out six distinct expression patterns during root development. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that genes involved in different processes were enriched at specific stages of root development. In contrast with the large number of shared expressed genes in root development, each stage or period of root development has only a small number of specific genes. In total, 712 (including 27 TFs) and 1,840 (including 115 TFs) genes were identified as root-stage and root-period specific, respectively at the level of microarray. Several of the specific TF genes are known regulators of root development, including DA1-related protein, SHORT-ROOT and BEL1-like. The remaining TFs with unknown roles would also play critical regulatory roles during sweetpotato tuberous root formation and development. CONCLUSIONS: The results generated in this study provided spatiotemporal patterns of root gene expression in support of future efforts for understanding the underlying molecular mechanism that control sweetpotato yield and quality.

Whole-Genome Sequencing and Comparative Genome Analysis of Fusarium solani-melongenae Causing Fusarium Root and Stem Rot in Sweetpotatoes.

Sweetpotato (Ipomoea batatas) is the eighth most important crop globally. However, the production and quality of sweetpotatoes are threatened by Fusarium diseases that are prevalent around the world. In this study, a Fusarium species that causes root and stem rot in sweetpotatoes was studied. The pathogenic fungus CRI 24-3 was isolated and sequenced using third- and next-generation sequencing techniques and a 49.6 Mb chromosome-level draft genome containing 15,374 putative coding genes were obtained. Molecular phylogenetic analysis showed that CRI 24-3 was an F. solani-melongenae strain within clade 3 of the F. solani species complex (FSSC). CRI 24-3 showed a relatively high number of virulence factors, such as carbohydrate-active enzymes (CAZymes), pathogen-host interaction (PHI) proteins, and terpene synthases (TSs), compared with the number of those identified in other sequenced FSSC members. Comparative genome analysis revealed considerable conservation and unique characteristics between CRI 24-3 and other FSSC species. In conclusion, the findings in the current study provide important genetic information about F. solani-melongenae and should be useful in the exploration of pathogenicity mechanisms and the development of Fusarium disease management strategies. IMPORTANCE Fusarium root and stem rot in sweetpotato are prevalent in the main sweetpotato-growing areas in China, and fungal isolation, morphological characteristics, and molecular phylogenetic analysis of the disease causal agent (F. solani-melongenae isolate CRI 24-3) were systematically studied. The genome sequence of F. solani-melongenae isolates CRI 24-3 was first reported, which should provide a basis for genome assembly of other closely related Fusarium species. Carbohydrate-active enzymes predicted in CRI 24-3 may be important to convert the substantial polysaccharides to sustainable and renewable energy. Moreover, other virulence factors facilitating Fusarium diseases, including effectors and toxic secondary metabolites, are ideal objects for pathogenicity mechanism research and molecular targets for fungicide development. The findings of comparative genome analysis of CRI 24-3 and 15 sequenced members of the F. solani species complex help promote an integral understanding of genomic features and evolutionary relationships in Fusarium.

Characterization and development of EST-derived SSR markers in cultivated sweetpotato (Ipomoea batatas).

BACKGROUND: Currently there exists a limited availability of genetic marker resources in sweetpotato (Ipomoea batatas), which is hindering genetic research in this species. It is necessary to develop more molecular markers for potential use in sweetpotato genetic research. With the newly developed next generation sequencing technology, large amount of transcribed sequences of sweetpotato have been generated and are available for identifying SSR markers by data mining. RESULTS: In this study, we investigated 181,615 ESTs for the identification and development of SSR markers. In total, 8,294 SSRs were identified from 7,163 SSR-containing unique ESTs. On an average, one SSR was found per 7.1 kb of EST sequence with tri-nucleotide motifs (42.9%) being the most abundant followed by di- (41.2%), tetra- (9.2%), penta- (3.7%) and hexa-nucleotide (3.1%) repeat types. The top five motifs included AG/CT (26.9%), AAG/CTT (13.5%), AT/TA (10.6%), CCG/CGG (5.8%) and AAT/ATT (4.5%). After removing possible duplicate of published EST-SSRs of sweetpotato, a total of non-repeat 7,958 SSR motifs were identified. Based on these SSR-containing sequences, 1,060 pairs of high-quality SSR primers were designed and used for validation of the amplification and assessment of the polymorphism between two parents of one mapping population (E Shu 3 Hao and Guang 2k-30) and eight accessions of cultivated sweetpotatoes. The results showed that 816 primer pairs could yield reproducible and strong amplification products, of which 195 (23.9%) and 342 (41.9%) primer pairs exhibited polymorphism between E Shu 3 Hao and Guang 2k-30 and among the 8 cultivated sweetpotatoes, respectively. CONCLUSION: This study gives an insight into the frequency, type and distribution of sweetpotato EST-SSRs and demonstrates successful development of EST-SSR markers in cultivated sweetpotato. These EST-SSR markers could enrich the current resource of molecular markers for the sweetpotato community and would be useful for qualitative and quantitative trait mapping, marker-assisted selection, evolution and genetic diversity studies in cultivated sweetpotato and related Ipomoea species.

Complete chloroplast genome of a novel chlorophyll-deficient mutant (clm) in sweetpotato (Ipomoea batatas L.).

The complete chloroplast genome of a novel chlorophyll-deficient mutant (clm) and its wild type (WT) in sweetpotato (Ipomoea batatas L.) was sequenced. The complete chloroplast genome of clm and WT was 161,393 bp and 161,429 bp in length, containing a large single copy (LSC) region of 87,561 bp and 87,597 bp, respectively, a small single copy (SSC) region with the same length of 30,890 bp and a pair of inverted repeat regions (IRs) with the same length of 12,052 bp. Both of them contained 132 genes including 87 protein-coding sequences, 37 tRNA, and eight rRNA. Comparing to the WT, four SNPs and three INDELs were detected and only one INDEL in the exon affecting the translation of rpoA gene. Phylogenetic analysis showed that clm and WT were closely related to Ipomoea tabascana. The complete chloroplast genome of clm and its WT will play a role in understanding the molecular mechanism of chlorophyll deficiency and developing molecular markers in sweetpotato.

De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas).

BACKGROUND: The tuberous root of sweet potato is an important agricultural and biological organ. There are not sufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying the tuberous root formation and development. Thus, high throughput transcriptome sequencing is needed to generate enormous transcript sequences from sweet potato root for gene discovery and molecular marker development. RESULTS: In this study, more than 59 million sequencing reads were generated using Illumina paired-end sequencing technology. De novo assembly yielded 56,516 unigenes with an average length of 581 bp. Based on sequence similarity search with known proteins, a total of 35,051 (62.02%) genes were identified. Out of these annotated unigenes, 5,046 and 11,983 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 17,598 (31.14%) unigenes were mapped to 124 KEGG pathways, and 11,056 were assigned to metabolic pathways, which were well represented by carbohydrate metabolism and biosynthesis of secondary metabolite. In addition, 4,114 cDNA SSRs (cSSRs) were identified as potential molecular markers in our unigenes. One hundred pairs of PCR primers were designed and used for validation of the amplification and assessment of the polymorphism in genomic DNA pools. The result revealed that 92 primer pairs were successfully amplified in initial screening tests. CONCLUSION: This study generated a substantial fraction of sweet potato transcript sequences, which can be used to discover novel genes associated with tuberous root formation and development and will also make it possible to construct high density microarrays for further characterization of gene expression profiles during these processes. Thousands of cSSR markers identified in the present study can enrich molecular markers and will facilitate marker-assisted selection in sweet potato breeding. Overall, these sequences and markers will provide valuable resources for the sweet potato community. Additionally, these results also suggested that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for gene discovery and molecular marker development for non-model species, especially those with large and complex genome.