Construction of high-density genetic map based on SLAF-seq and QTL analysis of major traits in sweetpotato [Ipomoea batatas (L.) Lam.].

Sweetpotato, Ipomoea batatas (L.) Lam., is an important worldwide crop used as feed, food, and fuel. However, its polyploidy, high heterozygosity and self-incompatibility makes it difficult to study its genetics and genomics. Longest vine length (LVL), yield per plant (YPP), dry matter content (DMC), starch content (SC), soluble sugar content (SSC), and carotenoid content (CC) are some of the major agronomic traits being used to evaluate sweetpotato. However limited research has actually examined how these traits are inherited. Therefore, after selecting 212 F1 from a Xin24 × Yushu10 crossing as the mapping population, this study applied specific-locus amplified fragment sequencing (SLAF-seq), at an average sequencing depth of 26.73 × (parents) and 52.25 × (progeny), to detect single nucleotide polymorphisms (SNPs). This approach generated an integrated genetic map of length 2441.56 cM and a mean distance of 0.51 cM between adjacent markers, encompassing 15 linkage groups (LGs). Based on the linkage map, 26 quantitative trait loci (QTLs), comprising six QTLs for LVL, six QTLs for YPP, ten QTLs for DMC, one QTL for SC, one QTL for SSC, and two QTLs for CC, were identified. Each of these QTLs explained 6.3-10% of the phenotypic variation. It is expected that the findings will be of benefit for marker-assisted breeding and gene cloning of sweetpotato.

Isolation, characterization, and functional verification of salt stress response genes of NAC transcription factors in Ipomoea pes-caprae.

Adverse environmental stress is a major environmental factor threatening food security, which is why improving plant stress resistance is essential for agricultural productivity and environmental sustainability. The NAC (NAM, ATAF, and CUC) transcription factors (TFs) play a dominant role in plant responses to abiotic and biotic stresses, but they have been poorly studied in Ipomoea pes-caprae. In this research, 12 NAC TFs, named IpNAC1-IpNAC12, were selected from transcriptome data. The homologous evolution tree divided IpNACs into four major categories, and six IpNACs were linearly associated with Arabidopsis ANAC genes. From the gene structures, protein domains, and promoter upstream regulatory elements, IpNACs were shown to contain complete NAC-specific subdomains (A-E) and cis-acting elements corresponding to different stress stimuli. We measured the expression levels of the 12 IpNACs under abiotic stress (salt, heat, and drought) and hormone treatment (abscisic acid, methyl jasmonate, and salicylic acid), and their transcription levels differed. IpNAC5/8/10/12 were located in the nucleus through subcellular localization, and the overexpressing transgenic Arabidopsis plants showed high tolerance to salt stress. The cellular Na+ homeostasis content in the mature and elongation zones of the four IpNAC transgenic sweetpotato roots showed an obvious efflux phenomenon. These conclusions demonstrate that IpNAC5/8/10/12 actively respond to abiotic stress, have significant roles in improving plant salt tolerance, and are important salt tolerance candidate genes in I. pes-caprae and sweetpotato. This study laid the foundation for further studies on the function of IpNACs in response to abiotic stress. It provides options for improving the stress resistance of sweetpotato using gene introgression from I. pes-caprae.

Resequencing of sweetpotato germplasm resources reveals key loci associated with multiple agronomic traits.

Sweetpotato is an important crop that exhibits hexaploidy and high heterozygosity, which limits gene mining for important agronomic traits. Here, 314 sweetpotato germplasm resources were deeply resequenced, and 4 599 509 SNPs and 846 654 InDels were generated, among which 196 124 SNPs were nonsynonymous and 9690 InDels were frameshifted. Based on the Indels, genome-wide marker primers were designed, and 3219 of 40 366 primer pairs were selected to construct the core InDel marker set. The molecular ID of 104 sweetpotato samples verified the availability of these primers. The sweetpotato population structures were then assessed through multiple approaches using SNPs, and diverse approaches demonstrated that population stratification was not obvious for most Chinese germplasm resources. As many as 20 important agronomic traits were evaluated, and a genome-wide association study was conducted on these traits. A total of 19 high-confidence loci were detected in both models. These loci included several candidate genes, such as IbMYB1, IbZEP1, and IbYABBY1, which might be involved in anthocyanin metabolism, carotenoid metabolism, and leaf morphogenesis, respectively. Among them, IbZEP1 and IbYABBY1 were first reported in sweetpotato. The variants in the promoter and the expression levels of IbZEP1 were significantly correlated with flesh color (orange or not orange) in sweetpotato. The expression levels of IbYABBY1 were also correlated with leaf shape. These results will assist in genetic and breeding studies in sweetpotato.

Comparative Metabolomic and Transcriptomic Analyses of Phytochemicals in Two Elite Sweet Potato Cultivars for Table Use.

To elucidate nutritional components in sweet potato cultivars for table use and to compare the phytochemicals of cultivars from different countries, 'Kokei No. 14' and 'Xinxiang' were selected. The physiological parameters and metabolites were determined using the colorimetric method and widely targeted metabolomics, respectively. Transcriptomic analysis was performed to explain the mechanism that resulted in phytochemical differences. 'Xinxiang' showed higher flavonoid and carotenoid contents. Metabolomics showed five upregulated flavonoids. Two essential amino acids (EAAs) and one conditionally essential amino acid (CEAA) were upregulated, whereas four EAAs and two CEAAs were downregulated. Unlike lipids, in which only one of thirty-nine was upregulated, nine of twenty-seven differentially accumulated phenolic acids were upregulated. Three of the eleven different alkaloids were upregulated. Similarly, eight organic acids were downregulated, with two upregulated. In addition, three of the seventeen different saccharides and alcohols were upregulated. In 'other metabolites,' unlike vitamin C, 6'-O-Glucosylaucubin and pantetheine were downregulated. The differentially accumulated metabolites were enriched to pathways of the biosynthesis of secondary metabolites, ABC transporters, and tyrosine metabolism, whereas the differentially expressed genes were mainly concentrated in the metabolic pathway, secondary metabolite biosynthesis, and transmembrane transport functions. These results will optimize the sweet potato market structure and enable a healthier diet for East Asian residents.

Metabolomic and Transcriptomic Analyses of the Flavonoid Biosynthetic Pathway for the Accumulation of Anthocyanins and Other Flavonoids in Sweetpotato Root Skin and Leaf Vein Base.

Sweetpotato [Ipomoea batatas (L.) Lam.] is a major tuberous root crop that is rich in flavonoids. Here, we discovered a spontaneous mutation in the color of the leaf vein base (LVB) and root skin (RS) in the Zheshu 81 cultivar. The flavonoid and anthocyanin metabolites and molecular mechanism were analyzed using metabolome and transcriptome data. Compared to the wild type, 13 differentially accumulated metabolites (DAMs) in the LVB and 59 DAMs in the RS were all significantly downregulated. Moreover, all the anthocyanin metabolites decreased significantly. The differentially expressed genes (DEGs) encoding the key enzymes in the later enzymatic reaction of anthocyanin and flavonoid were significantly downregulated in the mutant. The expression trends of the transcription factor MYB were evidently related to the anthocyanin content. These results offer insights into the coloration in the LVB and RS and a theoretical basis for determining the regulation of flavonoid and anthocyanin synthesis in sweetpotato.

Storage Property Is Positively Correlated With Antioxidant Capacity in Different Sweet Potato Cultivars.

Sweet potato decays easily due to its high respiration rate and reactive oxygen species (ROS) accumulation during postharvest storage. In this study, we explored the relationship between antioxidant capacity in leaves and storage properties in different sweet potato cultivars, the tuberous roots of 10 sweet potato cultivars were used as the experimental materials to analyze the storage property during storage at 11-15°C. According to the decay percentage after 290 days of storage, Xu 32 was defined as a storage-tolerant cultivar (rot percentage less than 25%); Xu 55-2, Z 15-1, Shangshu 19, Yushu, and Zhezi 3 as above-moderate storage-tolerant cultivars (rot percentage ranging from 25 to 50%); Sushu 16, Yanshu 5, and Hanzi as medium-storable cultivars (rot percentage 50-75%); and Yan 25 as a storage-sensitive cultivar (rot percentage greater than 75%). Meanwhile, analysis of the α-amylase activity in root tubers of the 10 sweet potato cultivars during storage indicated that α-amylase activity was lowest in the storage-tolerant cultivar Xu 32 and highest in the storage-sensitive cultivar Yan 25. Evaluation of antioxidant enzyme activities and ROS content in the leaves of these 10 cultivars demonstrated that cultivar Xu 32, which showed the best storage property, had higher antioxidant enzyme activity [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD)] but lower lipoxygenase (LOX) activity, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, and superoxide anion radical (O2⋅-) production rates compared with those of the storage-sensitive cultivar Yan 25 and the medium-storability cultivars Hanzi, Yanshu 5, and Sushu 16. Additionally, principal component analysis (PCA) suggested that sweet potato cultivars with different storage properties were clustered separately. Correlation and heat map analysis further indicated that CAT, APX, POD, and SOD activities were negatively correlated with α-amylase activity, while LOX activity and MDA and H2O2 contents were negatively correlated with the storage property of sweet potato. Combined, our findings revealed that storage property is highly correlated with antioxidant capacity in sweet potato leaves and negatively correlated with α-amylase activity in tuberous roots, which provides a convenient means for the screening of storage-tolerant sweet potato cultivars.

A single amino acid mutant in the EAR motif of IbMYB44.2 reduced the inhibition of anthocyanin accumulation in the purple-fleshed sweetpotato.

Purple-fleshed sweetpotato (Ipomoea batatas（L.）Lam.) is rich in anthocyanins. R2R3-type MYB transcription factors（TFs）with EAR motifs inhibiting anthocyanin biosynthesis have been reported, and there is still a lack of information on how mutations in the EAR motifs of MYBs affect anthocyanin accumulation. In this study, we obtained three IbMYB44 TFs by bioinformatics. Among these TFs, IbMYB44.1, IbMYB44.3 with a complete EAR motif and IbMYB44.2 with a single amino acid mutant in the EAR motif caused an amino acid substitution from leucine to valine. RT-qPCR analysis showed that IbMYB44s was expressed at lower levels in the purple-fleshed sweetpotato than in nonpurple-fleshed sweetpotato (P < 0.01). Transient expression assays showed that the inhibitory effect of IbMYB44.1/3 was stronger than IbMYB44.2 in tobacco leaves and red-skinned pears. RT-qPCR analysis further proved that IbMYB44.1/3 significantly inhibited the expression of anthocyanin biosynthesis-related genes compared with IbMYB44.2 in tobacco leaves and red-skinned pears. A dual luciferase reporter assay showed that IbMYB44s cannot directly activate the IbANS promoter, and the result was also verified by yeast one-hybrid (Y1H) experiments. Moreover, we identified the interaction of IbMYB340 with IbMYB44.1, IbMYB44.2 and IbMYB44.3 via yeast two-hybrid (Y2H) assays. Thus, IbMYB44.1/3 could interact with IbMYB340 to negatively regulate anthocyanin biosynthesis. This study enriched the regulatory network of anthocyanins and also provided a theoretical basis for a single amino acid mutant from leucine to valine in the EAR motif of IbMYB44.2 affecting anthocyanin biosynthesis in the purple-fleshed sweetpotato.

IbERF71, with IbMYB340 and IbbHLH2, coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potato (Ipomoea batatas L.).

KEY MESSAGE: The transcription factor (TF) IbERF71 forms a novel complex, IbERF71-IbMYB340-IbbHLH2, to coregulate anthocyanin biosynthesis by binding to the IbANS1 promoter in purple-fleshed sweet potatoes. Purple-fleshed sweet potato (Ipomoea batatas L.) is very popular because of its abundant anthocyanins, which are natural pigments with multiple physiological functions. TFs involved in regulating anthocyanin biosynthesis have been identified in many plants. However, the molecular mechanism of anthocyanin biosynthesis in purple-fleshed sweet potatoes has rarely been examined. In this study, TF IbERF71 and its partners were screened by bioinformatics and RT-qPCR analysis. The results showed that the expression levels of IbERF71 and partners IbMYB340 and IbbHLH2 were higher in purple-fleshed sweet potatoes than in other colors and that the expression levels positively correlated with anthocyanin contents. Moreover, transient expression assays showed that cotransformation of IbMYB340+IbbHLH2 resulted in anthocyanin accumulation in tobacco leaves and strawberry receptacles, and additional IbERF71 significantly increased visual aspects. Furthermore, the combination of the three TFs significantly increased the expression levels of FvANS and FvGST, which are involved in anthocyanin biosynthesis and transport of strawberry receptacles. The dual-luciferase reporter system verified that cotransformation of the three TFs enhanced the transcription activity of IbANS1. In addition, yeast two-hybrid and firefly luciferase complementation assays revealed that IbMYB340 interacted with IbbHLH2 and IbERF71 but IbERF71 could not interact with IbbHLH2 in vitro. In summary, our findings provide novel evidence that IbERF71 and IbMYB340-IbbHLH2 form the regulatory complex IbERF71-IbMYB340-IbbHLH2 that coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potatoes. Thus, the present study provides a new regulatory network of anthocyanin biosynthesis and strong insight into the color development of purple-fleshed sweet potatoes.

RNA-seq reveals the salt tolerance of Ipomoea pes-caprae, a wild relative of sweet potato.

Wild relatives of crops are often rich in genetic resources and provide great possibilities for crop improvement. Ipomoea pes-caprae is one of the wild relatives of sweet potato and has high salt tolerance. Transcriptomes in the treatment and control groups at various times were sequenced to identify salt tolerance genes and salt response pathways. A total of 40,525 genes were obtained, of which 2478 and 3334 were differentially expressed in the roots and leaves of I. pes-caprae under salt stress, respectively. Identification of candidate genes revealed that the mitogen-activated protein kinase (MAPK) signaling pathway of plants and plant hormone signal transduction participates in the salt signal of I. pes-caprae under salt stress. Homology to ABI2 (HAB2) and Clade A protein phosphatases type 2C (HAI1), which encode two protein phosphatases 2C (PP2C) in the abscisic acid (ABA) signal pathway, were continuously up-regulated upon salt stress, indicating their key role in the salt signal transduction pathway of I. pes-caprae. The expression of EIN3-binding F-box protein 1 (EBF1) in the ethylene signaling pathway was also up-regulated, revealing that the salt tolerance of I. pes-caprae was related to the scavenging of reactive oxygen species (ROS). This study provides insights into the mechanism of salt-tolerant plants and the mining of salt-tolerant genes in sweet potato for the innovation of germplasm resources.

MYB44 competitively inhibits the formation of the MYB340-bHLH2-NAC56 complex to regulate anthocyanin biosynthesis in purple-fleshed sweet potato.

BACKGROUND: Anthocyanins, which have important biological functions and have a beneficial effect on human health, notably account for pigmentation in purple-fleshed sweet potato tuberous roots. Individual regulatory factors of anthocyanin biosynthesis have been identified; however, the regulatory network of anthocyanin biosynthesis in purple-fleshed sweet potato is unclear. RESULTS: We functionally determined that IbMYB340 cotransformed with IbbHLH2 in tobacco and strawberry receptacles induced anthocyanin accumulation, and the addition of IbNAC56a or IbNAC56b caused increased pigmentation. Furthermore, we confirmed the interaction of IbMYB340 with IbbHLH2 and IbNAC56a or IbNAC56b via yeast two-hybrid and firefly luciferase complementation assays; these proteins could form a MYB340-bHLH2-NAC56a or MYB340-bHLH2-NAC56b transcriptional complex to regulate anthocyanin biosynthesis by binding to the IbANS promoter rather than the IbUFGT promoter. Furthermore, it was found by a transient expression system in tobacco leaves that IbMYB44 could decrease anthocyanin accumulation. Moreover, the interaction of IbMYB44 with IbMYB340 and IbNAC56a or IbNAC56b was verified. This result suggested that IbMYB44 acts as a repressor of anthocyanin in sweet potato. CONCLUSIONS: The repressor IbMYB44 affected anthocyanin biosynthesis by competitively inhibiting the IbMYB340-IbbHLH2-IbNAC56a or IbMYB340-IbbHLH2-IbNAC56b regulatory complex formation. Overall, the present study proposed a novel regulatory network whereby several vital TFs play key roles in regulating anthocyanin biosynthesis, and it provides strong insight into the potential mechanism underlying anthocyanin biosynthesis in sweet potato tuberous roots with purple color.