Sweetpotato, Ipomoea batatas (L.) Lam., is an important food crop worldwide.  Large scale evaluation of sweetpotato germplasm for genetic diversity is necessary to determine the genetic relationships between them and effectively use them in the genetic improvement.  In this study, the genetic diversity of 617 sweetpotato accessions, including 376 landraces and 162 bred varieties from China and 79 introduced varieties from 11 other countries, was assessed using 30 simple sequence repeat (SSR) primer pairs with high polymorphism.  Based on the population structure analysis, these sweetpotato accessions were divided into three groups, Group 1, Group 2 and Group 3, which included 228, 136 and 253 accessions, respectively.  Consistent results were obtained by phylogenic analysis and principal coordinate analysis (PCoA).  Of the three groups, Group 2 showed the highest level of genetic diversity and its accessions were mainly distributed in low-latitude regions.  The accessions from South China exhibited the highest level of genetic diversity, which supports the hypothesis that Fujian and Guangdong were the first regions where sweetpotato was introduced to China.  Analysis of molecular variance (AMOVA) indicated significant genetic differentiations between the different groups, but low levels of genetic differentiation existed between the different origins and accession types.  These results provide valuable information for the better utilization of these accessions in sweetpotato breeding.

Geranylgeranyl pyrophosphate synthase (GGPS) plays an important role in the biosynthesis of carotenoids.  In a previous study, the IbGGPS gene was isolated from a sweetpotato, Ipomoea batatas (L.) Lam., line Nongdafu 14 with high carotenoid contents, but its role and underlying mechanisms in carotenoid biosynthesis in sweetpotato were not investigated.  In the present study, the IbGGPS gene was introduced into a sweetpotato cv. Lizixiang and the contents of β-carotene, β-cryptoxanthin, zeaxanthin and lutein were significantly increased in the storage roots of the IbGGPS-overexpressing sweetpotato plants.  Further analysis showed that IbGGPS gene overexpression systematically up-regulated the genes involved in the glycolytic, 2-C-methyl-D-erythritol-4-phosphate (MEP) and carotenoid pathways, which increased the carotenoid contents in the transgenic plants.  These results indicate that the IbGGPS gene has the potential for use in improving the carotenoid contents in sweetpotato and other plants.

Nitrogen is an important nutrient for plant development.  Nitrogen and carbon metabolisms are tightly linked to physiological functions in plants.  In this study, we found that the IbSnRK1 gene was induced by Ca(NO₃)₂.  Its overexpression enhanced nitrogen uptake and carbon assimilation in transgenic sweetpotato.  After Ca(¹⁵NO₃)₂ treatment, the 15N atom excess, 15N and total N content and nitrogen uptake efficiency (NUE) were significantly increased in the roots, stems, and leaves of transgenic plants compared with wild type (WT) and empty vector control (VC).  After Ca(NO₃)₂ treatment, the increased nitrate N content, nitrate reductase (NR) activity, free amino acid content, and soluble protein content were found in the roots or leaves of transgenic plants.  The photosynthesis and carbon assimilation were enhanced.  These results suggest that the IbSnRK1 gene play a important role in nitrogen uptake and carbon assimilation of sweetpotato.  This gene has the potential to be used for improving the yield and quality of sweetpotato.

Simple sequence repeat (SSR) markers have been shown to be a powerful tool for varieties identification in plants.  However, SSR fingerprinting of sweetpotato varieties has been a little reported.  In this study, a total of 1 294 SSR primer pairs, including 1 215 genomic-SSR and 79 expressed sequence tag (EST)-SSR primer pairs, were screened with sweetpotato varieties Zhengshu 20 and Luoxushu 8 and their 2 F₁ individuals randomly sampled, and 273 and 38 of them generated polymorphic bands, respectively.  Four genomic-SSR and 3 EST-SSR primer pairs, which showed good polymorphism, were selected to amplify 203 sweetpotato varieties and gave a total of 172 bands, 85 (49.42%) of which were polymorphic.  All of the 203 sweetpotato varieties showed unique fingerprint patterns, indicating the utility of SSR markers in variety identification of this crop.  Polymorphism information content (PIC) ranged from 0.5824 to 0.9322 with an average of 0.8176.  SSR-based genetic distances varied from 0.0118 to 0.6353 with an average of 0.3100 among these varieties.  Thus, these sweetpotato varieties exhibited high levels of genetic similarity and had distinct fingerprint profiles.  The SSR fingerprints of the 203 sweetpotato varieties have been successfully constructed.  The highly polymorphic SSR primer pairs developed in this study have the potential to be used as core primer pairs for variety identification, genetic diversity assessment and linkage map construction in sweetpotato and other plants.

   The variant LM1 was previously obtained using embryogenic cell suspension cultures of sweetpotato variety Lizixiang by gamma-ray induced mutation, and then its characteristics were stably inherited through six clonal generations, thus this mutant was named LM1. In this study, systematic characterization of salt tolerance and Fusarium wilt resistance were performed between Lizixiang and mutant LM1. LM1 exhibited significantly higher salt tolerance compared to Lizixiang. The content of proline and activities of superoxide dismutase (SOD) and photosynthesis were significantly increased, while malonaldehyde (MDA) and H₂O₂ contents were significantly decreased compared to that of Lizixiang under salt stress. The inoculation test with Fusarium wilt showed that its Fusarium wilt resistance was also improved. The lignin, total phenolic, jasmonic acid (JA) contents and SOD activity were significantly higher, while H₂O₂ content was significantly lower in LM1 than that in Lizixiang. The expression level of salt stress-responsive and disease resistance-related genes was significantly higher in LM1 than that in Lizixiang under salt and Fusarium wilt stresses, respectively. This result provides a novel and valuable material for improving the salt tolerance and Fusarium wilt resistance of sweetpotato.

The somatic hybrid KT1 was previously obtained from protoplast fusion between sweetpotato (Ipomoea batatas (L.) Lam.) cv. Kokei No. 14 and its wild relative I. triloba L. However, its genetic and epigenetic variations have not been investigated. This study showed that KT1 exhibited significantly higher drought tolerance compared to the cultivated parent Kokei No. 14. The content of proline and activities of superoxide dismutase (SOD) and photosynthesis were significantly increased, while malonaldehyde (MDA) content was significantly decreased compared to Kokei No. 14 under drought stress. KT1 also showed higher expression level of well-known drought stress-responsive genes compared to Kokei No. 14 under drought stress. Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) analyses indicated that KT1 had AFLP and MSAP band patterns consisting of both parent specific bands and changed bands. Further analysis demonstrated that in KT1 the proportions of Kokei No. 14 specific genome components and methylation sites were much greater than those of I. triloba. KT1 had the same chloroplast and mitochondrial genomes as Kokei No. 14. These results will aid in developing the useful genes of I. triloba and understanding the evolution and phylogeny of the cultivated sweetpotato.

    A plastidic adenosine triphosphate (ATP)/adenosine diphosphate (ADP) transporter (AATP) is responsible for importing ATP from the cytosol into plastids. In dicotyledonous plants, increasing ATP supply is a potential way to facilitate anabolic synthesis in heterotrophic plastids. In this study, a gene encoding the AATP protein, named IbAATP, was isolated from sweetpotato (Ipomoea batatas (L.) Lam.). Transcripts of IbAATP were predominantly detected in the storage roots and leaves and were induced by exogenous sucrose and subjected to circadian rhythm. Transient expression of IbAATP in tobacco and onion epidermal cells revealed the plastidic localization of IbAATP. The overexpression of IbAATP in sweetpotato significantly increased the starch and amylose contents and led to enlarged starch granules. The IbAATP-overexpressing plants showed altered fine structure of amylopectin, which contained an increased proportion of chains with a degree of polymerization (DP) of 10–23 and a reduced number of chains with a DP of 5–9 and 24–40. In addition, starch from the transgenic plants exhibited different pasting properties. The transcript levels of starch biosynthetic genes, including IbAGP, IbGBSSI, IbSSI-IV, and IbSBE, were differentially regulated in the transgenic plants. These results revealed the explicit role of IbAATP in the starch biosynthesis of sweetpotato and indicated that this gene has the potential to be used to improve starch content and quality in sweetpotato and other plants.

Myo-inositol-1-phosphate synthase (MIPS) is a key rate limiting enzyme in the de novo biosynthesis of myo-inositol in plants. In the present study, the IbMIPS1 gene was introduced into sweetpotato cultivar Xushu 18 and the transgenic plants exhibited significantly enhanced salt tolerance compared with the wild-type (WT). Overexpression of IbMIPS1 up-regulated the salt stress responsive genes, including myo-inositol monophosphatase (MIPP), pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), psbA, phosphoribulokinase (PRK), and superoxide dismutase (SOD) genes, under salt stress. Inositol and proline content, SOD and photosynthesis activities were significantly increased, whereas malonaldehyde (MDA) and H2O2 contents were significantly decreased in the transgenic plants. These findings suggest that the IbMIPS1 gene may enhance salt tolerance of sweetpotato by regulating the expression of salt stress responsive genes, increasing the content of inositol and proline and enhancing the activity of photosynthesis.

Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). In the present study, a TPS gene, named IbTPS, was first isolated from sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lushu 3 by rapid amplification of cDNA ends (RACE). The open reading frame (ORF) contained 2 580 nucleotides encoding 859 amino acids with a molecular weight of 97.433 kDa and an isoelectric point (pI) of 5.7. The deduced amino acid sequence showed high identities with TPS of other plants. Real-time quantitative PCR analysis revealed that the expression level of IbTPS gene was significantly higher in stems of Lushu 3 than in its leaves and roots. Subcellular localization analysis in onion epidermal cells indicated that IbTPS gene was located in the nucleus. Transgenic tobacco (cv. Wisconsin 38) plants over-expressing IbTPS gene exhibited significantly higher salt tolerance compared with the control plant. Trehalose and proline content was found to be significantly more accumulated in transgenic tobacco plants than in the wild-type and several stress tolerance related genes were up-regulated. These results suggest that IbTPS gene may enhance salt tolerance of plants by increasing the amount of treahalose and proline and regulating the expression of stress tolerance related genes.

Sweetpotato (Ipomoea batatas (L.) Lam.) breeding is challenging due to its genetic complexity. In the present study, interval mapping (IM) and multiple quantitative trait locus (QTL) model (MQM) analysis were used to identify QTLs for starch content with a mapping population consisting of 202 F1 individuals of a cross between Xushu 18, a cultivar susceptible to stem nematodes, with high yield and moderate starch, and Xu 781, which is resistant to stem nematodes, has low yield and high starch content. Six QTLs for starch content were mapped on six linkage groups of the Xu 781 map, explaining 9.1-38.8% of the variation. Especially, one of them, DMFN_4, accounted for 38.8% of starch content variation, which is the QTL that explains the highest phenotypic variation detected to date in sweetpotato. All of the six QTLs had a positive effect on the variation of the starch content, which indicated the inheritance derived from the parent Xu 781. Two QTLs for starch content were detected on two linkage groups of the Xushu 18 map, explaining 14.3 and 16.1% of the variation, respectively. They had a negative effect on the variation, indicating the inheritance derived from Xu 781. Seven of eight QTLs were co-localized with a single marker. This is the first report on the development of QTLs co-localized with a single marker in sweetpotato. These QTLs and their co-localized markers may be used in marker-assisted breeding for the starch content of sweetpotato.

This paper reported firstly successful cloning of lycopene ε-cyclase (IbLCYe) gene from sweetpotato, Ipomoea batatas (L.) Lam. Using rapid amplification of cDNA ends (RACE), IbLCYe gene was cloned from sweetpotato cv. Nongdafu 14 with high carotenoid content. The 1 805 bp cDNA sequence of IbLCYe gene contained a 1 236 bp open reading frame (ORF) encoding a 411 amino acids polypeptide with a molecular weight of 47 kDa and an isoelectric point (pI) of 6.95. IbLCYe protein contained one potential lycopene ε-cyclase domain and one potential FAD (flavinadenine dinucleotide)/NAD(P) (nicotinamide adenine dinucleotide phosphate)-binding domain, indicating that this protein shares the typical characteristics of LCYe proteins. The gDNA of IbLCYe gene was 4 029 bp and deduced to contain 5 introns and 6 exons. Real-time quantitative PCR analysis revealed that the expression level of IbLCYe gene was significantly higher in the storage roots of Nongdafu 14 than those in the leaves and stems. Transgenic tobacco (cv. Wisconsin 38) expressing IbLCYe gene accumulated significantly more β-carotene compared to the untransformed control plants. These results showed that IbLCYe gene has an important function for the accumulation of carotenoids of sweetpotato.

The contents of carotenoids in the storage root of sweetpotato, Ipomoea batatas (L.) Lam. vary dramatically among different cultivars. However, so far little is known about the regulation of carotenoids synthesis in sweetpotato. In our laboratory, we identified a novel sweetpotato mutant, Nongdafu 14, which is a homogenous mutant derived from the wild type Kokei No. 14. The contents of carotenoids in the storage root of Nongdafu 14 were analyzed using high performance liquid chromatography (HPLC), and it was found that the amount of carotenoids, b-carotene, lutein and zeaxantion, three major types of carotenoids in sweetpotato storage roots, increased 2-26 folds in Nongdafu 14 compared to Kokei No. 14. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed in Nongdafu 14, and a differentially expressed cDNA library was constructed using the cDNA of Nongdafu 14 storage roots as tester and that of Kokei No. 14 storage roots as driver. Out of the 1 530 clones sequenced, we identified 292 nonredundant ESTs. GO and KEGG analyses of these differentially expressed ESTs indicated that diverse metabolism pathways were affected and candidate genes involved in regulation of carotenoids synthesis are suggested.

Sequence-related amplification polymorphism (SRAP) markers closely linked to stem nematode resistance gene were developed in sweetpotato, Ipomoea batatas (L.) Lam. Using bulked segregant analysis (BSA), 200 SRAP primer combinations were screened with the resistant and susceptible bulked DNA from the 196 progenies of an F1 single-cross population of resistant parent Xu 781×susceptible parent Xushu 18, 77 of them showed polymorphic bands between resistant and susceptible DNA. Primer combinations detecting polymorphism between the two bulks were used to screen both parents and 10 individuals from each of the bulks. The results showed that primer combination A9B4 produced 3 specific bands in the resistant plants but not in the susceptible plants, suggesting that the markers, named Nsp1, Nsp2 and Nsp3, respectively, linked to a gene for stem nematode resistance. Primer combination A3B6 also produced a SRAP marker named Nsp4 linking to the resistance gene. Amplified analysis of the 196 F1 individuals indicated that the genetic distance between these markers and the resistance gene was 4.7, 4.7, 6.3, and 9.6 cM, respectively.

Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. Based on the EST sequence selected from salt-stressed suppression subtractive hybridization (SSH) cDNA library constructed with a salt-tolerant mutant LM79, a NFU gene, termed IbNFU1, was cloned from sweetpotato (Ipomoea batatas (L.) Lam.) via rapid amplification of cDNA ends (RACE). The cDNA sequence of 1 117 bp contained an 846 bp open reading frame encoding a 281 amino acids polypeptide with a molecular weight of 30.5 kDa and an isoelectric point (pI) of 5.12. IbNFU1 gene contained a conserved Cys-X-X-Cys motif in C-terminal of the iron-sulfur cluster domain. The deduced amino acid sequence had 66.08 to 71.99% sequence identity to NFU genes reported in Arabidopsis thaliana, Eucalyptus grandis and Vitis vinifera. Real-time quantitative PCR analysis revealed that the expression level of IbNFU1 gene was significantly higher in the roots of the mutant LM79 compared to the wild-type Lizixiang. Transgenic tobacco (cv. Wisconsin 38) plants expressing IbNFU1 gene exhibited significantly higher salt tolerance compared to the untransformed control plants. It is proposed that IbNFU1 gene has an important function for salt tolerance of plants.

AFLP fingerprinting of the 98 main sweetpotato varieties planted in China has been constructed. Using 17 AFLP primer combinations which were selected from 1 208 primer combinations and generated the most amounts of polymorphic bands, AFLP analysis of the 98 main sweetpotato varieties gave a total of 410 clear polymorphic bands with an average of 24.12 polymorphic bands per primer combination. Each one of the 98 sweetpotato varieties could be clearly distinguished by EcoR I-cta/Mse I-ggc primer combination which generated the most polymorphic bands. AFLP-based genetic distance ranged from 0.0546 to 0.5709 with an average of 0.3799. The dendrogram based on AFLP markers indicated that sweetpotato varieties coming from the same regions or having same parents were clustered in the same groups. Analysis of molecular variance (AMOVA) revealed greater variations within regions (94.08%) than among regions (5.92%). Thus, the genetic variations mainly existed within regions, while the variations among regions were very low in the tested sweetpotato varieties. Significant genetic variations existed between “Northern” and “Southern” sweetpotato varieties when Yangtze River was used as the dividing line.

The production of transgenic sweetpotato (cv. Xushu 18) plants exhibiting enhanced salt tolerance using salt overlysensitive (SOS) genes was achieved through Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strainEHA105 harbors a binary vector pCAMBIA3301 with SOS genes (SOS1, SOS2 and SOS3) and bar gene. Selection culturewas conducted using 0.3 mg L-1 phosphinothricin (PPT). A total of 40 plants were produced from the inoculated 170 cellaggregates via somatic embryogenesis. PCR analysis showed that 37 of the 40 regenerated plants were transgenic plants.The in vitro assay demonstrated that superoxide dismutase (SOD) and proline were significantly more accumulated andmalonaldehyde (MDA) was significantly less accumulated in 21 transgenic plants than in control plants when they wereexposed to 86 mmol L-1 NaCl. Salt tolerance of these 21 plants was further evaluated with Hoagland solution containing 0,51, 86, and 120 mmol L-1 NaCl in the greenhouse. The results indicated that 6 of them had significantly better growth androoting ability than the remaining 15 transgenic plants and control plants. Expression of SOS genes in the 6 salt-toleranttransgenic plants was demonstrated by RT-PCR analysis. This study provides an alternative approach for improving salttolerance of sweetpotato.