LlU Cheng, ZHAO Ning, JlANG Zhi-cheng, ZHANG Huan, ZHAl Hong, HE Shao-zhen, GAO Shao-pei,LlU Qing-chang

Key Laboratory of Sweetpotato Biology and Biotechnology, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Crop Genetic Improvement/Laboratory of Crop Heterosis and Utilization, Ministry of Education/College of Agronomy & Biotechnology,China Agricultural University, Beijing 100193, P.R.China

Abstract 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.

Keywords: sweetpotato, genetic diversity, population structure, SSR

1.lntroduction

Sweetpotato,Ipomoeabatatas(L.) Lam., is an important food crop that is widely cultivated around the world.The total yield of sweetpotato ranks the eighth in world food production.China’s annual yield of sweetpotato accounts for approximately 55% of global production (FAO 2020).Sweetpotato originated in the South American lowlands and then spread to North America, Europe, Africa, Asia and Oceania (Gichukiet al.2003).In the late 16th century, sweetpotato was introduced from the Philippines to Fujian and Guangdong of China.Approximately 94% of Chinese sweetpotato bred varieties are related to the American variety ‘Nancy Hall’ and the Japanese variety ‘Okinawa 100’, so they exhibit a narrow genetic background (Liuet al.2012).Therefore, to broaden the genetic background of Chinese sweetpotato varieties, it is essential to assess the genetic diversity of the existing sweetpotato germplasm resources.

DNA molecular markers are powerful tools for germplasm characterization, variety identification,phylogenetic studies and diversity analysis in crops.Various molecular markers have been used to characterize sweetpotato germplasm.For example,using amplified fragment length polymorphism (AFLP)markers, Heet al.(2006) assessed the genetic diversity of 108 sweetpotato accessions and found that those accessions did not cluster according to their origins.Tanakaet al.(2007) studied the genetic diversity of 213 sweetpotato accessions using random amplified polymorphism DNA (RAPD) markers, which revealed only limited genetic differences in the sweetpotato accessions from different origins.Yanget al.(2015)analyzed the genetic diversity of 380 sweetpotato accessions and divided them into three groups based on simple sequence repeats (SSR) markers.Menget al.(2018) developed seven highly polymorphic SSR primer pairs and constructed the SSR fingerprinting of 203 sweetpotato varieties.Davidet al.(2018) analyzed 135 sweetpotato parents and six check clones using 31 SSR primer pairs and divided most African sweetpotato parents into two main subclusters, which exhibited no associations with morphology or geographical origins.Zhaoet al.(2019) divided 99 registered sweetpotato varieties into three subgroups with SSR markers.Using restriction site-associated DNA sequencing (RAD-seq),Fenget al.(2020) preliminarily evaluated the genomewide genetic diversity and population structure of sweetpotato.

More than 2 000 sweetpotato accessions are conserved in China.However, there are only a limited number of sweetpotato accessions with clear genetic diversity at present.Therefore, the large scale evaluation of sweetpotato germplasm for genetic diversity is still necessary to effectively use them in the genetic improvement of this crop.The objective of this study was to assess the genetic diversity and population structure of 617 sweetpotato accessions from China and 11 other countries, and to determine the relationships between them using SSR markers.The results of this study provide valuable information for the better utilization of these accessions in sweetpotato breeding.

2.Materials and methods

2.1.Plant materials

A total of 617 sweetpotato accessions, including 376 landraces and 162 bred varieties from China and 79 introduced varieties from 11 other countries, were employed in this study.These accessions were provided by the National Sweetpotato Genebank in Vitro (Xuzhou,China) and the National Sweetpotato Germplasm Resources Nursery (Guangzhou, China).Their origins are listed in Appendices A and B.Because most of the Chinese sweetpotato landraces originated from Guangdong, more Guangdong landraces were used in this study.

2.2.DNA extraction

Genomic DNA from the young leaves of each sweetpotato accession was extracted using the cetyltrimethylammonium bromide (CTAB) method(Saghai-Maroofet al.1984).The quality of the DNA was evaluated on a 1% (w/v) agarose gel.The concentration of DNA was determined by nanodrop2000c (NanoDrop,Waltham, MA, USA) and diluted to 50 ng μL–1.

2.3.SSR genotyping

A total of 30 SSR primer pairs with high polymorphism developed by Zhaoet al.(2013) were used to genotype the 617 sweetpotato accessions.The information for these SSR primer pairs is shown in Appendix C.The PCR amplification was performed in 20 μL volumes consisting of 2 μL 10× PCR buffer, 0.8 μL dNTPs (10 mmol L–1),0.2 μL EasyTaq?DNA polymerase (5 U μL–1), 2 μL SSR primer (7.5 μmol L–1), 3 μL genomic DNA (50 ng μL–1)and 12 μL deionized distilled water on a TaKaRa PCR Thermal Cycle (TP600, TaKaRa, Kusatsu, Japan).The thermal cycling conditions were as described by Menget al.(2018).The PCR products were analyzed on a 6%denaturing polyacrylamide gel using a Bio-Rad sequencer(PowerPacTM HV Power Supply, Bio-Rad, Hercules, CA,USA), and the electrophoresis results were detected by standard silver nitrate staining.Polymorphic bands were visually scored as binary data, with “1” for presence and “0”for absence, by a GeneRulerTM100 bp DNA Ladder.

2.4.Data analysis

The polymorphism information content (PIC) and gene diversity of each primer pair were calculated with PowerMarker v3.25 (Nei 1973; Liu and Muse 2005).Shannon’s information index and Nei’s unbiased genetic distance were calculated with Popgene v1.32 (Lewontin 1972; Nei and Roychoudhury 1974; Mohammadiet al.2008; Yaoet al.2009).Population structure was examined with Structure v2.3.4 (Falushet al.2007).The simulations were run with both “l(fā)ength of burnin period”and “number of MCMC reps after burnin” set at 100 000.The K values ranged from 1 to 10 and the runs for each K were replicated 10 times.The STRUCTURE HARVESTER website was used to determine the true value of K according to the method of Earl and VonHoldt(2012).The results of population structure analysis were imported into a map of Asia through the “rworldmap”package in R (South 2011).The UPGMA cluster analysis was carried out with Popgene v1.32 and the dendrogram was drawn with MEGA 7 (Kumaret al.2016).The principal coordinate analysis (PCoA) was done with GenAlEx v6.503 and the 3D scatter plot was drawn with Origin v9.0 (Peakall and Smouse 2006, 2012).Analysis of molecular variance (AMOVA) was completed with GenAlEx v6.503 (Excoffieret al.1992).

3.Results

3.1.Polymorphism of SSR primer pairs

Fig.1 LnP(D) (A) and ΔK (B) evaluations of the 617 sweetpotato accessions.

Thirty SSR primer pairs with high polymorphism were used to analyze the genetic diversity and population structure of 617 sweetpotato accessions from 12 countries.A total of 180 polymorphic bands were amplified, which ranged from 3 to 13 alleles per primer pair (Appendix C).The PIC values for each primer pair ranged from 0.1842 to 0.3593 with an average of 0.2861,and the gene diversity ranged from 0.2188 to 0.4704 with an average of 0.3596 (Appendix C).These results showed that the 30 SSR primer pairs were suitable for assessing the genetic diversity of the 617 sweetpotato accessions.

3.2.Population structure analysis

The population structure of the 617 sweetpotato accessions was inferred based on SSR markers.The LnP(D) score, an estimate of the posterior probability of the data for a given K (the number of populations),increased with an increase in K, and ΔKreached its maximum at K=3 (Fig.1-A and B), suggesting that each accession fell into one of three groups.Therefore, the 617 accessions were divided into three groups, Group 1,Group 2 and Group 3, which contained 228 (85 landraces,89 bred varieties and 54 introduced varieties), 136 (67 landraces, 48 bred varieties and 21 introduced varieties)and 253 (223 landraces, 26 bred varieties and 4 introduced varieties) accessions, respectively (Fig.2).

Fig.2 Population structure analysis of the 617 sweetpotato accessions.G1–G3, Group 1, Group 2 and Group 3, respectively.

Since the sweetpotato accessions used in this study were mostly from Asia, the results of the population structure analysis were introduced into Asian maps.The results showed that the accessions in Group 2 were mainly distributed in low altitude and coastal areas, while the accessions in Group 1 and Group 3 were widely distributed.The genetic diversity of the sweetpotato germplasm was abundant in the coastal areas of southeastern China, and it decreased gradually from low altitude to high altitude with the spread to inland areas.

3.3.Principal coordinate analysis

A PCoA was performed to validate the results of the population structure analysis.The first three principal coordinates explained 13.85, 5.42 and 3.80% of the molecular variance, respectively, amounting to 23.07%(Fig.3).All the accessions were labeled according to the population structure analysis.The three groups were basically separated from each other in three principal coordinates, which showed high consistency with the results of the population structure analysis (Fig.3).The PCoA revealed that Group 2 had richer genetic diversity.In addition, Group 1 and Group 3 had much overlap and the genetic distance between them was narrow, while both Group1 and Group 3 had large genetic distances from Group 2 (Fig.3).

3.4.UPGMA cluster analysis

Fig.3 The principal coordinate analysis of the 617 sweetpotato accessions.G1–G3, Group 1, Group 2 and Group 3,respectively.

The UPGMA cluster analysis showed that the sweetpotato accessions from the same group were clustered together(Fig.4-A), which is consistent with the results of the population structure analysis.Moreover, Group 1 and Group 3 were clustered together (Fig 4-B), indicating that these two groups were less genetically differentiated.

The sweetpotato accessions from the same origin did not exhibit obvious clustering.The accessions from southern locations of China were clustered together with those from Southeast Asian countries, America and Japan(Fig.5).The accessions of the same type were not clustered together.Chinese bred varieties were clustered together with introduced varieties.

3.5.Genetic differentiation and genetic diversity analysis

The genetic differentiations among the 617 sweetpotato accessions were analyzed using AMOVA.The results indicated that 11% of the total variation was attributed to the variation among groups, while 89% was attributed to the variation within groups.The variations among and within origins accounted for 5 and 95%, respectively, and those among and within germplasm types accounted for 3 and 97%, respectively.

Fig.4 Cluster analysis of the 617 sweetpotato accessions.A, phylogenic tree.B, model-based cluster analysis.G1–G3,Group 1, Group 2 and Group 3, respectively.

Shannon’s information index, first principal coordinate range and Nei’s unbiased genetic distance were used to assess the genetic diversity of the 617 sweetpotato accessions.Combining the results, the accessions of Group 2 exhibited the highest level of genetic diversity,followed by those of Group 3, and those of Group 1 had the lowest level (Fig.6-A).According to germplasm types, Chinese landraces showed the highest level of genetic diversity, followed by Chinese bred varieties,and introduced varieties had the lowest level (Fig.6-B).The accessions from South China (e.g., Fujian and Guangdong) exhibited the highest level of genetic diversity, and the genetic diversity declined from South China to the inland areas (Fig.6-C).

Fig.5 Cluster analysis of the 617 sweetpotato accessions based on their origins.

Fig.6 Comparisons of genetic diversity among the 617 sweetpotato accessions.A, comparison of genetic diversity between different groups.B, comparison of genetic diversity between different germplasm types.C, comparison of genetic diversity between different origins.

4.Discussion

The large scale evaluation of sweetpotato germplasm for genetic diversity is vital for exploiting the valuable alleles present in diverse landraces and varieties of this crop.However, the number of sweetpotato accessions with clear genetic diversity is still limited to date (Yanget al.2015; Davidet al.2018; Menget al.2018).SSR markers are widely used to assess the genetic diversity in crops because of their advantages of co-dominance,high polymorphism, reliability and reproducibility (Huanget al.2012; Xiaoet al.2012; Ngailoet al.2016).In this study, the genetic diversity and population structure of 617 sweetpotato accessions were analyzed using 30 SSR primer pairs.The PIC and gene diversity analyses revealed that these sweetpotato accessions have richer alleles and gene diversity (Appendix C).

The 617 sweetpotato accessions were divided into three groups by the Structure v2.3.4 model analysis,phylogenic analysis and PCoA, and there were no obvious correlations between the origins and types of sweetpotato germplasm (Figs.2–6).These results are consisted with those of Yanget al.(2015), possibly because of the cross incompatibility of sweetpotato (Yanget al.2017).Of the three groups, Group 2 exhibited the highest level of genetic diversity and its accessions were mainly distributed in low-latitude regions, while the genetic distances of Group 1 and Group 3 were relatively close(Figs.3 and 4).

The sweetpotato accessions from South China exhibited the highest level of genetic diversity, and the genetic diversity gradually declined from Fujian and Guangdong to the inland areas, which support the hypothesis that Fujian and Guangdong were the regions where sweetpotato was first introduced to China and it then spread to the inland areas (Fig.6).In addition, the accessions from the southern locations of China were clustered together with those from Japan, America and Southeast Asia, which is consistent with the history of the spread and breeding of sweetpotato (Fig.6).Furthermore,the genetic diversity of Chinese landraces was the highest,while that of the Chinese bred varieties was lower (Fig.6).The limited number of parents used in sweetpotato breeding may have led to the low genetic diversity of Chinese bred varieties, most of which generally have the genetic background of ‘Nancy Hall’ and ‘Okinawa 100’ (Liuet al.2012).In addition, direct selection for target traits in sweetpotato breeding may have also led to the low genetic diversity of the bred varieties.Therefore, the utilization of Chinese landraces should be given greater attention in sweetpotato breeding in China.

5.Conclusion

The genetic diversity and genetic structure of 617 sweetpotato accessions from China and 11 other countries were successfully assessed using 30 SSR primer pairs.These sweetpotato accessions were divided into three groups, and significant genetic differentiation existed between the different groups.Low levels of genetic differentiation were found between the different origins and types.The accessions from Fujian and Guangdong exhibited the highest level of genetic diversity, which supports the hypothesis that they were the first regions where sweetpotato was introduced to China.These results provide valuable information for the better utilization of these accessions in future sweetpotato breeding.

Acknowledgements

This work was supported by the National Key R&D Program of China (2019YFD1001301 and 2019YFD1001300), the earmarked fund for CARS-10-Sweetpotato and the Hebei Key R&D Program, China(20326320D).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2023.02.004