Application of simple sequence repeats in ...

Scientia Horticulturae 93 (2002) 215±224

Application of simple sequence repeats in determining the genetic relationships of cultivars used in sweet potato polycross breeding in Taiwan Shih Y. Hwanga,*, Yu T. Tsenga, Hsiao F. Lob a

Graduate Institute of Biotechnology, Chinese Culture University, 55 Hwagan Road, Yangmingshan, Taipei, Taiwan, ROC b Department of Horticulture, Chinese Culture University, 55 Hwagan Road, Yangmingshan, Taipei, Taiwan, ROC Accepted 16 August 2001

Abstract Simple sequence repeats (SSRs) were used to analyze the genetic diversity and genetic relationships among sweet potato [lpomoea batatas (L.) Lam.] cultivars. These cultivars consisted of Chinese and Japanese materials, landraces as well as cultivars derived either from hybrid or polycross breeding in Taiwan. SSR analysis from eight primer pairs revealed a total of 20 alleles of which 17 were polymorphic (85.0% polymorphism). An average of 2.5 alleles were obtained per SSR primer pair. On average 2.1 alleles per polymorphic SSR locus were ampli®ed. The construction of genetic relationships using unweighted pair group method with arithmetic mean (UPGMA) and principal coordinate analysis (PCA) demonstrated the capability of simple sequence repeats in sweet potato genotype identi®cation and classi®cation of genetic relationships. The UPGMA clustering and PCA revealed that polycross-derived cultivars possessed high levels of genetic diversity and originated from various genetic resources, and suggested the usefulness of polycross breeding strategy in spite of frequent cross-incompatibility. Moreover, high level of genetic variation in polycross breeding lines would assist in obtaining elite sweet potato materials in the future. In addition, most landraces were distantly related to the Chinese and Japanese materials and probably originated from Java and Brahman. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Genetic diversity; Genetic relationships; SSR; Sweet potato

1. Introduction Information on germplasm diversity and of the genetic relationships among elite breeding lines is critical in crop improvement. Sweet potato …2n ˆ 6X ˆ 90† (Ozias-Akins and * Corresponding author. Tel.: ‡886-2-28622331; fax: ‡886-2-28618266. E-mail address: [email protected] (S.Y. Hwang).

0304-4238/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 3 4 3 - 0

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Jarret, 1994) is the seventh important world's crop and is a major source of food and nutrition in developing countries (International Potato Center, 1996). Sweet potato in Taiwan was ®rst introduced from Java and Brahman as early as 17th century during Dutch colonization. Afterward, Japanese introduced sweet potato materials from Japan, USA, China, and also from Java and Brahman. Other sweet potatoes were introduced after World War II mainly from China, Japan, and USA. The breeding of sweet potato is usually carried out through hybrid or polycross breeding. Pre-screening of cross-compatibility is necessary, but time consuming, for the success of hybrid breeding. Jones (1965) proposed a polycross breeding scheme in which 20±30 parental lines are planted in an isolated plot and subjected to open pollination by insects and mass selection to eliminate the pre-screening of cross-compatibility. By using this polycross strategy, many elite sweet potato cultivars such as high yield, high b-carotene, and high protein cultivars have been generated in Taiwan. Despite common failure in positive identi®cation of parental lines using morphological markers that are subject to change due to environmental factors and cultural practices (Bernatzky and Tanksley, 1989), polycross breeding has been the major breeding program practiced in Taiwan since 1966. Sweet potatoes of Chinese, Japanese, and landraces in addition to hybrid- and polycross-derived cultivars formed the major composition of polycross breeding parental lines in Taiwan. However, no information has been obtained about the extent of genetic diversity in the polycross-derived cultivars since the practice of polycross breeding began in Taiwan. A variety of molecular techniques have been developed for measuring genetic variability. In sweet potato, RAPD had been used in cultivar ®ngerprinting (Connolly et al., 1994) and estimation of genetic diversity (Sagredo et al., 1998; Zhang et al., 1998). Huang and Sun (2000) used inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA in the study of genetic relationships of cultivated sweet potato and its wild relatives. Use of molecular markers in determination of the genetic relationship of sweet potato polycross breeding lines is useful for the improvement of the breeding ef®ciency. Micro-satellites are the region of DNA sequences consisting of arrays of a basic repeat unit that exhibit high levels of polymorphisms and have been successfully used in the study of genetic diversity and genotype identi®cation in barley (Saghai-Maroof et al., 1994), wheat (Plaschke et al., 1995), and rice (Xiao et al., 1996). Primers designed to ¯ank SSR loci in sweet potato were developed (Jarret and Bowen, 1994) and made this investigation possible. The information about the genetic diversity of sweet potato germplasm in Taiwan is particularly important for cultivar identi®cation and to enhance the classi®cation of germplasm collections. The objective of this investigation was to evaluate the genetic variation and the genetic relationship of elite sweet potato cultivars commonly used in polycross breeding in Taiwan by SSR assay with the emphasis on the genetic relationships among landraces, polycross-derived cultivars, and Chinese and Japanese materials. 2. Materials and methods 2.1. Plant materials and DNA extraction Twenty-two sweet potato cultivars were grown in a greenhouse for DNA isolation (Table 1). Stolons of these cultivars were provided by the Chiayi Agricultural Research

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Table 1 Name, origin, and classification of 22 sweet potato cultivars Cultivar number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 a

Cultivar name

Country of origin

Symbola

Characteristics

Changhua Hualien PF-1 Hualien PF-2 Kingmen Penhu RF Red flesh Yangmingshan PF Yongtsai Tainung 57 Tainung 67 Tainung 65 Tainung 66 Tainung 68 Tainung 70 Tainung 71 Taoyuan-2 Hsusu 18 Litzhsian Okinawa 100 Siemen 1 Beniazuma Satsumahikari

Taiwan Taiwan Taiwan Taiwan Taiwan Taiwan Taiwan

^ ^ ^ ^ ^ ^ ^

Virus-resistant

Taiwan Taiwan Taiwan Taiwan Taiwan Taiwan Taiwan Taiwan Taiwan China China Japan Japan Japan Japan

^

Leafy vegetable High vitamin A Better starch digestibility High protein Yield stability High starch High b-carotene High yield Leafy vegetable

& & & & & & * *

Pedigrees

High yield High yield

Tainung 27  Nancy Hall Nunglin 18  Centennial

Nancy Hall  Okinawa 100 Okinawa 100  Nancy Hall

Landrace (^), hybrid ( ), polycross (&), Chinese materials (*), and Japanese materials ( ).

Institute. Two cultivars were from China, four from Japan, two derived from hybrid breeding, six derived from polycross breeding, and eight were landraces. Tainung 67 was derived from a hybrid cross between Nunglin 18 and Nancy Hall, Nunglin 18 is Japanese material. Tainung 57 was derived from cross between Tainung 27 and Nancy Hall. Nancy Hall, introduced from USA, was the paternal parent for both Tainung 67 and Tainung 57. Although Taoyuan-2 was classi®ed as a polycross-derived cultivar, it is possibly a clonal variant of landrace Yongtsai (Liang Li, sweet potato breeder and former director of Chiayi Agricultural Research Institute, personal communication). Chinese Hsusu 18 was derived from a cross between Nancy Hall and Japanese Okinawa 100 (paternal parent). Chinese Litzhsian was derived from a cross between Okinawa 100 and Nancy Hall (paternal parent). From a sample of three plants for each sweet potato cultivar one leaf per plant was ground separately into powder in liquid nitrogen for the extraction of genomic DNA based on a modi®ed CTAB procedure (Doyle and Doyle, 1990). Ground leaf material (0.3 g) was placed in 5 ml of extraction buffer (2% CTAB, 0.1 M Tris±HCl (pH 8.0), 0.2 M EDTA, 1.4 M NaCl, 1% N-lauroylsarcosine, 1% SDS, 2% b-mercaptoethanol). The homogenate was incubated at 65 8C for 30 min, followed by another extraction with the addition of 50 mg/ml protease K in the extraction buffer and incubated at 55 8C for 30 min, followed by

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an equal volume extraction with chloroform:isoamylalcohol (24:1) and centrifuged for 20 min at 12,800g. The aqueous phase was transferred to a fresh centrifuge tube. Genomic DNA was precipitated with a two-thirds volume of absolute alcohol. Following centrifugation at 12,800g for 10 min, the alcohol was removed and the DNA pellet was rinsed twice with 75% (v/v) ethanol. The pellet was dried and suspended in 200 ml TE buffer (pH 8.0) and placed at 4 8C. DNA concentration was determined the following day using GeneQuant II RNA/DNA Calculator (Amersham Pharmacia Biotech). The DNA samples of the same cultivar were bulked for subsequent analysis. 2.2. SSR amplification Ten pairs of SSR primers designed for sweet potato were screened for single-locus microsatellite polymorphism (Jarret and Bowen, 1994). SSR primer pairs (Table 2) were ®rst subjected to annealing temperature screening using Robocycler GRADIENT 96 temperature cycler (Stratagene). The PCR ampli®cation procedure was mainly based on Welsh and McClelland (1990) and Williams et al. (1990). The PCR program was: step 1 at 94 8C for 3 min, step 2 35 cycles for 1 min at 94 8C for denaturation, 1 min at optimal temperature for annealing, and 1 min and 50 s at 72 8C for polymerization. The last step was 5 min at 72 8C for ®nal extension. For touchdown PCR ampli®cation, step 2 was changed to ®rst 10 cycles for 1 min at 94 8C, 1 min at 65 8C with 0.5 8C lower per cycle, and at 72 8C for 1 min and 50 s; and then 20 cycles for 1 min at 94 8C, 1 min at 60 8C, and 1 min and 50 s at 72 8C. Final volume of reaction mixture was 20 ml containing 500 mM KCl, 15 mM MgCl2, 0.01% gelatin, 100 mM Tris±HCl (pH 8.3), 0.2 mM dNTPs, 0.2 mM primer, 20 ng template DNA, 1 mg/ml RNase, and 0.5 U Taq polymerase. The ampli®cation products were electrophoresed in a 15% non-denaturing polyacrylamide gel and silver stained (Bassam et al., 1991). Table 2 Sequences of primers Sequence name

Sequence

Annealing temperature (8C)

SSR primers lb2/30 F lb2/30 R lb2/42 F lb2/42 R lb2/55 F lb2/55 R lb3/01 F lb3/01 R lb3/21 F lb3/21 R lb3/24 F lb3/24 R lb3/28 F lb3/28 R lb3/31 F lb3/31 R

50 -ACGCATAAGGGTATTGGTGAAG-30 50 -ACGGAGGATGGTTCAGGTG-30 50 -GCGGAACGGACGAGAAAA-30 50 -ATGGCAGAGTGAAAATGGAACA-30 50 -CGTCCATGCTAAAGGTGTCAA-30 50 -ATAGGGGATTGTGCGTAATTTG-30 50 -CCTTCATCACCTTCCATTCCT-30 50 -CTCCCAGTTAACCAAAACCTGA-30 50 -AATCCAAATGAGTCATACACCT-30 50 -CGAAAAATCTCTGGTTACGTT-30 50 -TTTGGCATGGGCCTGTATT-30 50 -GTTCTTCTGCACTGCCTGATTC-30 50 -TCGCCTTTCTCTTTGCACC-30 50 -CCCCTCTCTTCTACAACCCTTC-30 50 -TTCCCTTTCCTTTCCTTCCC-30 50 -ACCCCAAATCCCAACTCCA-30

65±60 65±60 65 62 65±60 65±60 65 62

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2.3. Data collection and analysis Each SSR fragment was treated as a unit character and scored as binary codes …1=0 ˆ ‡= †. Similarity matrices were constructed from the binary data with Jaccard's coef®cients (Jaccard, 1908). Jaccard's similarity ˆ Nab=Na ‡ Nb, where Nab represents the number of fragments shared by polycross breeding line a and b, Na represents ampli®ed fragments in sample a, and Nb represents ampli®ed fragments in sample b. A dendrogram was generated with the UPGMA algorithm as described by Sneath and Sokal (1973) with the SAHN (sequential, agglomerative, hierarchical, and nested clustering) routine. A graphical display of the genetic relationships was also constructed by principal coordinate analysis (Gower, 1966). These statistical analyses were performed by NTSYS-pc, Version 1.8 (Exeter Software, Setauket, NY). Expected heterozygosity was calculated from allele frequency using formula: n X Hˆ1 p2i iˆ1

where p is the frequency of the ith allele of each SSR marker (Nei, 1973) and n the number of alleles. 3. Results and discussion 3.1. Levels of SSR polymorphisms Ten pairs of SSR primers designed for sweet potato were tested for single-locus microsatellite polymorphism (Jarret and Bowen, 1994). Of the 10 SSR primer pairs, eight produced good resolution SSR bands (Table 2) while two pairs of SSR primers (lb2/27 and lb3/15) gave no ampli®cation products. These eight SSR primer pairs were then used for analysis of the genetic polymorphism and genetic relationships of Chinese and Japanese materials, landraces, hybrid-derived cultivars, and polycross-derived cultivars (Table 1). All eight SSR primer pairs ampli®ed single locus markers. Ampli®cation of 22 genotypes with these eight SSR primer pairs yielded a total of 20 alleles, of which 17 were polymorphic. Primer pair lb3/21 ampli®ed four alleles (100% polymorphic), the highest number, and lb3/01 ampli®ed only one monomorphic allele. An average of 2.5 alleles were obtained per SSR locus. In polymorphic SSR loci, an average of 2.1 polymorphic alleles were obtained. Four primer pairs, lb3/21, lb3/24, lb3/28, and lb3/ 31, ampli®ed 100% polymorphism. On average, 85.0% polymorphism was found for sweet potato genotypes based on SSR markers. An average of 0.55 expected heterozygosity excluding the monomorphic locus was comparable to 0.59 averaged from ®ve polymorphic SSR loci reported by Buteler et al. (1999). In this investigation, we learned that the germplasm used for polycross breeding in Taiwan possessed high levels of genetic variability. Only 1±4 alleles per SSR locus were ampli®ed in this study (Table 3), however, Buteler et al. (1999) reported that in sweet potato 3±10 alleles per SSR primer pair were ampli®ed. The discrepancy in the number of alleles ampli®ed between this study and the result of

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Table 3 Number of polymorphic bands produced and the number of alleles of the SSR locus Primer pairs lb2/30 lb2/42 lb2/55 lb3/01 lb3/21 lb3/24 lb3/28 lb3/31 Total Average a

No. of alleles

No. of polymorphic alleles

2 2 3 1 4 2 3 3

1 2 2 0 4 2 3 3

20 2.5

17 2.1

Expected heterozygositya 0.47 0.65 0.83 0.00 0.78 0.37 0.47 0.27 0.48 (0.55)

Number in parenthesis indicates expected heterozygosity excluding the monomorphic locus.

Buteler et al. (1999) was because the SSR primer pairs used were not all the same. One SSR primer pair lb2/55 ampli®ed three alleles both according to Buteler et al. (1999) and the present study. However, SSR primer pair lb2/42 ampli®ed ®ve alleles in their study but only two alleles in the present study. The reason for the smaller number of alleles ampli®ed with primer pair lb2/42 was probably the higher annealing temperature used in our study. The annealing temperature for the lb2/42 primer pair was 65±60 8C (touchdown annealing pro®le) in this study, however, Buteler et al. (1999) used 59 8C as the annealing temperature for the lb2/42 primer pair. The high level of SSR polymorphism in this study is comparable to the RAPD analysis in sweet potato (77.6%, Connolly et al., 1994; 51.7%, Zhang et al., 1998), and also higher than the 55.7% polymorphism of ISSR analysis on ®ve cultivars of allohexaploid sweet potato (Huang and Sun, 2000). The SSR studies in this investigation con®rmed that there is much genetic variation in sweet potato genomes. 3.2. Genetic similarity among cultivated sweet potato The frequency of pair-wise similarity coef®cients for SSR analysis of 22 sweet potato polycross breeding lines is shown in Fig. 1. The SSR-based Jaccard's coef®cient ranged between 0.400 and 0.938, with a mean of 0.645. Most similarity coef®cients were between 0.600 and 0.740, which explain 64.1% of the total frequency of pair-wise similarity coef®cient by SSR analysis. These results are similar to the results of Huang and Sun (2000), who analyzed ®ve sweet potato cultivars where the average similarity coef®cient was 0.658 based on ISSR analysis. The results in this study were also consistent with those of Connolly et al. (1994), who analyzed six sweet potato cultivars based on RAPD analysis, the similarity coef®cient ranged from 0.590 to 0.700. It is likely that the large genome size, allopolyploidy, and heterozygosity of sweet potato are the reasons for its high levels of polymorphisms. Moreover, genetic diversity is maintained due to self-incompatibility and vegetative reproduction. It was reported by He et al. (1995) that high levels of polymorphisms among sweet potato cultivars were ®xed

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Fig. 1. Frequency distribution of pair-wise SSR based similarity estimates among 22 polycross breeding lines of sweet potato.

through vegetative reproduction and maintained through high level of gene ¯ow due to self-incompatibility. However, the wide range of genetic variation in elite sweet potato polycross breeding lines could possibly lower the polycross breeding ef®ciency especially when the cultivars used as parental lines are cross-incompatible. 3.3. Genetic relationships among sweet potato cultivars Similarity matrices based on Jaccard's similarity coef®cient were used to conduct UPGMA analysis for SSR data in order to generate the dendrogram (Fig. 2). At a similarity coef®cient value of 0.680, three groups were evident in the UPGMA dendrogram. PCA performed on SSR data are shown in Fig. 3. PCA for SSR data did not separate polycross breeding lines very well. Based on SSR analysis, the ®rst and second principal coordinates explain 18.0 and 13.9% of the total variation, respectively. Three major clusters were found by UPGMA. The UPGMA clustering of sweet potato cultivars based on SSR markers showed that landraces Changhua, Kingmen, and Yangminshan PF were grouped into cluster A and had less genetic similarity with Chinese and Japanese materials. In cluster B, landraces Hualien PF-1 and Hualien PF-2 were almost identical to each other. It is not surprising that landraces Yongtsai and Taoyuan-2 had high genetic similarity since Taoyuan-2 was probably a clonal variant of landrace Yongtsai (Liang Li, personal communication). Japanese Okinawa 100 and Satsumahikari were also included in cluster B. Landrace red ¯esh was grouped into cluster C with Japanese Beniazuma and Chinese materials. Polycross-derived cultivars were scattered among these

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Fig. 2. UPGMA-based dendrogram of polycross breeding lines generated from SSR markers. The numerical scale indicates genetic similarity. Polycross breeding line numbers correspond to Table 1. The symbols for polycross breeding lines are landrace (^), hybrid ( ), polycross (&), Chinese materials (*), and Japanese materials ( ).

Fig. 3. Principal coordinate analysis of the 22 sweet potato polycross breeding lines included in this analysis based on SSR markers. Polycross breeding line numbers correspond to Table 1. The symbols for polycross breeding lines are landrace (^), hybrid ( ), polycross (&), Chinese materials (*), and Japanese materials ( ).

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three clusters with cluster A containing Tainung 66 and Tainung 68, Taoyuan-2 in cluster B, and cluster C containing Tainung 65, Tainung 70, and Tainung 71. Tainung 27 was maternal parent of hybrid-derived Tainung 57 with an unknown ancestor. Because the maternal parent of Tainung 67 is from Japan, it is not surprising that Tainung 67 was grouped into cluster B with closer genetic relationship to Japanese Okinawa 100 and Satsumahikari. The conclusion was con®rmed by a principal coordinate analysis showing that Tainung 67 was located around Japanese Okinawa 100 and Satsumahikari (Fig. 3). Chinese Litzhsian was derived from a cross between Okinawa 100 and Nancy Hall with Okinawa 100 the maternal parent. Although Litzhsian was grouped into sub-cluster of cluster C with Tainung 70, Tainung 65, and landrace red ¯esh by UPGMA, the PCA did not support this result. Nancy Hall, the paternal parent of Litzhsain may play a role in this case. Chinese Hsusu 18 was derived from Nancy Hall and Okinawa 100 cross with Nancy Hall the maternal parent. It is probable that Hsusu 18 with American Nancy Hall as maternal parent, had more distant relationships with landraces and most polycross-derived cultivars through UPGMA analysis, a result that was also supported by principal coordinate analysis. The structure of clusters revealed by PCA showed that landraces are more closely related to polycross- and hybrid-derived cultivars than to Chinese and Japanese material and probably originated from Java and Brahman, an aspect requiring further investigation. Interestingly, the scattering of polycross-derived cultivars among landraces and Japanese and Chinese materials indicated that polycross breeding is a useful breeding strategy in adopting various sources of sweet potato genomes. Therefore, the polycross breeding method generated highly polymorphic elite sweet potato cultivars in spite of possible existence of cross-incompatibility among parental lines. It is interesting to observe that PCA did not distinguish the 22 polycross breeding lines into separate groupings as such with UPGMA. However, the formula given by Jaccard (1908) could have introduced a certain bias in the estimation of genetic similarity because the absence of a fragment in two cultivars was not considered. Moreover, lack of correlation between UPGMA and PCA could be ascribed to the occurrence of selection and genetic drift during the development of cultivars (Graner et al., 1994; Kim and Ward, 1997). In contrast, the PCA result might be a re¯ection of the inherent genetic nature of these polycross breeding lines. In conclusion, micro-satellites possess several advantages over other molecular markers. They are abundant, hypervariable, and co-dominant. Therefore, SSR are particularly attractive for differentiating between plant varieties. The major result in this study is that SSR markers are appropriate for the genotyping and revealing genetic relationship of sweet potato cultivars. The ability to resolve genetic relationships among sweet potato cultivars may be more directly related to the highly polymorphic SSR marker technique. References Bassam, B.J., Caetano-AnolleÂs, G., Gressholf, P.M., 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196, 80±83. Bernatzky, R., Tanksley, S.D., 1989. Restriction fragment as molecular markers for germplasm evaluation and utilisation. In: Brown, A.H.D., Frankel, O.H., Marshall, D.R., Williams, J.T. (Eds.), The Use of Plant Genetic Resources. Cambridge University Press, New York, pp. 353±362.

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