Characterization of Tomentosa cherry (Prunus tomentosa Thunb.) genotypes using SSR markers and morphological traits

Scientia Horticulturae 118 (2008) 39–47

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Characterization of Tomentosa cherry (Prunus tomentosa Thunb.) genotypes using SSR markers and morphological traits Qijing Zhang a,b, Guijun Yan c, Hongyan Dai a, Xinzhong Zhang d,e, Chunmin Li e, Zhihong Zhang a,* a

College of Horticulture, Shenyang Agricultural University, Shenyang, Liaoning 110161, PR China Liaoning Institute of Pomology, Xiongyue, Liaoning 115214, PR China c School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, WA 6009, Australia d College of Agriculture and Biotechnology, China Agricultural University, Beijing 100094, PR China e Changli Fruit Research Institute, Hebei Academy of Agriculture and Forestry Sciences, Changli, Hebei 066600, PR China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 January 2008 Received in revised form 23 April 2008 Accepted 25 May 2008

A collection of 44 Tomentosa cherry (Prunus tomentosa Thunb.) accessions from 10 eco-geographical regions was evaluated using morphological descriptors. Ten quantitative and eight qualitative traits were analyzed and significant differences among populations were found for most traits. The highest variation observed was in fruit weight, fruit width and leaf width. P. tomentosa is distributed in at least five northern Chinese provinces and different accessions have their own specific morphological features. It was possible to identify several highly distinct accessions based on morphological characters. Genetic variation among the 44 Tomentosa cherries and 7 accessions from 3 related species (P. humilis, P. japonica and P. glandulosa) was characterized by simple sequence repeat (SSR) markers developed from peach, sweet cherry, sour cherry and apricot. Forty-four out of 110 SSR markers (40%) could be transferred to Tomentosa cherry and the other 3 species. A total of 250 alleles were detected with an average of 5.68 alleles per locus and an average polymorphism information content of 0.52. The results demonstrated the cross-species transferability of SSR primers developed in cultivated species to wild species in Prunus for the discrimination of different genotypes. Unweighted pair group method with arithmetic average (UPGMA) analysis of SSR data clustered the P. humilis, P. japonica and P. glandulosa into one group and the accessions of P. tomentosa into another parallel group with strong bootstrap support (93–100%). Within P. tomentosa, the weeping accessions formed a subgroup and the rest formed several groups that generally reflected their geographical origins. ß 2008 Elsevier B.V. All rights reserved.

Keywords: Tomentosa cherry Transferability Molecular markers SSRs Taxonomy

1. Introduction Prunus tomentosa Thunb. (2n = 16) belongs to the Prunus subgenus Lithocerasus in the family Rosaceae (Rehder, 1940; Ingram, 1948). P. tomentosa is native throughout temperate regions in China and the local people have been consuming its fruits for more than 2000 years (Yu¨ and Li, 1986). The Tomentosa cherry adapts to diverse environments, with an extensive germplasm resource for domestication and improvement. It is a very cold tolerant species and can endure temperatures as low as 40 8C. Its fruits are resistant to rain cracking and ripen synchronously. Fruits are also rich in vitamins and other antioxidative compounds, such as carotene, vitamins B1, B2, C, D, E and niacin (Gao et al., 2000). It has the potential to be

* Corresponding author. Tel.: +86 24 88487143; fax: +86 24 88487144. E-mail address: [email protected] (Z. Zhang). 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.05.022

cultivated as an ornamental shrub or for fresh fruit production in harsh and cold areas. It may also be used to improve other Prunus species as a gene donor because the genus is capable of wide hybridization. Seed lots were taken to America where it is known as Nanking cherry. Limited breeding has been carried out in the USA and the former Soviet Union to produce cultivars and rootstocks for various Prunus species (Kash, 1989). A breeding program on Tomentosa cherry in China started recently and several cultivars such as ‘Jixiang’, an ornamental weeping type and a white fruit type were released for commercial production, however, they are direct selections from naturally pollinated populations. Although it has been widely used as rootstock for peaches, plums and sweet cherries, the species has not been developed into a commercial fruit crop worldwide. It still remains in the realm of home gardeners in North America and local markets in Asia. The potential of this species in the breeding of new cherry cultivars for fresh production needs further investigation. The species might be a valuable germplasm to increase cultivation in

Q. Zhang et al. / Scientia Horticulturae 118 (2008) 39–47

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the colder regions of the Urals and Siberia, North America, Canada, the far east region of Russia as well as northern China. An understanding of its genetic diversity among and within the wild accessions from its natural distribution is essential for the formulation of strategies for their conservation and utilization. Simple sequence repeat (SSR) or microsatellite markers are becoming a useful tool for genotyping, germplasm characterization and fingerprinting in many plant species because they are PCR-based, highly polymorphic, codominant, abundant and highly reproducible (Powell et al., 1996). Moreover SSR markers have been developed in almost every cultivated fruit species of Prunus, including peach (Cipriani et al., 1999; Testolin et al., 2000; Dirlewanger et al., 2002), apricot (Lopes et al., 2002; Messina et al., 2004), sour cherry (Downey and Iezzoni, 2000), Japanese plum (Mnejja et al., 2004), and sweet cherry (Cantini et al., 2001; Clarke and Tobutt, 2003; Struss et al., 2003). By comparison of genetic linkage maps obtained from different species, the genome structure was found to be highly conserved in Prunus (Joobeur et al., 2000; Yamamoto et al., 2001). This provided the possibility of transferring these developed SSR markers between related species. Primers designed for peach SSR loci have been used widely in other relatives of Prunus (sweet and sour cherry, plum, almond, apricot and black cherry) as demonstrated by many researchers. Downey and Iezzoni (2000) used SSR markers identified from sweet cherry, peach and sour cherry to study genetic diversity in black cherry (P. serotina). Lambert et al. (2004) use primers developed from peach and sour cherry to construct genetic maps of apricot cultivars (P. armeniacea). However, there have been no reports on the use of molecular markers to study the genetic diversity among Tomentosa cherry germplasm. The biodiversity of Tomentosa cherry and its relationship to other related species remain uncertain. Therefore, it is necessary to conduct a morphological and molecular study to understand the genetic diversity among Tomentosa cherries and their relationship with other related species. The overall objective of this study was to investigate the existing genetic resources of Tomentosa cherry, collect different accessions and evaluate the collection by morphological and DNA studies. The specific aims were to test the transferability of SSR markers developed in other related species in Tomentosa cherry and to analyze the genetic relationships within Tomentosa cherry accessions and their relationships with several other Prunus species. Furthermore this research was intended to offer information on the taxonomy and geographic distribution of this important wild germplasm to facilitate its development as a crop plant in the future.

Fig. 1. Geographic location of collection sites of P. tomentosa accessions used in this study.

2. Materials and methods 2.1. Plant material A total of 51 accessions (Table 1) were used in this study. All were in subgenus Lithocerasus of genus Prunus (Rehder, 1940; Ingram, 1948), including 44 accessions of P. tomentosa, 4 accessions of P. humilis, 2 accessions of P. glandulosa and 1 accession of P. japonica. The Tomentosa cherry accessions were collected from 10 locations from 5 different provinces in China. The accessions of P. humilis were collected from Taigu in Shanxi province. The rest were from the Arboretum of Xiongyue in Liaoning Province. The accession numbers and their geographical distributions are shown in Table 1 and Fig. 1, respectively. 2.2. Morphological characters Characterization of vegetative material and fruits was based on almond descriptors developed by the International Plant Genetic Resources Institute (IPGRI) (Gulcan, 1985) with minor modifications. Eight qualitative traits were recorded by visual inspection of

Table 1 Plant materials used in the study and their origin Taxa.

Collection site (County, Province)

Longitude 8E

Latitude 8N

Code of the accessions

P. tomentosa

Qianan, Hebei Xiongyue, Liaoning

118.73 122.24

40.01 40.28

Dengta, Liaoning

123.34

41.43

Shenyang, Liaoning Yixian, Liaoning Gongzhuling, Jilin Shulan, Jilin Suiling, Heilongjiang

123.57 121.22 124.82 126.95 127.11

41.82 41.55 43.51 44.39 47.24

Mudanjiang, Heilongjiang Kouxian, Shanxi

129.61 111.50

44.58 36.08

HebeiQA(1), HebeiQB(2), HebeiQC(3), HebeiQD(4) LiaoningXA(5), LiaoningXB(6), LiaoningXC(7), LiaoningXD(8), LiaoningXE(9), LiaoningXF(10) LiaoningDA(11), LiaoningDB(12), LiaoningDC(13), LiaoningDD(14), LiaoningDE(15), LiaoningDF(16), LiaoningDG(17) LiaoningSA(18), LiaoningSB(19), LiaoningSC(20) LiaoningYA(21), LiaoningYB(22), LiaoningYC(23), LiaoningYD(24) JilinGA(25), JilinGB(26), JilinGC(27), JilinGD(28), JilinGE(29), JilinGF(30) Jixiang(31), JilinSA(32), JilinSB(33) HeilongjiangSA(34), HeilongjiangSB(35), HeilongjiangSC(36), HeilongjiangSD(37), HeilongjiangSE(38), HeilongjiangSF(39), HeilongjiangSG(40), HeilongjiangSH(41) HeilongjiangMA(42) ShanxiKA(43), ShanxiKB(44)

P. humilis P. japonica

Taigu, Shanxi Xiongyue, Liaoning

112.53 122.24

37.42 40.28

OuliA(45), OuliB(46), OuliC(47), OuliD(48) ChanggengYuli(49)

P. glandulosa

Xiongyue, Liaoning Xiongyue, Liaoning

122.24 122.24

40.28 40.28

Maili (Pink)(50) Maili(White)(51)

Q. Zhang et al. / Scientia Horticulturae 118 (2008) 39–47 Table 2 Morphological diversity of some important traits for 44 wild Tomentosa cherry accessions Attribute

Minimum

Maximum

Mean

Standard deviation

Flower size (cm) Sepal length (cm) Length of sepal tube (cm) Fruit length (cm) Fruit width (cm) Stalk length (cm) Leaf length (cm) Leaf width (cm) Fruit weight (g) Stone weight (g)

1.64 0.26 0.45

2.90 0.48 0.67

2.18 0.35 0.57

0.26 0.04 0.06

67.98** 43.39** 36.98**

0.88 0.84 0.10 3.24 1.82 0.41 0.08

1.51 1.82 0.54 6.70 4.27 2.85 0.28

1.20 1.21 0.24 4.76 2.82 1.13 0.14

0.12 0.18 0.11 0.76 0.60 0.45 0.05

102.66** 240.41** 136.40** 103.66** 145.26** 502.16** 23.37**

**

F

Indicates significant difference at 0.01 level.

the Tomentosa cherry trees, which included branch type, flower color, petal number, fruit color, fruit shape, stone shape, leaf shape and leaf tip shape. Ten quantitative traits namely: flower size, length of sepal tube, sepal length, fruit weight, fruit length, fruit width, stalk length, stone weight, leaf length and leaf width were based on 10 measurements of each trait (Table 2). 2.3. Genomic DNA extraction Young expanded terminal leaves were collected for DNA isolation. Total genomic DNA was extracted using the CTAB procedure (Doyle and Doyle, 1990) with minor modifications. About 100 mg leaves were ground in liquid nitrogen. The powder was then transferred into a 1.5-ml Eppendorf tube with 650 ml of extraction buffer [3% (w/v) CTAB, 1.4 M NaCl, 100 mM Tris–HCl, 20 mM EDTA pH 8.0, 2% (v/v) b-mercaptoethanol and 1% (w/v) polyvinylpyrrolidone] and incubated at 65 8C in a water bath for 40 min. Protein was removed by extraction with chloroform– isoamyl alcohol (24:1), and DNA was precipitated with isopropanol and washed with 70% ethanol. The pellet was dissolved in TE buffer (10 mM Tris–HCl, 1 mM EDTA pH 8.0), treated with RNase (1 mg ml1, 60 min, 37 8C). DNA was reprecipitated by the addition of cold absolute ethanol and redissolved in 0.1 ml TE buffer. DNA concentrations were measured using a spectrophotometer (UV– vis, Beckman DU 800), checked on 1% TAE agarose gels and diluted to a final concentration of 250 ng ml1 before PCR amplification. 2.4. PCR amplification and DNA electrophoresis Extracted genomic DNA was PCR-amplified using 44 pairs of SSR primers (Table 3). PCR reactions were performed in 20 ml volume containing 20 mM Tris–HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.4 mM each primer, 40 ng genomic DNA and 0.45 units Taq polymerase. Reactions were carried out on a PTC-200 (MJ Research, USA) thermocycler using the following temperature profile: an initial step of 4 min at 94 8C, 28–31 cycles of 45 s at 94 8C, 45 s at an appropriate annealing temperature depending on the primers used (57.5–62 8C) and 45 s at 72 8C, and a final step of 8 min at 72 8C. The amplification products were loaded on 6% nondenatured polyacrylamide gels in TBE buffer at a constant current of 40 mA. The gels were stained with silver nitrate according to the protocol of Promega (Promega, Madison, USA). Each primer combination was amplified twice to verify reproducibility. 2.5. SSR data analysis The polymorphic information content (PIC) of SSR patterns were estimated using the formula (Powell et al., 1996):

41

PIC = 1  Pij2, where Pij is the frequency of the jth pattern for the ith marker locus and summation extends over n patterns. Each SSR band was considered as a character and the presence or absence of the band was scored in binary code (present = 1, absent = 0). A data matrix was assembled and analyzed using phylogenetic analysis using parsimony (PAUP; Swofford, 1998) and a pairwise distance matrix was generated based on total character differences. The genetic relatedness was analyzed using unweighted pair group method with arithmetic average (UPGMA) based on distance measure of total character differences. Bootstrap analysis with 1000 replicates was performed to obtain the confidence of branches of the UPGMA tree. 3. Results 3.1. Morphological assessments Analysis of variance revealed highly significant differences (p < 0.01) among accessions for all of the quantitative traits indicating that there was a high degree of phenotypic diversity among the accessions (Table 2). The branch type was evaluated by direct observation, only 2 accessions collected from Shanxi province showed weeping branches. Flower diameters ranged from 1.64 cm (LiaoningYC) to 2.90 cm (LiaoningXE), the average flower diameter of LiaoningXE was significantly (p < 0.01) larger than all other accessions examined. Multi-petals were found in several accessions (HebeiQA, LiaoningDD, JilinGB, JilinGD and JilinGF), although most accessions had standard 5 petals. Flower color was also different, light pink (59%) was the most common color followed by white (30%). Pink color was only observed in 5 accessions, LiaoningDD, JilinGA, JilinGC, HeilongjiangSA and HeilongjiangSC. Sepal length ranged from 0.26 cm (JilinGD) to 0.48 cm (LiaoningYB) and length of sepal tube ranged from 0.45 cm (JilinGA) to 0.67 cm (LiaoningXA, HeilongjiangSC). Fruit weight was most variable among the quantitative traits (F value at 502.16). Average fruit weight ranged between 0.41 g (LiaoningYB) and 2.85 g (HeilongjiangSH). The average fruit weight of HeilongjiangSH was significantly (p < 0.01) higher than all other accessions examined. The highest average value of fruit length was 1.51 cm (HeilongjiangSH), while the lowest was 0.88 cm (LiaoningYB). Likewise, the highest average value of fruit width was 1.82 cm (HeilongjiangSH), while the lowest value was 0.84 cm (LiaoningYB). Fruits were mostly round in shape, oblate fruit shape was the most common in accessions from Heilongjiang province. Oblong shape was only found in 4 accessions including LiaoningYB, LiaoningSA, ShanxiKA and ShanxiKB. Fruit color was less variable with only red and white types. HebeiQA had the longest stalk (0.54 cm) and HeilongjiangSC the shortest (0.10 cm). Generally, accessions in Heilongjiang group had shorter stalks than those from other provinces. Stone weight was the least variable among quantitative traits (F value at 23.37). Round and elliptical stone shapes were most common, while 3 accessions (LiaoningXD, LiaoningDB and LiaoningSB) had long-elliptical stones. The longest leaf length (6.70 cm) and longest leaf width (4.27 cm) was found on different accessions of HebeiQD and LiaoningXB respectively, while the shortest ones (3.24 cm and 1.82 cm) were found on JilinGA. Leaf color was dark green in most accessions, except for HebeiQB and LiaoningDF, which had red veins, and HeilongjiangSA, HeilongjiangSB, HeilongjiangSC, which had light red leaves. Therefore, the Heilongjiang province has unique germplasm with red leaf color. Most accessions had elliptical leaves except for HebeiQB, HebeiQC, LiaoningDA and all the Jilin accessions had narrow-elliptical leaves with the exception of JilinGA, which had elliptical leaves as the rest. Elliptical leaves commonly have apiculate tips and narrow-elliptical leaves have

42

Table 3 List of SSR primers used and their amplified products in Tomentosa cherry and its related species Primers

Primer sequence (50 ! 30 )

Motif

Source

Expected size range (bp)

UDP96-001

AGTTTGATTTTCTGATGCATCC TGCCATAAGGACCGGTATGT

(CA)17

Cipriani et al. (1999)

100–140

UDP96-003

TTGCTCAAAAGTGTCGTTGC ACACGTAGTGCAACACTGGC

(CT)11(CA)28

Cipriani et al. (1999)

UDP96-008

TTGTACACACCCTCAGCCTG TGCTGAGGTTCAGGTGAGTG

(CA)23

UDP96-013

ATTCTTCACTACACGTGCACG CCCCAGACATACTGTGGCTT

UDP97-401

Related species

Total

PIC values Tomentosa cherry

Related species

6

5

4

0.75

0.71

0.61

85–95

2

2

1

0.07

0.08

0

Cipriani et al. (1999)

135–155

5

4

2

0.42

0.42

0.35

(AG)22(TG)8TT(TG)10

Cipriani et al. (1999)

175–185

5

5

0

0.77

0.77

–

TAAGAGGATCATTTTTGCCTTG CCCTGGAGGACTGAGGGT

(GA)19

Cipriani et al. (1999)

115–130

4

3

1

0.41

0.25

0

UDP97-403

CTGGCTTACAACTCGCAAGC CGTCGACCAACTGAGACTCA

(AG)22

Cipriani et al. (1999)

105–120

7

6

3

0.75

0.75

0.40

UDP98-408

ACAGGCTTGTTGAGCATGTG CCCTCGTGGGAAAATTTGA

(CT)14

Cipriani et al. (1999)

130–145

2

1

2

0.08

0

0.44

UDP98-021

AAGCAGCAATTGGCAGAATC GAATATGAGACGGTCCAGAAGC

(GA)22(CA)11

Testolin et al. (2000)

100–150

6

4

4

0.69

0.67

0.61

UDP98-022

CTAGTTGTGCACACTCACGC GTCGCAGGAACAGTAAGCCT

(TG)12(AG)24

Testolin et al. (2000)

110–170

8

8

0

0.77

0.77

–

UDP98-025

GGGAGGTTACTATGCCATGAAG CGCAGACATGTAGTAGGACCTC

(CA)19

Testolin et al. (2000)

95–125

8

4

5

0.73

0.69

0.74

UDP98-410

AATTTACCTATCAGCCTCAAA TTTATGCAGTTTACAGACCG

(AG)23

Testolin et al. (2000)

135–185

5

5

3

0.78

0.65

0.45

UDP98-412

AGGGAAAGTTTCTGCTGCAC GCTGAAGACGACGATGATGA

(AG)28

Testolin et al. (2000)

95–140

14

9

9

0.87

0.86

0.82

UDP98-414

AAAAGGCACGACGTTGAAGA TTCAGATTGGGAATTTGCAG

(TC)24

Testolin et al. (2000)

165–185

8

4

4

0.65

0.56

0.53

UDP98-416

TTTTCTCAGCAGCCAAACAA ATGTTTCGTGCTTCTGCTCC

(AG)18

Testolin et al. (2000)

80–90

4

3

1

0.47

0.33

0

pchCms4

CTCACGCTATTTCTCGG CCTCGACGAAGAGCTCG

(GA)9

Sosinski et al. (2000)

220–240

4

2

3

0.58

0.50

0.60

pchgms2

GTCAATGAGTTCAGTGTCTACACTC AATCATAACATCATTCAGCCACTGC

(CT)24

Sosinski et al. (2000)

125–165

6

5

4

0.78

0.76

0.65

pchgms3

ACGGTATGTCCGTACACTCTCCATG CAACCTGTGATTGCTCCTATTAAAC

(CT)14

Sosinski et al. (2000)

175–200

6

6

5

0.79

0.77

0.72

Pchgms4

ATCTTCACAACCCTAATGTC GTTGAGGCAAAAGACTTCAAT

(CT)21

Sosinski et al. (2000)

160–185

9

4

8

0.75

0.68

0.84

Pchgms5

CCAGTAGATTTCAACGTCATCTACA GGTTCACTCTCACATACACTCGGAG

(GA)9

Sosinski et al. (2000)

165–180

6

2

4

0.37

0.15

0.72

pchgms25

GCCAGGAGGCTTTAACCTGT TCAGACCCCCTTTCATCATC

(TG)13(AG)22

Sosinski et al. (2000)

360–440

8

7

2

0.52

0.54

0.24

Q. Zhang et al. / Scientia Horticulturae 118 (2008) 39–47

Alleles no. Tomentosa cherry

Total

AGAAGGGCAAGCCCAAGTGC TGCAAAGCCAGAGCCCACAA

(AG)14

Yamamoto et al. (2002)

190–200

5

3

3

0.51

0.49

0.61

BPPCT025

TCCTGCGTAGAAGAAGGTAGC CGACATAAAGTCCAAATGGC

(GA)29

Dirlewanger et al. (2002)

155–175

5

3

2

0.39

0.20

0.41

CPPCT23

CATGGTTTGCAACTGTCTTCA GACACAGGTGTGTAGATCATTGG

(CT)9

Aranzana et al. (2002)

165–185

2

1

1

0.12

0

0

CPPCT25

TGTATCCCCTCCTCGGATAA GGCATTAATGGATGATGATGAA

(CT)23

Aranzana et al. (2002)

115–130

5

2

4

0.30

0.11

0.65

CPPCT26

AGACGCAGCACCCAAACTAC CATTACATCACCGCCAACAA

(CT)22

Aranzana et al. (2002)

100–170

7

7

0

0.72

0.72

–

CPPCT27

GAGCAGTTCATAAGTTGGAACAA CGATAAAGATTTTGACTGCATGA

(CT)30

Aranzana et al. (2002)

50–105

7

5

4

0.65

0.57

0.64

CPPCT35

CTACCCATTAGCCACCAAGC TCCCAATTCGTTGCAATCTT

(CT)21

Aranzana et al. (2002)

155–190

9

8

4

0.80

0.76

0.72

CPPCT36

TTCTATCCCGGAAGCTGTTG CACATGTATGTCTATGCTTCTGTG

(CT)23

Aranzana et al. (2002)

185–200

2

2

1

0.18

0

0.41

UCD-CH11

TGCTATTAGCTTAATGCCTCCC ATGCTGATGTCATAAGGTGTGC

(CT)15

Struss et al. (2003)

115–150

10

8

4

0.78

0.72

0.65

UCD-CH12

AGACAAAGGGATTGTGGGC TTTCTGCCACAAACCTAATGG

(CA)14

Struss et al. (2003)

170–200

6

4

3

0.71

0.68

0.57

UCD-CH13

ACCCGCTTACTCAGCTGAAC TTAGCACTAAGCCTTTGCTGC

(CA)10

Struss et al. (2003)

140

1

1

1

0.04

0

0.26

UCD-CH15

TCACTTTCGTCCATTTTCCC TCATTTTGGTCTTTGAGCTCG

(CT)15

Struss et al. (2003)

85–95

3

1

2

0.27

0

0.22

UCD-CH19

GTACAACCGTGTTAACAGCCTG ACCTGCACTACATAAGCATTGG

(CA)12

Struss et al. (2003)

125–150

4

4

0

0.72

0.72

–

UCD-CH31

TCCGCTTCTCTGTGAGTGTG CGATAGTTTCCTTCCCAGACC

(CT)26

Struss et al. (2003)

170-200

4

4

1

0.68

0.69

0

UCD-CH39

CACTGTCTCCCAGGTTAAACTC CCTGAGCTTTTGACACATGC

(CT)28

Struss et al. (2003)

95–140

8

1

7

0.35

0

0.85

PMS2

CACTGTCTCCCAGGTTAAACT CCTGAGCTTTTGACACATGC

(TA)24GA(TA)3(TG)24

Cantini et al. (2001)

100–120

7

1

6

0.35

0

0.81

PMS30

CTGTCGAAAGTTTGCCTATGC ATGAATGCTGTGTACATGAGGC

Cantini et al. (2001)

135–175

5

4

2

0.45

0.41

0.49

PMS40

TCACTTTCGTCCATTTTCCC TCATTTTGGTCTTTGAGCTCG

Cantini et al. (2001)

95–110

4

1

4

0.11

0

0.56

PMS67

AGTCTCTCACAGTCAGTTTCT TTAACTTAACCCCTCTCCCTCC

Cantini et al. (2001)

150–170

6

6

1

0.48

0.52

0

PS8e8

CCCAATGAACAACTGCAT CATATCAATCACTGGGATG

Sosinski et al. (2000)

170–180

2

1

2

0.04

0

0.24

PceGA59

AGAACCAAAAGAACGCTAAAATC CCTAAAATGAACCCCTCTACAAAT

Cantini et al. (2001)

140–175

8

6

4

0.65

0.61

0.58

PacA10

TGAGCATAATTGGGGCAG GCCAGAGAAGCCATTTCAGT

(GA)14

Decroocq et al. (2003)

75–100

3

2

2

0.11

0.08

0.35

PacA33

TCAGTCTCATCCTGCATACG CATGTGGCTCAAGGATCAAA

(GA)16

Decroocq et al. (2003)

185–220

5

5

2

0.61

0.62

0.24

Q. Zhang et al. / Scientia Horticulturae 118 (2008) 39–47

M6a

43

0.47 0.64 5 4 9

Source

Decroocq et al. (2003) (CA)14(GA)11

215–295

PIC values Tomentosa cherry Total Related species Alleles no. Tomentosa cherry Total

acuminate tips. However, JilinGC and JilinGD with narrowelliptical leaves had apiculate tips and LiaoningDC and JilinGA with elliptical leaves had acuminate tips. Leaf type in Jilin group was most variable. 3.2. Genetic diversity

Motif

Expected size range (bp)

0.77

Q. Zhang et al. / Scientia Horticulturae 118 (2008) 39–47 Related species

44

Very high levels of polymorphisms were detected among the 44 Tomentosa cherries and seven genotypes from the related species (Table 3, Fig. 2). A total of 250 alleles were revealed by 44 SSRs selected from the published 110 SSR markers based on clarity and specificity, with an average of 5.68 alleles per SSR. The marker UDP98-412 generated 14 alleles, the largest number found among the 44 markers, while markers UCD-CH13 were least polymorphic with only 1 allele. Within the 44 Tomentosa cherries, 44 primers yielded 173 polymorphic bands, with an average of 3.93 alleles per SSR. Within the 7 accessions of other species, 40 primers out of the 44 yielded 130 bands with an average of 2.95 per locus. Of the total number of alleles, 20.8% were shared between the Tomentosa cherry and its related species, 69.2% were unique to the Tomentosa cherry accessions and 52.0% were unique to its related species. Fig. 2A shows the amplification patterns from primer UDP96-001. Across all 51 accessions analyzed, the PIC values for individual loci ranged from 0.04 (UCD-CH13, PS8e8) to 0.87 (UDP98-412) with an average of 0.52. PIC values were different between Tomentosa cherry accessions and its related species. In the population of Tomentosa cherry, PIC values ranged from 0 (UDP98-408, CPPCT23, CPPCT36, UCD-CH13, UCD-CH15, UCDCH39, PMS2, PMS40 and PS8e8) to 0.86 (UDP98-412) with an average of 0.44, while in the related species, PIC values ranged from 0 (UDP96-003, UDP97-401, UDP98-416, CPPCT23, UCD-CH31 and PMS67) to 0.85 (UCD-CH39) with an average of 0.47. Locus UDP98412 was the most informative with the highest value of PIC and number of alleles. Unique bands or band patterns were generated by 27 accessionspecific primers on 34 Tomentosa accessions. The weeping accessions had both unique bands and unique band patterns amplified by 11 primers, UDP96-001, UDP96-003, UDP97-401, UDP98-412, UDP98-414, pchgms3, CPPCT26, PceGA59, PacA10, PacA33 and PacB35 (Fig. 2B). The other 4 primers of UDP98-021, Pchgms4, BPPCT025 and CPPCT35 produced unique band patterns on weeping accessions. The other 32 accessions had their own specific band patterns instead of unique bands, among which LiaoningXA, LiaoningXC and LiaoningDB showed unique band patterns for 4 primers. HebeiQB, LiaoningXD, LiaoningSB, LiaoningYD, HeilongjiangSH and JilinSA showed unique band patterns for 3 primers. The other accessions either had 2 or 1 unique band patterns (data not shown).

Primer sequence (50 ! 30 )

ATTGCGATTTCGGTCTGTT CCATCCCAAATTGCTTACTT

Primers

PacB35

Table 3 (Continued )

3.3. Genetic relationship among Tomentosa cherry and related species UPGMA analysis based on total character difference grouped the 44 Tomentosa cherry accessions and 7 accessions of related species into 13 groups designated as A to M (Fig. 3). Bootstrap analysis using 1000 replicates showed that four forks had 100% bootstrap support and 14 forks had greater than 50% bootstrap support. Group A to Group L included all the P. tomentosa populations which were clearly divergent from the included accessions of P. humilis, P. japonica and P. glandulosa. Group A included genotypes from Hebei with 60% bootstrap support forming a distinct Hebei ecotype. Group B contained accessions from both Hebei province and Liaoning province forming a Hebei– Liaoning overlap ecotype. Group C comprised 8 accessions collected from Xiongyue, Dengta and Shengyang in Liaoning province forming a distinct Liaoning ecotype, for which most

Q. Zhang et al. / Scientia Horticulturae 118 (2008) 39–47

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Fig. 2. (A) Amplification products using SSR primer pair UDP96-001 on 44 Tomentosa cherry accessions. M = DNA size markers, 1–44 = Tomentosa cherry accessions as listed in Table 1. (B) Amplification products using SSR primer pair PacA10 on 22 Tomentosa cherry accessions. Allele b were shared by all accessions, alleles a and c were unique to the whipping types ‘ShanxiKA’ and ‘ShanxiKB’ as arrowed.

accessions had round red fruit, elliptical big green leaves. Group D contained only 2 accessions LiaoningDF and LiaoningDG. Group E containing 5 accessions showed a distinct Jilin ecotype. Major traits from this group included narrow-elliptical and small leaf types. Group F containing 10 accessions formed a distinct Heilongjiang ecotype, the obvious morphological character in this group was oblate fruit shape with short stalk length. Three redleafed accessions were also grouped in this cluster. The accessions of JilinGB and HeilongjiangSH clustered together forming a Jilin– Heilongjiang overlap ecotype into group G. LiaoningDD formed one cluster as group H, and LiaoningXA and JilinGD formed the Liaoning–Jilin ecotype into group I. The populations of LiaoningYA, LiaoningYB and LiaoningYC formed a specific Liaoning–Yixian ecotype into group J. Group K contained only one accession of LiaoningYD. A distinct cluster was formed (group L) for the two weeping accessions of ShanxiKA and ShanxiKB. In group M, 4 accessions of P. humilis were clustered together, then clustered with 1 accession of P. japonica and finally with 2 accessions of P. glandulosa, which formed a distinct sister group to Tomentosa cherry. 4. Discussion The present study has revealed enormous morphological diversity among the accessions of Tomentosa cherries. Apart from the common traits shared by many accessions, genotypes from each geographical region had their own unique characteristics: Shanxi province is likely the centre of origin of weeping cherry; red-leafed genotypes were only found in Heilongjiang province; small leaf and narrow-elliptical leaf types were found only in Jilin province; Hebei province had an accession with the longest fruit stalk and longest sepal; and Liaoning province had 3 accessions with long-elliptical shaped stones. The results here complement a previous report in Flora Reipublicae Popularis Sinicae (Yu¨ and Li, 1986) in several aspects. Flower diameter was more variable than previously reported (1.5–2.0 cm). Our study showed that there was

considerable variation within the 44 accessions with regard to this important trait. Also sepal length and length of sepal tube were slightly larger than those previously reported for P. tomentosa. The heaviest fruit weight found in this study was in HeilongjiangSH, which has the greatest potential commercial value. Fruit length and fruit width were also larger than previously recorded. Only round fruit had been recorded previously, however, oblate and oblong shapes were observed in our study. Stalk length was also more variable than previously reported, and since this was significantly different among accessions, measurement of stalk length could be used to distinguish these accessions. Among morphological traits, fruit weight, fruit width, leaf width, stem length, branch type, fruit color and fruit shape were the most useful traits to assess accessions of Tomentosa cherries. In Prunus L., SSR markers have been used widely for cultivar identification and genetic mapping (Aranzana et al., 2003; Cipriani et al., 1999; Testolin et al., 2000), and are a powerful tool for phylogenetic studies. The number of alleles detected per locus in Tomentosa cherry ranged from 1 to 9 with a mean value of 3.9. This value was similar to the reported 4.2 per locus in 27 peach cultivars with 41 SSR markers (ranging from 1 to 9), while it was considerably higher than 2.8 (ranging from 1 to 6) in 21 sweet cherry cultivars with 33 SSR markers (Dirlewanger et al., 2002). When considering all the accessions tested (including the related species), the number of alleles detected per locus ranged from 2 to 14 with a mean of 5.7 which was much higher than that within Tomentosa cherry genotypes. The PIC values generated from each marker showed a large variation with a mean number of 0.52. These results suggest that SSR markers are very useful in the discernment of genetic relationships among accessions. Normally, high number of alleles and PIC values come from a more diverse germplasm resource. All the genotypes included in this study could be differentiated unambiguously with only three SSRs: UDP96-013, UDP98-412 and UCD-CH11. This high discrimination occurs even between closely related genotypes, for example, ShanxiKA and ShanxiKB were

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Q. Zhang et al. / Scientia Horticulturae 118 (2008) 39–47

Fig. 3. Dendrogram of 44 Tomentosa cherry accessions and 7 relative accessions based on UPGMA analysis of SSR polymorphisms. The accessions (Table 1) clustered into groups A to M (numbers on left). Numbers below the lines indicate bootstrap values (percentage of 1000 replicates). Bootstrap values greater than 50% are shown.

indistinguishable when screened by 43 pairs of primers and only the UDP96-013 primer could separate them. This suggests that the usefulness of the SSR technique can be improved by increasing the number of primers analyzed (Reale et al., 2006). 34 accessions each had a unique fingerprint allowing unequivocal identification of each Tomentosa cherry genotype. Precise characterization of the existing Tomentosa cherry accessions is essential for efficient germplasm management, and selection of parent material within breeding programs. Our results confirmed that SSR techniques are not only efficacious in revealing genetic distances among different accessions, but are useful in solving cases of homonyms and synonyms. Amplification with 44 out of 110 primers cloned from peach, sweet cherry, sour cherry and apricot demonstrated the crossspecies transferability of these markers as shown in previous studies (Cipriani et al., 1999; Downey and Iezzoni, 2000). The average frequency for the transferability observed here was 40%, among which the UDP group of markers from peach revealed the highest level of transferability at 56%, followed by the UDC group from cherry at 50% and Pac group from apricot at 38%. The other groups (pchgms, CPPCT and PMS) of markers showed less transferability to Tomentosa cherry and its related species tested in this study. Our results from the UDP group of markers agreed well with those of Cipriani et al. (1999), who reported that 59% of

peach SSR primer pairs amplified products of the expected size in other Prunus species. The overall transferable rate was lower than previous reports of 80% from cherry to plum, apricot and peach (Struss et al., 2003), and 71% from peach to sweet cherry (Wu¨nsch and Hormaza, 2002). This indicates that Tomentosa cherry and its related species used in this study probably have a slightly greater genetic distance than within the group of peach, sweet cherry and apricot. Weeping type was a wild mutant found in 1991 in Kouxian, Shanxi province (Feng et al., 1999). Fifteen primers could classify them as a special group with their own unique banding patterns among which 11 primers could produce specific alleles. Cluster analysis showed that the weeping genotypes exhibited a greater genetic distance from the other variants (with 100% high bootstrap). The molecular data obtained in this study suggest that this variant can probably be placed as an independent subspecies from the rest of P. tomentosa. Although the two weeping genotypes were morphologically different, they were very closely related at the molecular level. The results showed that weeping types may have recently evolved and thus their genetic diversity is limited. Within Tomentosa cherry accessions, cluster analysis generally reflects accession separation based on geographical origin. While some red-leafed and multi-petal types were found, they did not cluster into distinct groups suggesting that these traits may only be

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controlled by a few genes. Accessions from P. humilis, P. japonica and P. glandulosa showed a reasonably close relationship with Tomentosa cherry. Similar close relationships were found by Cai et al. (2006) who used RAPD method to analyze relationships among different cherry species including P. tomentosa and P. humilis. A close relationship was found between P. glandulosa and P. japonica, however, they were observed a distant relationship with P. tomentosa based on RAPD analysis (Shimada et al., 2001). Further research involving more species is needed to reveal the systematic position of Tomentosa cherry within Prunus. We conclude that this is the world first study of such a kind on Tomentosa cherry from China, which deals with the morphological variation and the molecular basis of genetic diversity. Information gained from this investigation will contribute to the set-up of the ‘descriptors for Tomentosa cherry’. Results will assist identification, selection and combination of some useful traits from the wild types to improved cultivated varieties. The research also highlights the need to preserve wild germplasm as some earlier identified wild genotypes are more and more difficult to find because of urbanization and other human activities. Now is time to start a conservation project while a large level of genetic diversity can still be found. The high levels of morphological and molecular diversity found in this study are encouraging and lead to the prediction that domestication and use of this wild germplasm for the improvement of Prunus fruits will be beneficial to the world stone fruit industry. Acknowledgements This work was financially supported by Program for Innovation Team in University of Liaoning Province (2007T161). The authors acknowledge Mr. Wei Sun in the Berry Institute of Heilongjiang Province and Mr. Fen Li in Fruit Institute of Jilin Province for providing plant materials. Our appreciation is also extended to Mr. Guangwen Shang at Fruit Releasing Center in Yixian and Mr. Lin Li at Horticultural and Forestry Department in Liaoyang for assistance in germplasm collection. References Aranzana, M.J., Garcia-Mas, J., Carbo, J., Arus, P., 2002. Development and variability analysis of microsatellite markers in peach. Plant Breed. 121, 87–92. Aranzana, M.J., Pineda, A., Cosson, P., Dirlewanger, E., Ascasibar, J., Cipriani, G., Ryder, C.D., Testolin, R., Abbott, A., King, G.J., Iezzoni, A.F., Arus, P., 2003. A set of simple-sequence repeat (SSR) markers covering the Prunus genome. Theor. Appl. Genet. 106, 819–825. Cai, Y.L., Li, S., Cao, D.W., Qian, Z.Q., Zhao, G.F., Han, M.Y., 2006. Use of amplified DNA sequences for the genetic analysis of the cherry germplasm. Acta Hortic. Sin. 33, 249–254 (in Chinese). Cantini, C., Iezzoni, A.F., Lamboy, W.F., Bortizki, M., Struss, D., 2001. DNA fingerprinting of tetraploid cherry germplasm using simple sequence repeats. J. Am. Soc. Hortic. Sci. 126, 205–209. Cipriani, G., Lot, G., Huang, W.-G., Marrazzo, M.T., Peterlunger, E., Testolin, R., 1999. AC/GT and AG/CT microsatellite repeats in peach [Prunus persica (L.) Batsch]: isolation, characterisation and cross-species amplification in Prunus. Theor. Appl. Genet. 99, 65–72. Clarke, J.B., Tobutt, K.R., 2003. Development and characterization of polymorphic microsatellites from Prunus avium ‘Napoleon’. Mol. Ecol. Notes 3, 578–580.

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