Genetic Characterization of Brassica rapa chinensis L., B. rapa ...

Philippine Journal of Science 

138 (2): 141-152, December 2009 

ISSN 0031 - 7683 

Genetic Characterization of Brassica rapa chinensis L., 

B. rapa parachinensis (L. H. Bailey) Hanelt, and B. oleracea 

alboglabra (L. H. Bailey) Hanelt Using Simple Sequence 

Repeat Markers 

Stephanie U. Celucia 1 , Robert C de la Peña 2 and Neilyn O. Villa 1* 

1 Institute of Biological Sciences, College of Arts and Sciences, 

University of the Philippines Los Baños, College, Laguna, Philippines 

2 AVRDC-The World Vegetable Center, Shanhua, Tainan, Taiwan 

Genetic diversity of 39 accessions of Brassica rapa chinensis L., 28 accessions of B. rapa 

parachinensis (L. H. Bailey) Hanelt, and 29 accessions of B. oleracea alboglabra (L. H. Bailey) 

Hanelt was studied. Fifty-four SSR primers were used and produced 122 scorable bands in 

which 77 were polymorphic. The average rate of polymorphic loci was 71.08% which indicates 

high genetic diversity among the accessions. Phylogenetic analysis showed that B. rapa chinensis 

and B. rapa parachinensis are genetically closely related to each other. B. oleracea alboglabra 

accessions grouped together in a separate cluster. Groupings also reflected geographical 

similarities and may suggest misidentification of certain accessions in the germplasm collection. 

Thus, SSR analysis proved to be a useful tool in assessing the genetic diversity of leafy Brassica 

germplasm. 

Key Words: Brassica oleracea, Brassica rapa, dendrogram, genetic diversity, geographical origin, 

microsatellites, simple sequence repeats, 

INTRODUCTION 

Brassica is a highly diverse genus of plants belonging to 

the family Brassicaceae or the mustard or cabbage family. 

It contains species that are of great economic importance 

since most species are some of the world’s oilseed, forage, 

ornamental and vegetable crops. Almost all parts of many 

species have been developed to be edible including roots, 

stems, buds, leaves, flowers, and seeds (AVRDC 2000). 

Brassica crops consist of three primary species, 

namely Brassica rapa or Chinese cabbage (n=10), 

B. oleracea or Kale (n=9), and B. nigra Koch (n=8) 

and three amphidiploids, B. juncea (n=2x=18), B. 

carinata (n=2x=17) and B. napus (n=2x=19) (Ren et 

*Corresponding author: neilyn24@yahoo.com 

al. 1995). The three amphidiploids arose from crossing 

and paleopolyploidization among the primary species. 

Although most Brassica crops originated from Western 

Europe and the Mediterranean, East Asia is the major 

secondary center of diversity for leafy Brassica like pak 

choi (B. rapa chinensis), choysum (B. rapa parachinensis), 

and kailaan (B. oleracea alboglabra). Since their 

introduction to China, these species significantly changed 

in structure, form, and productivity by domestication. 

Because of the allogamous breeding system in Brassica, 

morphological and botanical variability in the many 

subspecies and cultivar groups of B. rapa and B. juncea 

has increased (Lee 1982; Li 1981; Opena et al. 1988). 

B. rapa has a wide range of varieties and contains 11 

subspecies that are classified into two distinctly different 

groups, namely Pekinensis, which is the more common 

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Vol. 138 No. 2, December 2009 

group, and Chinensis. B. r. chinensis is commonly called 

Pak choi. It is also called “bok choy”, in English, and 

“xiao baicai” ("small white vegetable") in Mandarin. 

Chinensis varieties do not form heads. They have smooth, 

dark green leaf blades forming a cluster suggestive of 

mustard. B. rapa parachinensis is a commercial variant 

of B. rapa chinensis. It is a small, delicate version of bok 

choy or simply the flowering heart of any Chinese cabbage 

(Kessler 1989; Evans et al. 1988). 

On the other hand, the species B. oleracea contains a 

wide array of vegetables, including broccoli, cauliflower, 

and brussel sprouts. B. oleracea alboglabra is a form of 

cabbage in which the central leaves do not form a head. 

It is considered to be closer to wild cabbage than most 

domesticated forms. Kai-lan, a separate cultivar of B. 

oleracea much used in Chinese cuisine, is somewhat 

similar to kale in appearance and is occasionally called 

"kale" in English (Guerena 2006; Hanson 2006; van der 

Vossen 1993). 

Genetic diversity is very important to a successful 

crop improvement. It helps protect our food supply by 

broadening the range of genes available to meet agricultural 

production challenges. The evaluation of genetic diversity 

present in germplasm collections promotes the efficient 

use of genetic variation in establishing a breeding program 

(Paterson 1991). 

There have been studies on identifying genetic relationships 

of Brassica species using different genetic markers. A 

study by Ren et al. (1995) used RAPD markers to assess 

the diversity of Chinese vegetable brassicas. It also 

suggested that Chinese cabbage is more likely to have 

been produced from the hybridization of pak choi and 

turnip than as a selection of pak choi and turnip alone. In 

another study (An et al. 2000), the genetic relationships 

among Brassica species based on RAPD markers were 

used to provide information for properly selecting 

parents of crosses and improving breeds of Brassica 

species. Furthermore, Zhao et al. (2005) inferred genetic 

relationships within B. rapa using AFLP fingerprints. 

In 2004, Lowe et al. mentioned that microsatellite or SSR 

(simple sequence repeat) markers have been developed 

and characterized for use with genetic studies of Brassica 

species. More recently, Louarn et al. (2007) used database 

derived SSR markers for cultivar differentiation in B. 

oleracea. SSRs are tandem repeat sequences having less 

than six base pairs. They are very polymorphic due to the 

high mutation rate affecting the number of repeat units. 

They are also very abundant and randomly distributed in 

the genome. Polymorphisms of SSR can be easily detected 

on high-resolution gels (Gianfranceschi 1998). SSR is 

advantageous over other DNA-based markers because it 

Villa et al.: Genetic Characterization of Leafy Brassica 

Species Using SSR Markers 

is co-dominant, evenly distributed in the genome, and it 

allows the identification of many alleles at a single locus. 

It also requires only a small amount of DNA for PCR 

(polymerase chain reaction) analysis. 

In this investigation, SSR markers were used to assess the 

relationships among economically important leafy Brassica 

species, namely B. rapa chinensis, B. rapa parachinensis 

and B. oleracea alboglabra. Since the genetic diversity of 

leafy Brassica is not well characterized, this study would 

be able to benefit researchers from around the world who 

work with these species. 

MATERIALS AND METHODS 

Plant materials. This study included 39 accessions 

of B. rapa chinensis (Figure 1a), 28 accessions of B. 

rapa parachinensis (Figure 1b), and 29 accessions of B. 

oleracea alboglabra (Figure 1c). The accessions were 

obtained from the germplasm collection of the Genetic 

Resources and Seed Unit (GRSU) of the Asian Vegetable 

Research and Development Center (AVRDC) in Shanhua, 

Taiwan. The collection includes conventional and modern 

cultivars originating from different geographical locations. 

The accessions used in the study are listed in Table 1. 

Seeds were sown in a soil bed 10 cm apart within a row 

and 15 cm between rows on the bed. One four-row plot 

was made in 1m-soil bed. There were 3 replications for 

every accession. 

Primers. Fifty-four simple sequence primer pairs were 

used to generate the DNA fingerprints. Their name, motif 

type, forward, and reverse primers are listed in Table 2. 

Primers 1-13 and 51 were designed based on their location 

in the genetic map of B. napus; primers 14, 15 and 52 

were found on a B. nigra genetic map; primers 16-35, 53 

and, 54 were located in B. oleracea; and primers 36-50, 

55, and 56 were located in the genetic map of B. rapa. 

The primers used in this study are developed by Lowe 

et. al. in 2004. 

Genomic DNA extraction. Three months after sowing, 

the youngest healthy leaves were harvested from 10 plants 

of each accession. They were placed in small, transparent, 

resealable plastic bags and were immediately placed in 

-80°C freezer until use. 

Leaves were frozen individually by dipping into liquid 

nitrogen and were ground using mortar and pestle. The 

powdered sample was collected in a 1.5 mL plastic 

tube with cap and kept in -20°C refrigerator while the 

extraction buffer (Echevarria-Machado et al. 2005) was 

being prepared. 

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Figure 1. The three Brassica species used in this study (a) B. rapa chinensis, (b) B. rapa parachinensis and (c) B. oleracea 

alboglabra. 

Table 1. List of accessions used in this study, their variety names, and geographical origins. 

Accession Number Variety name Subspecies Geographical Origin 

Brassica rapa 

CR001 Zhu Pob Tsai chinensis Guangzhou / China 

CR004 Xia Hua (F1) chinensis Clover Seeds / Hong Kong 

CR009 Lvxiu91-1 chinensis Qingdao / China 

CR011 Hangzhou Long-Petioled chinensis Hangzhou / China 

CR012 Hangzhou Shiny Winter chinensis Hangzhou / China 

CR013 Suzhou Green chinensis Hangzhou / China 

CR014 Shanghai May Late chinensis Hangzhou / China 

CR015 Shanghai April Late chinensis Hangzhou / China 

CR017 Black-Leaved May Late chinensis Shanghai / China 

CR019 Bottle Long-Petioled White chinensis Zhejiang / China 

CR020 Black Shiny Winter chinensis Zhejiang / China 

CR021 Savoy Pakchoi chinensis Zhejiang / China 

CR022 Thailand Fast chinensis Jiansu / China 

CR027 Clover Pakchoi (128) chinensis Clover Seeds / Hong Kong 

CR030 Shen-Bao Green No.2 chinensis Shanghai / China 

CR031 Heat Resistant 605 Green chinensis Shanghai / China 

CR034 Long-Petioled White chinensis Hangzhou / China 

CR039 Qing You Si Hao chinensis Jiansu / China 

CR040 Fu-Bao Heat Resistant Green chinensis Jiansu / China 

CR041 Aijiao Datou Qingjiang Pakchoi chinensis Fujian / China 

CR044 Dwarf Black Behay chinensis Guangzhou / China 

CR048 Four Season Sweet Pakchoi chinensis Guangdong / China 

CR049 Golden Early No.1 Sweet Pakchoi chinensis Guangdong / China 

CR050 Yellow Leaved Pakchoi chinensis Guangzhou / China 

CR051 Dwarf Pakchoi chinensis Guangzhou / China 

CR052 Late Dwarf Black-Leaved chinensis Guangzhou / China 

CR068 Czuen-Shuei Pakchoi chinensis Guangzhou / China 

CR070 Hongkong Dwarf Milky White chinensis Good Earth Seeds / Hong Kong 

CR075 Yellow Leaf (Local Variety) chinensis Vegetable and Fruit Seeds / Vietnam 

table continued next page . . . 

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table 1 continuation 


CR076 Yellow Stem (Local Variety) chinensis Local Variety / Vietnam 

CR077 Green Stem (Local Variety) chinensis Local Variety / Vietnam 

CR079 Selected Spoon Pakchoi chinensis Shantong / China 

CR091 Dwarf Erect Pakchoi chinensis Guangzhou / China 

CR092 Early Huang-Jing Pakchoi chinensis Guangdong / China 

CR094 Pakchoy White chinensis Tropica Vegetable Seeds / Vietnam 

Bcc11 KSL Nylon Pakchoi chinensis KSL / Taiwan 

Bcc19 WYS Huang-Jing Pakchoi chinensis WYS / Taiwan 

Bp05 (KY0606)Ching Chiang Pai-Tsai chinensis Known-You Seeds / Taiwan 

Bp07 (KY0603)Gracious Pai-Tsai chinensis Known-You Seeds / Taiwan 

CR003 Early White (IV) (F1) parachinensis Hunan / China 

CR005 Shiny Green No.12 parachinensis Guangzhou / China 

CR006 No.49-19 Choysum parachinensis Guangzhou / China 

CR007 Liu Bao 701 parachinensis Guangzhou / China 

CR010 Giant Seeds’ Choysum(A-12) parachinensis Guangzhou / China 

CR023 Gai Liang Shi Yue Hong parachinensis Wuhan / China 

CR024 Red Hybrid 60 (F1) parachinensis Wuhan / China 

CR025 Early White Choysum (II) (F1) parachinensis Hunan / China 

CR026 Early White Choysum (III) (F1) parachinensis Hunan / China 

CR035 Guang-Yan #31 Shiny Green parachinensis Guangzhou / China 

CR037 Hsin-Liu Waxy Choysum parachinensis Shanghai / China 

CR042 Autumn Red No.2 parachinensis Sichuan / China 

CR058 Early Hybrid White Choysum parachinensis Guangzhou / China 

CR071 Hua-Tsuei Choysum parachinensis Beijing / China 

CR073 Giong Cai Ngot Tosakan parachinensis East West Seeds / Vietnam 

CR082 Shiny Green 60 Days parachinensis Guangzhou / China 

CR083 Late #2 Choysum parachinensis Guangzhou / China 

CR085 Cui Qing No. 26 parachinensis Guangdong / China 

CR087 Zao Shu Cu Tiao You Qing Tian Cai Xin parachinensis Guangzhou / China 

Bc07 (KY0605)Yu-Tsai-Sum parachinensis Known-You Seeds / Taiwan 

Bc57 Late Shiny Green #8 parachinensis Guangzhou / China 

B00468 Yayoi Komatsuna parachinensis Kyowa Noen / Japan 

B00469 Uzuki Komatsuna parachinensis Kyowa Noen / Japan 

B00472 Natsudora Komatsuna parachinensis Yamato Noen / Japan 

B00473 Fukuha Komatsuna parachinensis Yamato Noen / Japan 

B00474 Maruba Wase Komatsuna parachinensis Yamato Noen / Japan 

B00475 Goseki Komatsuna parachinensis Yamato Noen / Japan 

B00590 I. B. – 1737 parachinensis IARI / India 

B. oleracea 

CR046 Guang-Dong Large-Stemmed Jie Lan alboglabra Guangzhou / China 

CR059 Fast Grow All Season alboglabra Guangzhou / China 

CR060 Early Point-Leaved Kailaan alboglabra Guangzhou / China 

CR061 Moderate-Flowered Kailaan alboglabra Guangzhou / China 

CR097 (KY1314)Veg-Gin(F1) alboglabra Known-You Seeds / Taiwan 


144







CR098 Nova alboglabra P.T. East West Seed / Indonesia 

Ba04 GN Black-leaf Kailaan alboglabra Kung Nong Seeds / Taiwan 

Ba05 GN White-leaf Kailaan alboglabra Kung Nong Seeds / Taiwan 

Ba08 DG Green Kailaan alboglabra Cheng Chi Seeds / Taiwan 

Ba09 DG Yellow Kailaan alboglabra Cheng Chi Seeds / Taiwan 

Ba11 EX Savoy Black Kailaan alboglabra Excellent Seeds / Taiwan 

Ba17 FT Savoy-leaf Black Kailaan alboglabra Fong Tien Seeds / Taiwan 

Ba18 Kailaan L1 (Chrysanthemum) alboglabra FTHES / Taiwan 

Ba19 Kailaan L2 (Round-leaf) alboglabra FTHES / Taiwan 

Ba20 Kailaan L3 (Big-flower) alboglabra FTHES / Taiwan 

Ba24 Round-leaf Black Kailaan alboglabra Excellent Seeds / Taiwan 

Ba25 High-stem Round-leaf Kailaan alboglabra Nien Fong Seeds / Taiwan 

Ba27 Kailaan (White-flower) alboglabra Nien Fong Seeds / Taiwan 

Ba32 Stem Kailaan BBT 35 alboglabra East-West Seed / Philippines 

Ba33 Kailaan alboglabra Ramgo Seeds / Philippines 

Ba34 Chinese Kale: Khana Yot alboglabra Chia Tai Seeds / Thailand 

Ba35 Large Leaf Kailaan alboglabra Chia Tai Seeds / Thailand 

Ba38 Yellow-Flower White Kailaan alboglabra Fu Nong Seeds / Taiwan 

Ba39 Savoy-Leaf Kailaan 117 alboglabra Leckat Co. / Malaysia 

Ba41 Yellow-Flower Large-Stem Kailaan alboglabra Fong Tien Seeds / Taiwan 

TB00216 Taoyuan #3 (B01127) alboglabra Taoyuan DAIS / Taiwan 

TB00217 Taoyuan #4 (B01128) alboglabra Taoyuan DAIS / Taiwan 

TB00799 Mbeya Green A alboglabra RCA / Tanzania 

TB00800 Mbeya Green B alboglabra RCA / Tanzania 

Table 2. Summary of simple sequence repeat primer pairs used in the genetic characterization of Brassica. 

Primer 

Count 

Primer 

Name 

Motif Type Forward Primer Reverse Primer 

1 Na10-E02 di GA/CT TCGCGCATGTAATCAAAATC TGTGACGCATCCGATCATAC 

2 Na10-F06 tri GGC/CCG CTCTTCGGTCGATCCTCG TTTTTAACAGGAACGGTGGC 

3 Na12-C03 di GA/CT ATCGTTGCCATTAGGAGTGG ACCAAATTAACCCTCTTTGC 

4 Na12-C06 di GA/CT AACGGATGAAGAACACATTGC TAGGGCCTGTTATTCGATGG 

5 Na12-C07 di GA/CT ACTCAACCCCACAAACCTG AGTTCCCCGGACCGATTAG 

6 Na12-C08 di GA/CT GCAAACGATTTGTTTACCCG CGTGTAGGGTGATCTATGATGGG 

7 Na12-D03 di GA/CT GGTAAGCCAAAAACCCTTCC GAAACCGGTAACAAAGTCGG 

8 Na12-F12 tri GGC/CCG CGTTCTCACCTCCGATAAGC TCCGATGTAGAATCAGCAGC 

9 Na14-B03 di GA/CT GATGGTGCCGATTCAATGA CCCATCAGCACTAGAAACCA 

10 Na14-C12 di GA/CT CACATTTTGGTTCAATTCGG TACGACCTGGTTTCGATTC 

11 Na14-D07 tri GGC/CCG GCATAACGTCAGCGTCAAAC CTGCGGGACACATAACTTTG 

12 Na14-E02 com GA/CT-GT/CA ACTGGCTACATGAGTTTCAGTG GAGGGAAGACAACTGGTCTCA 

13 Na14-E08 tri GGC/CCG TTACTATCCCCTCTCCGCAC GCGGATTATGATGACGCAG 

14 Ni2-B01 dis di GT/CA AAGGAGATTGTTTTGGGGC AAGACTAATAAACACACGGCG 

15 Ni4-E08 di GA/CT GATTTTGAGGAAGCGGAGG CAAAGCACTGAGAGAGAGAGAGAG 


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Primer 

Count 

Primer 

Name 

Motif Type Forward Primer Reverse Primer 

16 Ol10-A05 di GA/CT TGTAATAACCCGACCCATCC CTCTCTCGCTCTCTCGATCC 

17 Ol10-B01 di GA/CT CCTCTTCAGTCGAGGTCTGG AATTTGGAAACAGAGTCGCC 

18 Ol10-F11 tri GGC/CCG TTTGGACGTCCGTAGAAGG CAGCTGACTTCGAAAGGTCC 

19 Ol10-G08 di GA/CT TGCTTAATTGATTAGGGCAG TTACCTCATCAGGTGGAGGC 

20 Ol10-G09 di GA/CT TGCTTCCTTTTCTTCGCTC GAAGCACGAACGCGAGAG 

21 Ol10-H04 di GA/CT TCACCCCTCTATATCCACCC CAGAATCTGCCTGAACATCG 

22 Ol10-H07 di GA/CT TAGAGATGTCACCCGAAGGC AGCTTCATTTCAGTCGGTGG 

23 Ol11-B03 tri GGC/CCG ATGAAAACCAATCCAGTGCC GATAGCAGATGGAAGAGCCG 

24 Ol11-B05 di GA/CT TCGCGACGTTGTTTTGTTC ACCATCTTCCTCGACCCTG 

25 Ol11-G11 tri GGC/CCG GTTGCGGGCGAAACAGAGAAG GAGTAGGCGATCAAACCGAG 

26 Ol11-H02 tri AAT/AAG TCTTCAGGGTTTCCAACGAC AGGCTCCTTCATTTGATCCC 

27 Ol11-H06 di GA/CT TCCGAACACTCTAAGTTAGCTCC TTCTTCACTTCACAGGCACG 

28 Ol12-B05 di GA/CT GGAAAGCGAAGAGTGACGAC ATTGGGTAAAGCTGTGCTCG 

29 Ol12-E03 tri GGC/CCG CTTGAAGAGCTTCCGACACC GACGGCTAACAGTGGTGGAC 

30 Ol12-F02 di GA/CT GGCCCATTGATATGGAGATG CATTTCTCAATGATGAATAGT 

31 Ol12-F11 di GT/CA AAGGACTCATCGTGCAATCC GTGTCAGTGGCTACAGAGAC 

32 Ol12-G04 di GA/CT CGAACATCTTAGGCCGAATC GGTTAACCTGCGGGATATTG 

33 Ol13-C03 di GA/CT GATCGGAGATGCGATGAGAG GACTGCACCAGTGAAAAACTC 

34 Ol13-C12 di GA/CT AGAGGCCAACAAAGAACACC GAAGCAGCACCAGTGACAAG 

35 Ol13-E08 di GA/CT TTCGCAACTCCTCCTAGAATC AAGGTCTCACCACCGGAGTC 

36 Ra2-A01 di GA/CT TTCAAAGGAAAGGGCATCG TCTTCTTCTTTTGTTGTCTTCCG 

37 Ra2-A04 di GA/CT AAAAACTCCTCTCAACG CCCAAAGTTAGGTTTTAATGTAATCTC 

38 Ra2-A11 di GA/CT GACCTATTTTAATATGCTGTTTTACG ACCTCACCGGAGAGAAATCC 

39 Ra2-E01 di GT/CA TCTATATTAACGCGCGACGG GCACACACACACTCAAACCC 

40 Ra2-E03 di GA/CT AGGTAGGCCCATCTCTCTCC CCAAAACTTGCTCAAAACCC 

41 Ra2-E04 di GA/CT ACACACAACAAACAGCTCGC AACATCAAACCTCTCGACGG 

42 Ra2-E07 di GA/CT ATTGCTGAGATTGGCTCAGG CCTACACTTGCGATCTTCACC 

43 Ra2-E11 di GA/CT GGAGCCAGGAGAGAAGAAGG CCCAAAACTTCCAAGAAAAGC 

44 Ra2-E12 di GA/CT TGTCAGTGTGTCCACTTCGA AAGAGAAACCCAATAAAGTAGAACC 

45 Ra2-F11 di GA/CT TGAAACTAGGGTTTCCAGCC CTTCACCATGGTTTTGTCCC 

46 Ra2-G09 di GA/CT ACAGCAAGGATGTGTTGACG GAGAGCCTCTGGTTCAAGC 

47 Ra3-C04 tri GGC/CCG CTAACCTCAGACGGAGACGG CTTTAAACTCCGACCAACCG 

48 Ra3-D02B di GA/CT CACAGGAAACCGTGGCTAGA AACCCAACCTCAACGTCTTG 

49 Ra3-E05 di GT/CA TTCTCATGCTCCAACCACAG GTTTCTTCCAAGCCAAGGCTG 

50 Ra3-H10 di GA/CT TAATCGCGATCTGGATTCAC ATCAGAACAGCGACGAGGTC 

51 Na12-H04 di GA/CT TTTATCGTCTTTCCCCTCCC ACAAGGAACTAGAGAGAGAGAG 

52 Ni4-B10 di GA/CT GTCCTTGAGAAACTCCACCG CCGATCCCATTCTAATCCC 

53 Ol11-C02 di GT/CA GCATTGCAATCTTGTTGGTC CGTTTCCATACAGACGTAAGAC 

54 Ol12-D05 di GA/CT TCCATGCCAACGACAAGGTC AAGAGGCGACTCTATTGCG 

55 Ra2-A10 di GT/CA CCAGTGTGTGTGTGTGTGTG TTTAACAGATAGCGCAGTGGTC 

56 Ra2-D04 di GT/CA TGGATTCTCTTTACACACGCC CAAACCAAAATGTGTGAAGCC 

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Wet, powdery leaf material measuring 2.5 mL was then 

homogenized with 1 mL extraction buffer using a standard 

mini vortex and transferred to a 5 ml tube. 0.25 ml of 20% 

SDS was added. After mixing, the tube was incubated at 

65°C for 10 min. An amount of 1.25 mL 5 M potassium 

acetate was added. The tube was shaken vigorously and 

incubated for 20 min on ice. Tubes were centrifuged at 

4500 rpm for 20 min. The supernatant was transferred 

to new 5 mL tubes and 3.5 mL cold isopropanol was 

added. The tubes were mixed manually by inverting 

the tubes gently for three to five minutes. Tubes were 

then centrifuged at 4500 rpm for 30 s. Supernatant was 

discarded. The pellet was washed twice or thrice with 

70% ethanol and was left to air dry. The pellet was then 

resuspended in 50 μL of sterile distilled water, incubated 

at 55°C for 5 min and spun at 4500 rpm for 2 min. The 

solution was transferred to a new 500 μL eppendorf tube. 

An aliquot was used for quantification of DNA in gel 

electrophoresis in 1.2% agarose. Four concentrations of 

lamda DNA (200, 100, 50, and 25 ng/µL) were used to 

quantify the DNA. All DNA samples were diluted 30x 

from the original concentration to be 10 ng/µL before use 

as template DNA in the PCR analysis. 

SSR assay. Following an initial screening of 56 primers, 

only 54 primers were selected for use in amplification 

since the two other primers did not show any amplified 

DNA. Reaction mixtures of 15 µL contained 6.3 µL MQ 

H 2 

O, 1.5 µL 10x PCR buffer, 1.35 µL 2.5 nM dNTP, 0.375 

µL each of forward and reverse primers, 0.1 µL of 0.5 

U/µL Taq polymerase that was originally 5 U/µL, and 5 

µLDNA sample. Reaction mixtures were incubated in MJ 

Research PTC-200 Peltier Thermal Cycler DNA Engine 

programmed for 34 cycles of 30 s in 94°C (denaturation), 

1 min in 45°C (annealing temperature), and 45 s at 72°C 

(extension). The same reaction mixture without genomic 

DNA was run with each amplification to serve as negative 

control. Amplified products were resolved by high 

resolution gel electrophoresis using 4% acrylamide gel 

made with 1x TBE. Each piece of gel contains all samples 

with the same primer. Acrylamide gels were ran using a 

dual double-wide mini-vertical gel kit. Each gel was run 

at 50 volts at approximately 48 min depending on the size 

of PCR product. A total of 56 acrylamide gels were run 

corresponding to 56 primers used for the study. Using 

1-kb DNA ladder, band sizes of amplification products 

were estimated. Gels were submerged in a solution 

containing 10,000X Invitrogen SyBr Safe DNA gel stain 

for 20 min with continuous shaking and photographed 

under UV light. 

Data Analysis. SSR acrylamide gel images were scored 

visually. Clearly distinguishable bands were scored as 

present (1) or absent (0). Weak and poor bands were not 



recorded. The rate of polymorphic loci was computed by 

dividing the number of polymorphic bands with the total 

number of amplified bands. The UPGMA method and 

Proc Cluster of SAS system was used to compute for the 

distance values and to generate the phylogenetic tree. 

Bootstrap values were not obtained. 

RESULTS AND DISCUSSION 

A total of 122 distinct bands were generated using the 54 

primers. From these, 77 were polymorphic. An average of 

six polymorphic bands per primer was observed. Having 

generated the top polymorphic rates, twelve primers 

were found to be most useful for the analysis. It can be 

observed from Table 3 that these primers produced diverse 

numbers of amplified and polymorphic bands. Na11-C12 

produced the least number of amplified bands (with only 

three bands) while Ra2-E11 produced the greatest number 

of amplified bands (22 bands). Na10-E02 and Na11-C12 

produced the least number of polymorphic bands (three 

bands) while Ol11-G11 and Ra2-E03 produced the 

greatest number of polymorphic bands (10 bands). The 

same table also indicates the percent polymorphic loci 

of the 12 primers. The percentage ranged from 30% - 

100%, with a mean of 71.08%. Na10-E02 showed the 

lowest rate of polymorphic loci (30%) while Na12-C08, 

Na14-C12, Ol12-E03, Ol12-F11, and Ra2-E03 showed 

a very high polymorphic rate of 100%. This supports 

Table 3. Average rate of polymorphic loci of accessions of Brassica sp. 

using 12 different primers. 

PRIMER 

No. of 

Amplified 

Bands 

No. of 

Polymorphic 

Bands 

Rate of 

Polymorphic 

Loci (%)* 

1 Na10-E02 10 3 30 

6 Na12-C08 4 4 100 

10 Na14-C12 3 3 100 

25 Ol11-G11 15 10 67 

26 Ol11-H02 10 7 70 

29 Ol12-E03 8 8 100 

30 Ol12-F02 11 6 55 

31 Ol12-F11 8 8 100 

34 Ol13-C12 11 5 45 

40 Ra2-E03 10 10 100 

43 Ra2-E11 22 8 36 

56 Ra2-D04 10 5 50 

TOTAL 122 77 

AVERAGE 10.17 6.42 71.08 

Number __________________________ 

of polymorphic bands 

* Rate of Polymorphic Loci = 

Number of amplified bands 

X 100 

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previous reports by Agrama and Tuinstra (2003), Smith 

et al. (1997), and Powell et al. (1996) that SSR markers 

are highly polymorphic. This information is important 

especially for future studies to distinguish which primers 

are appropriate for assessing genetic diversity and 

relationships among accessions in a germplasm collection. 

In this study, primers Na12-C08, Na14-C12, Ol12-E03, 

Ol12-F11, and Ra2-E03 are the most recommended for 

such purposes because they were able to demonstrate the 

highest polymorphism. 

The representative gel in Figure 2 shows bands amplified 

using primer Ra2-D04. Banding pattern in B. rapa 

chinensis and B. r. parachinensis were more similar than 

those of B. o. alboglabra (Figure 2). B. o. alboglabra 

accessions gave more distinctive SSR banding patterns. 

This is expected because the two subspecies chinensis 

and parachinensis are members of the same species. 

Moreover, B. rapa ((n=10) and B. oleracea (n=9) 

contain very different genomes, namely the C and the A 

genome, respectively. Both are believed to be two of the 

three ancestral genomes (the third being the B genome 

of B. nigra) from which the amphidiploid species B. 

carinata (BC genome, n=17), B. juncea (AB genome, 

n=18) and B. napus (AC genome, n=19) arose through 

paleopolyploidization (U 1935). 

It is also noticeable that three accessions of B. r. chinensis 

(CR048, CR049, and CR050) and three accessions of B. 

r. parachinensis (CR024, CR025, and CR026) showed 

banding patterns that were very similar with that of five 

accessions of B. o. alboglabra (CR061, CR097, TB00217, 

TB00799, and TB00800). These B. o. alboglabra 

accessions exhibited banding patterns that were different 

from the rest of the subspecies and are more different 

from B. r. chinensis and B. r. parachinensis at a distance 

of 5.5. This was consistently observed using other primers. 

This observation may support the close evolutionary 

relationship between B. rapa and B. oleracea as reported 

by Chyi et al. (1992). However, it is also possible that 

these accessions of B. oleracea have been misidentified 

and are really members of B. rapa. Further analysis should 

be done to clarify the identity of these accessions. 

According to the consensus tree in Figure 3, accessions 

belonging to B. o. alboglabra clustered together while 

those belonging to B. r. chinensis and B. r. parachinensis 

formed another cluster. Meanwhile, all varieties of B. 

o. alboglabra belong to cluster 1. No accessions from 

other subspecies clustered with B. o. alboglabra. The 

observation in the band patterns of B. o. alboglabra 

accessions CR061, CR097, TB00217, TB00799, and 

TB00800 can also be confirmed in the consensus 

dendrogram since these accessions are highly different 



compared with other accessions of B. o. alboglabra. It 

can be observed that B. o. alboglabra accessions TB00799 

and TB00800 are very closely related to each other having 

a genetic distance of 1.0, which is the closest distance 

in the dendrogram. Both of these accessions came from 

Tanzania, which is far from the Asian origin of the other 

accessions used in the study. 

Accessions of B. r. chinensis and B. r. parachinensis 

grouped together in Cluster 2. This is expected because 

they belong to the same species. Within this cluster, two 

subclusters were formed. In subcluster 1, B. r. chinensis 

accessions CR048, CR049, and CR050 clustered with B. r. 

parachinensis accessions CR024, CR025, and CR025. All 

these accessions came from China. Also, these accessions 

showed similar banding patterns in most of the gels which 

were produced using different primers. It can be confirmed 

that these accessions are really closely related. 

In subcluster 2, many smaller subclusters were formed. It 

was evident that B. r. parachinensis accessions B00468, 

B00472, B00473, B00474, and B00475 clustered together. 

The most possible reason for this result is that these 

accessions all came from Japan. Out of the six accessions 

that came from Japan, only accession B00469 did not 

cluster with the other accessions. This accession clustered 

together with the B. r. chinensis accessions and not with 

B. r. parachinensis. This indicates that B. r. parachinensis 

accession B00469 could possibly be a member of B. 

r. chinensis and was just mistakenly identified as a B. 

r. parachinensis. In this subcluster of B. r. chinensis 

accessions, another B. r. parachinensis accession (CR042) 

clustered with them. This accession came from China like 

the other accessions with which it clustered together. This 

is controversial since this accession clustered together 

with many B. r. chinensis accessions. These B. r. chinensis 

accessions came from the same geographical origin which 

is China. Moreover, B. r. chinensis accession 28 clustered 

with B. r. parachinensis accessions CR005, CR006, 

CR007, CR010, CR035, CR082, CR083, CR085, CR087, 

and Bc57. All of these B. r. parachinensis accessions 

came from Guangzhou, China. This may indicate that their 

geographical origin is the main cause of their clustering. 

This confirms that geographic affinity would contribute 

to the similarity between accessions (Ren et al. 1995). 

As discussed above, accessions belonging to different 

subspecies but were collected from the same region were 

more similar to each other than to accessions of the same 

species but of different origin. Zhao et al. (2005) made the 

same observation as they studied different morphotypes 

of B. rapa using AFLP markers. According to them, this 

suggests an independent origin in both sites of collection 

and/or a long and separate domestication and breeding 

history in both regions. 

148





Figure 2. Representative gel showing polymorphic SSR bands of Brassica rapa chinenesis, B. rapa parachinensis and B. oleracea alboglabra accessions amplified by primer Ra2-D04. 

149





Figure 3. Consensus tree showing intra- and interspecific relationships among leafy Brassica species as determined by the UPGMA method and 

Proc Cluster of the SAS system. 

CONCLUSION 

From the data obtained, several conclusions can be 

drawn: (1) Genetic differences were observed within 

and between species of Brassica. There was high genetic 

variation among the accessions of B. rapa chinensis, 

B. r. parachinensis, and B. oleracea alboglabra. There 

was greater genetic diversity shown within B. rapa than 

in B. oleracea alboglabra. (2) B. r. chinensis and B. r. 

parachinensis produced bands more similar to each other 

compared with B. o. alboglabra. (3) The accessions of B. 

o. alboglabra clustered together and showed difference 

with the accessions of B. rapa. (4) Generally, there was 

a very close relationship between genetic diversity and 

150



geographical origin. Accessions from the same geographic 

origin belonging to the same subspecies are similar to each 

other. For instance, two B. o. alboglabra accessions that 

came from Tanzania are very much genetically similar 

to each other but were found to be highly different from 

other B. oleracea alboglabra species that came from Asia. 

(5) SSR markers proved to be useful in the germplasm 

characterization of B. rapa and B. oleracea species. 

ACKNOWLEDGEMENT 

The authors wish to thank the Asian Vegetable Research and 

Development Center for providing the plant materials and 

equipment for the conduct of this study, Mr. L-C Chang for 

the Brassica photographs, and Ms. S-M Huang for technical 

assistance. This research was partially supported by grant 

from the National Science Council of Taiwan. 

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