Study on Classification and Genetic Diversity of Kentucky Bluegrasses by Using RAPD Markers

Dec. 2012

ScienceDirect

Vol. 19 No. 4 37-41

Journal of Northeast Agricultural University (English Edition)

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Study on Classification and Genetic Diversity of Kentucky Bluegrasses by Using RAPD Markers Wang Chao, Li Xin, Gao Li-na, Zhang Lu, Liu Wei, Liu Hui-min, and Chen Ya-jun* College of Horticulture, Northeast Agricultural University, Harbin, 150030, China

Abstract: A total of 69 random primers were screened by using random amplified polymorphic DNA (RAPD) markers to analyze the genetic bands of 32 Kentucky bluegrass cultivars. A total of 197 bands were amplified from 46 primers, among which 195 bands were polymorphic. Each primer could amplify one to nine polymorphic bands with an average of 4.3 per primer. Based on similarity coefficient analysis of RAPD results and by using NTSYS software to cluster analyze with the average UPGMA method, the result showed that 18 cultivars of the 32 were in group 1, three cultivars were in group 2, two cultivars were in group 3, eight cultivars were in group 4, and only one cultivar in group 5. Key words: Kentucky bluegrass, RAPD, genetic diversity, clustering analysis CLC number: S688.4; Q943.2

Document code: A

Article ID: 1006-8104(2012)-04-0037-05

taxonomic research on the introduced Kentucky

Introduction

bluegrass cultivars and to understand the genetic

Kentucky bluegrass (Poa pratensis L.) is one of the

different cultivars.

most widely used cool-season turf grasses in the

　This study adopted the introduced European and

world. This species originated in Europe, north of

American Kentucky bluegrass varieties in addition

Asia and Africa, before it was introduced in North

to native materials from China, and then the genetic

America. The USA had improved on the Kentucky

diversity was analyzed by using the RAPD (Random

bluegrass since 1950s, and gradually industrialized

Amplified Polymorphic DNA) analysis. The objective

varieties worldwide. China introduced many Kentucky

of this study was to classify Kentucky bluegrass

bluegrass varieties from abroad in recent 20 years,

cultivars to estimate the genetic diversity and variety

which enriched the turfgrass cultivar resources in

of range in cultivars, and also to detect the genetic

China, but at the same time, resulted in the degradation

relationship among different Kentucky bluegrass

of those that couldn't adapt to the local climate

cultivars by RAPD markers, which would provide

environment. Because of the blind introduction of

theatrical basis for further reasonable utilization

some cultivars and unfamiliarity with their genetic

of introduced Kentucky bluegrass cultivars and

background and bionomics, serious economic loss

the development of domestic Kentucky bluegrass

ensued. In view of this, it's now necessary to make

resources and breeding work in China.

relationship and the genetic background among

Received 26 February 2012 Supported by the National Natural Science Foundation of China (30871735; 31272191); the National Key Technology R&D Program (2006BAD01A19-4-2); the Natural Science Foundation of Heilongjiang Province (C0207; C200619) Wang Chao (1987-), female, Master, engaged in the research of landscape plant resources and stress physiology. E-mail:[email protected] * Corresponding author. Chen Ya-jun, professor, supervisor of Ph. D student, engaged in the research of physiology and ecology of landscape plant. E-mail: [email protected] http: //publish.neau.edu.cn

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Vol. 19 No. 4 2012

Journal of Northeast Agricultural University (English Edition)

followings: 1 µL template DNA (10-20 ng • µL-1), 2 µL

Materials and Methods

10× reaction buffer, 1.8 µL MgCl2 (25 mmol • L-1), 0.4 µL

Materials

(5 U • µL-1), 1.5 µL RAPD primer (10 pmol • µL-1),

The materials of Kentucky bluegrass were introduced

added deionized water until reaching 20 µL. The

from Europe and America, and one wild cultivar

RAPD primer was synthesized by Bioasia.

origined from Heilongjiang, China. The cultivars code,

　The reaction process of the RAPD was as the follow-

name and origin area are shown in Table 1.

ings: 94℃ fore-degeneration 5 min, 94℃ degenera-

dNTP (2.5 mmol • L-1), 0.2 µL TaqDNA polymerase

tion 30 s, 37℃ annealing 1 min, 72℃ extention 1 min, Methods

30 s circulation 34, 72℃ extention 7 min, then stopped

DNA extraction methods

at 4℃. After staining with bromphenol blue, PCR

Every sample was randomly selected from three plants

production was electrophoresised for 1.5 h in 1.5%

and their tender leaves. We abstracted con-genome

(w/v) sepharose. Pictures were taken using the gelatin

DNA by using SDS methods and then detected the

system after electrophoresis, and then the data was

integrality and density of DNA.

recorded. Some of the bad results or those with

The RAPD methods

missing primers and samples should be electrophoresis

The reaction system of the RAPD was as the

and amplified.

Table 1　Kentucky bluegrass cultivars and their origins Name

Code

Origin

Name

Code

Origin

Freedomll Total Eclipse America Liberator NuGlade Award Impact Nassau Conni Merit Brootlawn Brown Sapphire Baron Nova

Z1 C1 M1 J1 X1 J2 L2 N1 K3 Y1 F1 B2 L4 D3 X2

America America America America America America America America Denmark America America America America America America

F2 Q1 B1 G4 L5 K1 W1 H2 C2 O1 L3 N2 B4 P3 U1

America America Denmark America America America America America America America America America America America America

Rugby2

G3

America

Fortuna Bluechip Barlin Park Kenblue Kentucky Midnight Blackstone Supermerit Opel Bluemoon Baronie Broadway Pumium Sobra Poa pratensis var. anceps

A

Heilongjiang, China

Data processing

of resolution.

In electrophoresis map, we represented a site where

　Electrophoresis of the same primer and different

primer and template DNA were complementary, which

templates showed that the RAPD band with the same

a molecular marker estimated the molecular weight

mobility rate had the same molecular weight. Bands

of amplified product according to the corresponding

presented in all the templates were public bands,

location of molecular weight in the gelatin and on

expressing no polymorphism, while others unique

the basis that the weight of DNA was inversely

to the template were polymorphic bands, expressing

proportional with electrophoresis mobility on the range

polymorphism. The RAPD was dominant marker,

E-mail: [email protected]

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Wang Chao et al. Study on Classification and Genetic Diversity of Kentucky Bluegrasses by Using RAPD Markers

the individuals with the bands were marked 1, those

the band's amount that was within material j but not in i.

without were marked 0, and those that had lost the bands were marked –1. 　Genetic parameters calculation:

Results

　 The similar coefficients between germplasm

Primer screening

were simple matching coefficient (SM), SM=(a+d)/

Took four different Kentucky bluegrass varieties as

(a+b+c+d). a was the amount when both of the

templates, used the best reaction system to carry out

compared varieties were 1, which was called correct

PCR amplification to 500 item primers, and screened

matching; b and c respectively indicated the amount

primers according to the amplification strap results.

when compared varieties were 1 and 0, which was

The principle was that amplification strap of primer

called incorrect matching; d was the amount when

was more, clear and having polymorphism.

compared varieties were –1, which was called negatively matching.

RAPD polymorphism analysis

　The similar coefficients used in the experiment were

This experiment proceeded to polymorphism screen-

as the followings:

ing of 500 random primers made from Bioasia Com-

　Jacquard's coefficient (JACCA, 1908):

pany, 431 primers (account 86.2%) had not produced

　Jcij=a/(a+b+c)

products which could be detected. A total of 69 pri-

　 Jcij represented the genetic similar coefficients

mers could produce polymorphism band, and we

between i and j in this formula, a was the quantitative

could only choose 46 primers to amplify to get clear

band within two materials, b was the band's amount

amplification bands. The results are shown in Figs. 1

that was within material i but not in material j, c was

and 2. U03

2 000 bp 1 000 bp 500 bp 250 bp 100 bp M J2 F2 Q1 K1 Y1 H2 L4 F1 Q1 D3 X2 G3 L2 L3 L5 M1 C1 Z1 N2 N1 P3 G4 C2 J1 A U1 B4 W1 K3 B1 B2 X1

Fig. 1　The RAPD map amplified by U03 primer M, Marker DL2000.

BF09

2 000 bp 1 000 bp 500 bp 250 bp 100 bp M J2 F2 Q1 K1 Y1 H2 L4 F1 Q1 D3 X2 G3 L2 L3 L5 M1 C1 Z1 N2 N1 P3 G4 C2 J1 A U1 B4 W1 K3 B1 B2 X1

Fig. 2　The RAPD map amplified by BF09 primer M, Marker DL2000. http: //publish.neau.edu.cn

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Vol. 19 No. 4 2012

Journal of Northeast Agricultural University (English Edition)

　 From Figs. 1 and 2 we knew that every primer

cultivars.

got genome DNA finger printing, the polymorphism behaved differently with a different primer, and

Genetic relationships of cultivars

different primers showed significant differences with

Pair-wise similarity coefficient of Jaccard cultivars'

different materials' finger printings and amounts of

coefficient of similarity was between 0.125 and

amplified bands; the same primers had different finger

0.714, the result showed that there was significant

printing with different materials. Amplified straps

heterogeneity difference among different cultivars; the

were tested from different primers ranging from 1 to

highest pairs of cultivars similar coefficient were O1

9, with every primer amplifying 4.3 strap on average.

and X2; the highest average coefficient of similarity

The primers which amplified the most straps were

was H2, the lowest one was C2, it indicated that H2 had

BC10, W12, X02 etc.; the primers which amplified

the minimum genetic distance to the other cultivars,

the fewest straps were U14, BB17, V15, and W20.

and had the closest genetic relationships; C2 had the

Forty-six primers totally produced 197 different

maximum genetic distance to other cultivars, therefore,

molecular weight RAPD markers in 32 samples,

the farthest genetic relationship.

among which mono-morphs bands were two, which

　 Utilizing NTSYS-PC2.11a software, data was

accounted for 1.01%, polymorphism bands were 195,

clustered according to 1, 0, –1 which inverted from

which accounted for 98.5%. It explained the genetic

the RAPD amplification results. Jaccard amplification

background complexity among Kentucky bluegrass

results are shown in Fig. 3 and Table 2. J2 Q1 O1 X2 D3 G3 L2 L3 Y1 H2 L4 F1 L5 M1 C1 Z1 N2 N1 F2 K1 P3 G4 A J1 U1 B4 W1 B1 B2 X1 K3 C2

0.20

0.33

0.46 Coefficient

0.59

0.71

Fig. 3　Statistics results of Jaccard genetic similarity among 32 Kentucky bluegrass cultivars by the RAPD E-mail: [email protected]

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Wang Chao et al. Study on Classification and Genetic Diversity of Kentucky Bluegrasses by Using RAPD Markers

The genetic relationship of European and American Table 2　Cluster result of 32 Kentucky bluegrass cultivars by

introduced varieties was closer and hereditary basis

the RAPD

was much narrower. The morphological marker method was used as the main method of identifying

Class

Cluster result

Ⅰ

J2, Q1, O1, X2, D3, G3, L2, L3, Y1, H2, L4, F1, L5, M1, C1,

Ⅱ

F2, K1, P3

tucky bluegrass cultivars, the morphological difference

Ⅲ

G4, A

among different cultivars was becoming smaller and

Ⅳ

J1, U1, B4, W1, B1, B2, X1, K3 C2

smaller, making it very difficult to distinguish them

Ⅴ

Z1, N2, N1

the authenticity and purity of cultivars. With the increasing in the production and development of Ken-

solely from morphology. Using the RAPD marker along with other markers to study genetic diversity

　 We could see from Fig. 3 that the experimental

of germplasm resources of Kentucky bluegrass to

samples were divided into five groups with coefficient

identify varieties would be more accurate, fast, simple

similarity of 0.434: the first group included 18

and plentiful; therefore, the RAPD is an effective

cultivars: J2, Q1, O1, X2, D3, G3, L2, L3, Y1, H2, L4, F1,

method for variety classification and identification.

L5, M1, C1, Z1, N2, and N1; the second group included three cultivars: F 2, K 1, and P 3; the third group

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http: //publish.neau.edu.cn