9 - MP-Vol.03-No.03页

Molecular Pathogens 2012, Vol.3, No.1, 1

p://mp.sophiapublisher.com

Table 3 Sample information of apple, pear, apricot and peach trees for this experiment

Trees

Cultivar

Gathering place and the serials number of samples

Total

Yanqi

Hejing

Bohu

Korla

Heshuo

Xinhe

Aksu

Apple

Ralls

Aa1-

Aa30

Aa31-

Aa60

Aa61-

Aa90

Aa91-

Aa120

Aa121-

Aa150

—

Aa151-

Aa180

570

Red delicious

Ab1-

Ab30

—

Ab31-

Ab60

—

Ab61-

Ab90

Golden delicious

Ac1-

Ac30

—

Ac31-

Ac60

—

Ac61-

Ac90

Red Fuji

Ad1-

Ad30

Ad31-

Ad60

—

Ad61-

Ad90

—

Ad91-

Ad120

Starkrimson

Ae1-

Ae30

—

Ae31-

Ae60

Ae61-

Ae90

Peer

Korla pear

Pa1-

Pa30

—

Pa31-

Pa60

Pa61-

Pa90

Pa91-

Pa120

—

480

Jinfeng pear

Pb1-

Pb30

Pb31-

Pb60

Pb61-

Pb90

—

Ya pear

Pc1-

Pc30

—

Pc61-

Pc90

Pc91-

Pc120

—

Dang shan pear

—

Pd1-

Pd30

—

Apple pear

Pe1-

Pe30

Pe31-

Pe60

Pe61-

Pe90

Pe91-

Pe120

Pe121-

Pe150

—

Peach

P. persica

Sieb. et Zucc

Ta1-

Ta30

—

Ta31-

Ta60

Ta61-

Ta90

—

Ta91-

Ta120

P. persica.

var.nectariana

Tb1-

Tb30

—

Tb31-

Tb60

Tb61-

Tb90

—

Tb91-

Tb120

P. persica.

var.densa

Tc1-

Tc30

—

Tc31-

Tc60

Tc61-

Tc90

—

Tc91-

Tc120

Apricot

X1-

X50

—

X51-

X100

X101-

X150

X151-

X200

X201-

X250

X251-

X300

300

3.3

Cloning and sequencing

The amplification fragments were separated by

electrophoresis on a 2% agarose gel in 1×TAE buffer,

excised from the gel under ultraviolet light and

recovered using UNIQ-10 Gel Extraction Kit (Sangon)

according to manufacturer’s protocol. The recovered

fragments were cloned into pUCm-T vector, which is

2 773

nt in length with Amp resistance and enables

blue/white color screening (Shanghai Biological

Engineering Technology & Service Corp.), followed

by transformation into competent

coli

cells. The

desired clones were identified by restriction digestion

and PCR after plasmid DNA isolated from the white

colonies. Selected clones were then submitted to

Shanghai Biological Engineering Technology &

Service Corp. for sequencing. Sequences were analyzed

by Basic Local Alignment Search Tool (BLAST) on

www.ncbi.nlm.nih.gov

and the DNAMAN software.

3.4

In situ

RT-PCR

The distribution of ASSVd within leaf tissues of these

four kinds of fruit trees was investigated by

in situ

RT-PCR. Paraffin sections were prepared according to

the method of Niu et al

(2007).

Complementary DNA

synthesis was performed according to the method of

Zhao and Niu (2006) and

in situ

RT-PCR

amplification and immunological detection used the

methods of Niu et al

(2007).

Authors’ Contributions

YTW and YZ designed this experiment and carried out the

experiment; YTW also took part in the sequence analysis and

wrote manuscript; JXN directed the experimental and

modified the manuscript. All authors have read and approved

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