2. Materials and methods2.1. Plant

2. Materials and methods
2.1. Plant material and DNA isolation
Given its geography, six representative M. wufengensis populations
were selected based on field investigations and their distribution
in and around Wufeng in Hubei (Fig. 1). The largest population,
i.e., Dushuping (DSP), was further divided into five subpopulations on
the basis of slope aspect and elevation. A range of 18–33 samples of
each population were collected for analysis for a total sample size of
252 (Table 1). During spring blooming, twigs from adult trees, at least
30 to 50 years old, with buds were cut and cultured in 25°C water at
our laboratory. Young, 3 to 5 cm long leaves were spread out and
selected for DNA isolation.
Genomic DNA of single individuals was extracted following the
improved CTAB protocol [7]. The concentration and quality of DNA
samples were checked on 0.8% agarose gel by a comparison with
lambda DNA standards and with UV spectroscopy. The DNA samples
were diluted to approximately 10 ng/μL with a 0.1% TE buffer.
2.2. ISSR analysis
The sequence and codes of 56 ISSR primers followed by NAPS
Standard Unit Primers (UBC, Canada) were screened. Of these, 10
primers with clear and polymorphic bands were used for amplification
of all 252 accessions from the six populations. The annealing
temperature of each primer was optimized by a gradient PCR (Table 2).
2.3. SRAP analysis
Our SRAP protocol followed the method established by Li and Quiros
[8]. Forty eight SRAP primer pair combinations (em1~em6 × me1~me8)
were screened by separate PCR products on a 1.5% agarose gel for
successful amplification. Ten of these 48 primer pairs produced clear
and stable polymorphic bands and labeled as 252 accessions of the six
populations (Table 2). The 10 primer pair combinations are em1–me1,
em1–me3, em1–me5, em2–me5, em4–me1, em5–me3, em5–me4,
em5–me5, em5–me8 and em6–me5. All ISSR and SRAP primers were
synthesized by the Sangon Biotech (Shanghai) Co, Ltd.
2.4. Bands scoring and data analysis
Bands were scored by hand. Bands with the same weight were
scored as a line according to the weight of the DNA ladder (100 bp).

ผลลัพธ์ (ไทย) 3:

2 . วัสดุและวิธีการ
2.1 . วัสดุโรงงานและการสกัดดีเอ็นเอ
ได้รับของภูมิศาสตร์ เป็นตัวแทน 6 เมตร wufengensis ประชากร
ถูกเลือกตามสนามและการกระจาย
ใน และ รอบ ๆ Wufeng ในมณฑลหูเป่ย์ ( รูปที่ 1 ) ที่ใหญ่ที่สุดของประชากร ,
) dushuping ( DSP ) ถูกแบ่งออกเป็นห้าสองบน
พื้นฐานด้านความลาดชันและระดับความสูง A range of 18–33 samples of
each population were collected for analysis for a total sample size of
252 (Table 1). During spring blooming, twigs from adult trees, at least
30 to 50 years old, with buds were cut and cultured in 25°C water at
our laboratory. Young, 3 to 5 cm long leaves were spread out and
selected for DNA isolation.
Genomic DNA of single individuals was extracted following the
improved CTAB protocol [7]. The concentration and quality of DNA
samples were checked on 0.8% agarose gel by a comparison with
lambda DNA standards and with UV spectroscopy. The DNA samples
were diluted to approximately 10 ng/μL with a 0.1% TE buffer.
2.2. ISSR analysis
The sequence and codes of 56 ISSR primers followed by NAPS
Standard Unit Primers (UBC, Canada) were screened. Of these, 10
primers with clear and polymorphic bands were used for amplification
of all 252 accessions from the six populations. The annealing
temperature of each primer was optimized by a gradient PCR (Table 2).
2.3. SRAP analysis
Our SRAP protocol followed the method established by Li and Quiros
[8]. Forty eight SRAP primer pair combinations (em1~em6 × me1~me8)
มีผลิตภัณฑ์ที่เป็นเจล โดยแยก PCR , 1.5% สำหรับ
ประสบความสำเร็จขยาย . สิบเหล่านี้ 48 ไพรเมอร์คู่ผลิตชัดเจน
และวงดนตรี polymorphic ที่มั่นคงและติดป้ายว่าเป็น 252 ตัวอย่างของหก
ประชากร ( ตารางที่ 2 ) 10 คู่ไพรเมอร์ผสม em1 – me1
em1 me3 , – , - me5 em1 , me5 em2 – em4 me1 , – , - me3 eM5 eM5 me4 , – , me5
eM5 ––– me8 em6 eM5 , และ me5 . โดย
srap issr ทั้งหมดและที่สังเคราะห์จากแสงอ่อน BIOTECH ( เซี่ยงไฮ้ ) จำกัด
2.4 . วงดนตรีและวงดนตรี
ข้อมูลการวิเคราะห์คะแนนคะแนนโดยมือ วงดนตรีกับน้ำหนักเดิม
คะแนนเป็นเส้นตามน้ำหนักของบันไดดีเอ็นเอ ( 100 BP )