Pengfei LIU,Qu CHEN,Muheng ZENG,Xiaoming WANG,Feng JIANG

Crop Research Institute,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China

Responsible editor:Tingting XU Responsible proofreader:Xiaoyan WU

Molecular biology has been developed rapidly.Molecular markers (RFLP,SSR,RAPD),genetic engineering technology,cell engineering technology,DNAsequencing technology,DNA chip technology and enzyme engineering technology have been widely applied in plant genetic diversity analysis,genetic map and physical map construction,germplasm evaluation,plant DNA fingerprinting,genetic trait marking and assisted selection[1-8].For the researches of molecular biology,the highly efficient extraction of DNA is the premise and basic link.But for DNA extraction from plants,the grinding of plant leaves is the first step,and is also the most time and effort-consuming step.

There are currently two main grinding methods in laboratory,con ventional grinding method[8-9]and electric drill grinding method[10].For the conventional grinding method,the sample is mixed with quartz sand in a mortar,and then ground by adding liq uid nitrogen or β-mercaptoethanol.For the electric drill grinding method,the sample is mixed with DNA extraction buffer in a centrifuge tube,and then ground by a high-speed running electric drill.The running electric drill has disadvantages of damaging tubes,wasting samples and consuming a large amount of efforts.Both of the two grinding methods are characterized by time and effort consuming and non-u niformty,which are particularly prominent in DNA extraction from a large number of samples in molecular marker-assisted breeding.

The inbred lines Zheng 58,PH6WC,Yu 87-1 and Zi 330 were selected for constructing a four-way cross population (Zheng 58/Yu 87-1//PH6WC/Zi330).Based on SSR molecular marker analysis,the genetic linkage map of maize was constructed[10].A new rapid and batch-oriented crushing method was developed for DNA extraction from plant leaves.Moreover,the practicability of extracted DNA in SSR molecular marker analysis was verified.

Materials and Methods

Test materials and design

In autumn of 2010,two cross combinations,Zheng 58/Yu87-1 and PH6WC/Zi 330 were developed in the teaching and research base of Zhongkai University of Agriculture and Engineering in Guangzhou.The F1generations of the two single-cross combinations were cultivated in spring of 2011.The full-sib mating was carried out.The four-way cross population (Zheng 58/Yu 87-1//PH6WC/Zi 330) was constructed.The F2generation (F2:3family)of the four-way cross population was planted in the teaching and research base in Panyuzhongcun Village in spring of 2012.During the seedling stage,the single plant DNA of the F2population was extracted for genotyping and constructing genetic linkage map of molecular marker.

Crushing of leaves

According to requirements by test,certain amount (50-100 mg) of fresh plant leaves was weighted and placed in a 2 ml round-bottomed centrifuge tube.A clean steel ball weighing more than 0.5 g was placed in the centrifuge tube.Then the lid of centrifuge tube was covered.All the prepared centrifuge tubes were placed in an antifreeze container and flooded by liquid nitrogen.The antifreeze container was shocked 1-5 min 10-90 s after covering the lid of the container.After then,the lids of the centrifuge tubes were opened,and the steel balls in the centrifuge tubes were sucked out using a magnet.Thus the powder samples were prepared,and they were preserved at 4 ℃.

DNA extraction

The DNA extraction was performed using the kit.The plant DNA kit was provided by the Beijing Dingguo Changsheng Biotechnology Co.,Ltd.

SSR molecular marker detection

The SSR primer,of which the sequence was uniformly distributed in maize genome,was screened out from the Maize GDB (www.maizgdb.org).The primers,Taq polymerase,dNTP,ddWater,TE buffer were all purchased from the Beijing Dingguo Changsheng Biotechnology Co.,Ltd.

The PCR reaction system was composed of 1 μl DNA Template (10-20 ng/μl),1 μl Forward Primer (5 μmol/μl),1 μl Reverse Primer (5 μmol/μl),3 μl 10× Taq buffer (containing 20 mM Mg2+),0.6 μl Taqase(2 U/μl),0.6 μl dNTPs (10 mM/μl) and 2.8 μl ddH2O.

The PCR reaction program was as follows:hot lid temperature of 96 ℃,95 ℃5 min;95 ℃45 s,45-65 ℃45 s,72 ℃1 min,30-40 cycles;72 ℃5-10 min.The amplification products were preserved at 4 ℃.

The PCR amplification products were examined on a 30% non-denaturing polyacrylamide gel[10]for 30 min-3 h in electrophoresis buffer of 1TBA at a constant voltage of 200-220 V.After the electrophoresis,the gel staining was carried out according to the following procedures:fixation with 10%ethanol and 0.5% acetic acid for 10-15 min,permeating in 0.2% silver nitrate solution for 10-15 min,rinsing twice for 10-30 s with distilled water and staining with 1.5% sodium hydroxide and 0.4%formaldehyde.

Molecular marker analysis

Atotal of three types (1:1,1:2:1 and 1:1:1:1) of segregating populations of F2generation of the four-way cross population were marked.The marking results were represented with different lowercase letters.If the band type of the first parent was marked as a,all the same bands were marked as a; the firstly-shown second different band type was marked as b,and all the same bands were marked as b;the firstly-shown third different band type was marked as c,and all the same band were marked as c,and so on.The segregating marking could be divided into four types,back-cross type(aa×ab,aa×ba,ab×aa,ab×bb),F2type (ab×ab,ab×ba),three-way type(ab×cc,aa×bc,ab×ac,ab×ca,ab×bc,ab×cb)and four-way type (ab×cd).In the marking of band types of segregating populations,the homozygous band types were represented as aa,bb,cc and dd,and the heterozygous band types were represented as ab,ac,ad,bc and bd.

Results and Analysis

Screening results of SSR primer polymorphism

The polymorphism screening of the four parents of sweet maize was conducted using SSR primer.As shown in Fig.1,from left to right,every four adjacent bands were divided into one group.The products in each group were amplified using the same SSR primer pair.After the DNA extracted from leaf powder was amplified,clear DNA bands were obtained for SSR primer polymorphism analysis.As shown in Fig.1,the SSR primers were bnlg2086,umc1917,umc1603,umc-1590,bnlg615,bnlg1564,um1261,umc1961,bnlg1621,umc1185,umc-1465 and umc1555,respectively(from left to right).

Isolating results of populations using SSR molecular marker

The DNA of the four-way cross population was amplified by bnlg2046.Aseries of clear DNA bands were obtained(Fig.2).

Discussion

The rapid and batch-oriented crushing technology of maize leaves will accelerate DNA extraction,and the rapid crushing of maize leaves is also an important part of molecular markerassisted breeding.Currently,the DNA extraction kit has been developed continuously.However,none convenient and rapid crushing program has been provided.For DNA extraction with kit,well-crushed plant sample is the premise.Therefore,the establishment of rapid and batch-oriented crushing method of leaves plays an important role in numerous molecular marker tests.Compared with current technologies,the developed crushing method has following advantages:①Some leaf samples can be crushed simultaneously,and the number of simultaneously crushed leaf samples is related to volume of antifreeze container.②The developed crushing method consumes less time and effort.③The developed crushing method is safe and non-toxic,and does not require toxic biological antioxidants (i.e.β-mercaptoethanol).The operation is also simple.④The DNA bands amplified by SSR primer are clearer,and they fully meet test requirements.

The results suggest that this method can be applied in different molecular marker-assisted breeding works.The developed method has simple operation,and it is also characterized by rapidness,bulk,low cost and non-toxicity.In a word,the developed rapid and batch-oriented crushing method of leaves is feasible in laboratory tests.

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