Yongan LIU,Binrong PAN,Gaohong YUE,Xixue MEI,Likui XU*,Zongchen ZHANG,Zhihui ZHOU

1.South-Zhejiang Key Laboratory of Crop Breeding,Wenzhou 325006,China;2.Wenzhou Academy of Agricultural Sciences,Wenzhou 325006,China

Molecular marker is an important technique in wheat genetics and breeding.It can be used to study the genetic diversity of wheat germplasms,as well as to assist the selection of agronomic traits.So molecular maker is a targeted and efficient technique,and it is not restricted by use time.Usually,the DNA for molecular marker is extracted from young wheat leaves.During the study,although young wheat leaves can be obtained by seed germination in time of need,the growth of wheat seedlings is restricted by seasons to a certain extent.In unsuitable seasons,the selected target plants will not grow normally,except in greenhouse.The cultivation facilities are just the lack of basic scientific units.Thus,DNA extraction from half-grain wheat seeds far away from embryos is an effective way to solve this problem.After extracting DNA from half-grain wheat seeds for molecular marker,the remaining half seeds can still be used for sowing without the aid of greenhouses and other facilities.

Currently,there have been many reports on extraction of DNA from wheat seeds[1-2].In the extraction process,phenol,chloroform,isoamyl alcohol and other reagents are usually used.However,chloroform is controlled by the public security department,and its purchase needs the approval by public security department[3].For a number of basic scientific units,the purchase of chloroform is not only relatively difficult but also time-consuming.Therefore,a chloroform-free DNA extraction method from wheat seeds was established in this study so as to provide technical support for molecular marker of wheat in basic scientific units.

Materials and Methods

Materials

The Yangmai 18 containingPm21 gene was used as research object.The wheat cultivar was introduced by the Jiangsu Lixiahe Region Institute of Agricultural Sciences. The main reagents used in this study included Tris,concentrated hydrochloric acid,EDTA,SDS,NaCl,ethanol,ammonium acetate,etc(of analytical grade).

Methods

DNA extraction from young wheat leavesThe DNA in young wheat leaves was extracted according to the method described by Chaiet al.[4],and it was used as the control for DNA extracted from wheat seeds.The RNA enzyme was not added into the extracted DNA.

DNA extraction from wheat seeds using chloroformThe DNA in wheat seeds was extracted according to the method described by Donget al.[1],and it was used as the control for DNA extracted from wheat seeds without using chloroform.

DNA extraction from wheat seeds without using chloroformUsing a razor blade,the wheat seeds were all cut into two parts,one with embryo and the other without embryo.The half-grain wheat seeds were placed on a weighing paper and concentrated in the center by folding the weighing paper.The seeds were ground with a pestle and transferred to 1.5-ml centrifuge tubes.Subsequently,700 μl of DNA extraction buffer (100 mmol/L Tris·HCl pH 8.0,50 mmol/L EDTA,500 mmol/L NaCl,2%SDS)was added to each of the tubes.The tubes were bathed in water at 65℃for 30 min.During the bathing,the mixtures in tubes were mixed several times.The tubes were cooled to room temperature.They were centrifuged at 13 000 rpm for 15 min.Then,certain amounts (500 μl for each)of supernatants were transferred to new 1.5-ml tubes,and ammonium acetate(50 μl,10 mol/L)and ethanol(1 ml,95%)were added successively.After then,the tubes were turned upside down 7-8 times and centrifuged at 13 000 rpm for 10 min.The supernatants were discarded. After adding certain amounts (500 μl for each)of ethanol,the tubes were turned upside down 7-8 times and centrifuged at 13 000 rpm for 2 min.The supernatants were discarded,too.The precipitates in tubes were dried at 37℃for 1 h,and then dissolved by certain amounts(50 μl for each)of TE buffer.

DNA quality inspectionA certain amount(2 μl)of each DNA sample was first mixed with a certain amount(1 μl)of 6 × DNA Loading Buffer and then examined by 1%agarose gel electrophoresis (containing EB,15 min).

PCR testThe three subunits(Wx-A1,Wx-B1 andWx-D1)ofWxprotein and powdery mildew-resistant genePm21 in Yangmai 18 were detected by multi-PCR[5].The sequences of primers and their expected amplification fragments were shown in Table 1.

Results and Analysis

DNA quality inspection

As shown in Fig.1,the bands of DNA extracted by the three different methods were all shown on the electrophorogram.However,the quantities of DNA differed among different extraction methods.The concentrations of DNA extracted from wheat leaves and wheat seeds using chloroform were relatively high,and the concentration of DNA extracted from wheat seeds without using chloroform was relatively low.It has been reported that there are significantdifferences in RNA concentration among different extraction methods and different wheat seed parts.The concentrations of RNA extracted from wheat leaves and half-grain wheat seeds with embryos were higher,and the concentration of RNA extracted from half-grain wheat seeds without embryos was lower.In addition,the size of RNA fragment extracted from wheat leaves was smaller,and the size of RNA fragment extracted from half-grain wheat seeds with embryos using chloroform was larger.However,the size of RNA fragmentextracted from half-grain wheat seeds with embryos without using chloroform was relatively uniform.

Multi-PCR test

As shown in Fig.2,the target bands ofWx-D1 (204 bp),Wx-A1(265 bp),Wx-B1(854 bp)andPm21(1 400 bp)of the amplified DNA extracted by three differentmethods were all basically shown on the electrophorogram.However,there were still some differences in amplification results,especially in amplification results ofWx-D1 (204 bp)andPm21(1 400 bp).The amplification results of DNA extractedfrom wheatleaves were best,and the target bands were all bright;the amplification results of DNA extracted from half-grain wheat seeds using chloroform ranked second,but the band ofWx-D1(204 bp)in lane two (DNA extracted from halfgrain wheat seeds with embryos using chloroform)wasnotobvious;the amplification results of DNA extracted from half-grain wheat seeds without using chloroform were the poorest,and the bands of amplifiedPm21(1 400 bp)from DNA extracted from half-grain wheat seeds with and without embryos were all relatively dark,which might be due to low DNA template concentration.

Discussion

The established chloroform-free DNA extractionmethodforwheat seeds omits the step of chloroform-isoamyl alcohol or phenol-chloroformisoamyl alcohol extraction,so it reduces the operation steps.Moreover,it has no requirement for chloroform.The established method is characterized by conventional reagents and simple operation.Compared with the conventional chloroform extraction method,although the concentration of DNA extracted by the established chloroform-free method is relatively low,the extracted DNA can still be used as template for PCR.

Table 1 Sequences of primers and their expected amplification fragments

In this study,multiple PCR is used to inspect the quality of extracted DNA.Comparedwiththegeneral PCR,multi-PCR has advantages of less reagents,higher throughput and economy[9].However,multi-PCR has higher requirements for template DNA quality,its primers are difficult to be designed,and there is competition among different amplified fragments[10].The DNA extracted by chloroform-free method can be used to amplify all the target fragments,indicating that the established extraction method has a good practicability.However,using the DNA extracted by the chloroform-free method as the template,the band ofPm21 (1 400 bp)is relatively dark.This problem can be solved by adding loading quantity of DNA sample or adopting polyacrylamide gelelectrophoresis.Therefore,the chloroformfree DNA extraction method can be used when there are no special requirements for DNA quality or chloroform cannot be acquired timely,and it is especially practicable for basic scientific units.

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