Huiquan Zheng?Hongjing Duan?Dehuo Hu?Ruping Wei?Yun Li

Sequence-related amplified polymorphism primer screening on Chinese fir(Cunninghamia lanceolata(Lamb.)Hook)

Huiquan Zheng?Hongjing Duan?Dehuo Hu?Ruping Wei?Yun Li

Chinese fir(Cunninghamia lanceolata(Lamb.) Hook)is one of the most important coniferous tree species used for timberproduction in China.Here,we conducted a sequence-related amplified polymorphism(SRAP)primer screening assay with a total of 594 primer combinations, using 22 forward and 27 reverse primers on four representative Chinese fir genotypes.The obtained results indicated that Chinese fir genomic DNA has a notable amplification bias on the employed forward or reverse primer nucleotides(30selection bases).Out of the tested primer sets,35 primer combinations with clearly distinguished bands,stable amplification,and rich polymorphism were selected and identified as optimal primer sets.These optimal primer pairs gave a total of 379 scorable bands, including 265 polymorphic bands,with an average of 10.8 bands and 7.6 polymorphic bands per primer combination. The produced band number for each optimal primer set ranged from 7 to 14 with a percentage of polymorphic bands spanning from 33.3 to 100.0%.These primer combinations could facilitate the next SRAP analysis assays in Chinese fir.

Chinese firSRAPPrimerScreening assayPolymorphism

Introduction

The rapid development of molecular biology techniques has provided new approaches to plant breeding.One of the most promising is the DNA marker technique as it offers great potential to the dissection of plant genetic structure, diversity,and functionality that are required for markerassisted breeding schemes(Walter et al.1998;Agarwal et al.2008;Poczai etal.2013).Nowadays,DNA markers have been integrated in conventionalbreeding programs or used as a substitute for conventional phenotypic selection on the assumption that efficiency and precision of the genetic improvement could be greatly increased(Collard and Mackill 2008).

The available DNA marker techniques include random amplified polymorphic DNA(RAPD),restriction fragment length polymorphism(RFLP),amplified fragment length polymorphism(AFLP),inter simple sequence repeat (ISSR),and simple sequence repeat(SSR),sequence-related amplified polymorphism(SRAP),and single nucleotide polymorphism(SNP)(Agarwal et al.2008;Bracci et al. 2011;Rana et al.2013).Of these,the SRAP technique is recognized as a simple,efficient,and cost-effective marker system that could be used in multiple molecular biology studies,including genetic-diversity analysis,genomic and cDNA fingerprinting,map construction,gene tagging,and map-based cloning(Liand Quiros 2001;Budak etal.2004; Baloch et al.2010).Compared to other marker systems, this technique is specifically targeted to genome openreading frame(ORF)sequences,which provide more genetic information associated with phenotypes(Ferrioletal.2003;Uzun etal.2009;Castonguay etal.2010;Rana et al.2013).For successful application of SRAP,the most critical step is the selection of optimal forward and reverse primer pairs that permit an effective polymorphism characterization of target plant species.

Chinese fir(Cunninghamia lanceolata(Lamb.)Hook)is a coniferous evergreen tree species(2n=2x=22 chromosomes)that is widely spreading throughout southern China.The reason is that this species has great afforestation and commercialvalues due to its desirable attributes in adaptability,growth rate,and the versatility and usefulness of its wood for industrial applications(Shi et al.2010; Huang etal.2012).Over the past50 years,much efforthas been made to improve the Chinese firthrough conventional breeding:remarkable successes have already been achieved(e.g.large-scale collection of elite germplasms; establishment of first-,second-,and third-generation seed orchards;and significantgains in superior clones).

To accelerate the Chinese fir breeding process,some organizations emphasized the genetic,transcriptomic,and proteomic aspects(Mu¨ller-Starck and Liu 1988;Yeh et al. 1994;Wang et al.2007;Shi et al.2010;Shen et al.2011; Wang et al.2011;Huang et al.2012;Wang et al.2013). However,current knowledge concerning the genetic information associated with phenotypes or multiple biology traits in Chinese fir is still rather limited,which has seriously hindered the breeding progress of this species depending on conventional improved technologies alone. To address this issue,we conducted a primer screening assay with a total of 594 primer combinations,aiming to identify the authoritative primer sets suitable for SRAP marker-assisted breeding schemes of Chinese fir.

Materials and methods

Plantmaterials

The case study materials were four C.lanceolata clones (c707,c718,c763 and c779)that represent different genotypes in the Chinese fir germplasm bank of Xiaokeng State ForestFarm(Shaoguan,Guangdong,China),which were the grafted age of3 years.These clonesallbelong to the Lechan provenance(China)but differ in their families and morphological traits(e.g.leaf colors,flowering and cone characteristics)thatauthorized their divergence in genetics.

DNA extraction and SRAP-PCR

Genomic DNA was extracted from mature Chinese fir leaves,using a Plant Genomic DNA Kit(TIANGEN, Beijing,China).The SRAP-PCR assay was carried outin a 25 l l reaction system containing 0.5 l l of genomic DNA (about 50 ng),0.5 l l forward primer(10 l mol/l),0.5 l l reverse primer(10 l mol/l),12.5 l l 29 Taq Plus PCR MasterMix(TIANGEN,Beijing,China),and 11 l l double distilled water.The thermal cycling conditions were as described by Li and Quiros(2001).All the reactions were performed on a VeritiTM96-WellThermalCycler(Applied Biosystems,Foster City,CA,USA).

The PCR products were observed on 2%agarose gel stained with ethidium bromide,using a Universal Hood II imaging system(Bio-Rad,Hercules,CA,USA).Each PCR assay for the same DNA sample was repeated three times in separate experiments.The primers used in this study were synthesized by Sangon Biotech(Shanghai)Co.,Ltd., Shanghai,China.Forward and reverse primer sequences are listed in Table 1.

Results and discussion

A totalof594 differentSRAP primercombinations,using 22 forward and 27 reverse primers,were employed(Table 1). These primer sets were evaluated on four representative genotypes(c707,c718,c763 and c779)for their ability to prime PCR amplification in Chinese fir.Each primerpairhad a distinctive banding profile,and the amplification efficiency seemed to be somewhat different(data not shown).The amplification profiles could be classified into four types as represented by Me1-Em19,Me3-Em6,Me13-Em23,and Me8-Em11 respectively(Fig.1);the separated bands in the top three types appeared to be detectable.

A total of 4,090 SRAP bands were observed herein,of which 1,948(47.6%)were monomorphic and 2,142 were polymorphic(52.4%).These preliminary results indicated that the SRAP technique could be used as an alternative molecular tool on Chinese fir.Several other DNA-based makersystems,such as RAPD,AFLP,ISSR and SSR,have previously been applied in the molecular biology studies (e.g.genetic diversity analysis,map construction,association analysis,and molecular typing and identification)of Chinese fir(You and Hong 1998;He et al.2000;Chen etal.2001;Tong and Shi2004;Lietal.2007;Yang etal. 2009;Shen et al.2011;Ouyang etal.2014).

However,these techniques provided limited functional gene information associated with the traits of interest.The SRAP technique may be helpfulin deciphering the genomic basis ofcomplex traits thatare related to the economic and ecologicalvalue of Chinese fir.In other plantspecies,such as Cucurbita pepo,the information given by SRAP markers was more concordant to the morphological variability and to the evolutionary history of the morphotypes than thatof AFLP markers(Ferriol et al.2003).Youssef et al.(2011) further reported that the SRAP technique could generate approximately threefold more specific bands than AFLPproving to be a proficient tool in Musa species discrimination.Smutkupt et al.(2006)demonstrated the higher discriminatory power of SRAP over RAPD in legumes. Herein,the SRAP technique also resulted in a considerable number(2,142)of polymorphic bands indicating a discriminatory potential on Chinese fir genomes.

Table 1 List of the tested sequence-related amplified polymorphism primers.The selective nucleotides for each primer are underlined

Fig.1 The representative SRAP profiles in this study.The electrophoretograms from Me1-Em19,Me3-Em6,Me13-Em23,and Me8-Em11 primer pairs using four different Chinese fir genomic DNA as templates are employed as representative of:1 clear,distinguished, stable and rich polymorphism profiles,2 clear,distinguished,stable butlow(none)polymorphism profiles,3 reproducible profiles only at a detectable level,and 4 null amplification,respectively.M,DNA marker.The number of 1,2,3,4 represents the Chinese fir genotype c707,c718,c763 and c779 respectively

Fig.2 Amplification efficiency of the tested SRAP primers in Chinese fir.The total number of SRAP bands produced by each forward orreverse primerdirected primercombinations is shown.The total number of polymorphic bands for each primer directed sets is indicated by black column.PPB,percentage of polymorphic band

By scoring the bands from forward or reverse primer directed primer combinations(e.g.Me1-Em1 to Me1-Em27),we found thatthe amplification ability ofeither the forward or reverse primers varied significantly with each other,revealing a genomic bias of Chinese fir on different forward-and reverse-primer nucleotides(30selection bases)(Fig.2).In fact,primer preference for successful SRAP-PCR assays has been widely recognized in many plant species,including the woody plants Prunus persica (Ahmad etal.2004),Diospyros kaki(Guo and Luo 2006), and Bixa orellana(Valdez-Ojeda et al.2008),Paeonia suffruticosa(Han etal.2008),Pinus koraiensis(Feng etal. 2009),and Morus(Zhao et al.2009),Citrus(Uzun et al. 2009),Prunus armeniaca(Ai et al.2011),Coffee L. (Mishra etal.2011),and Punica granatum(Soleimanietal. 2012),as wellas Vitis(Jing etal.2013).Among these tree species,the following primers were used in most of the cases:forward primer Me1,Me2,and Me4 with 30selection bases ATA,AGC,and ACC respectively,and the reverse primer Em3(30selection bases GAC)and Em5(30selection bases AAC).The primer Me1,Me4 and Em5 were also found to be helpfulfor the SRAP experiments in Chinese fir as evidenced in Fig.2 and Table 2.Intriguingly,our data further revealed a set of other useful forward or reverse primers including Me11,Me9,Me17, Me16,and Me10,and Em19,Em1,Em26,Em21,and Em17.These primers offer potential for the selection of suitable SRAP forward-reverse primer combinations specific for Chinese fir.

According to the electrophoresis results,35 SRAP primer pairs with clearly distinguished bands,stable amplification, and rich polymorphism were selected(Table 2),and identified as authoritative primer sets for SRAP experiments in Chinese fir.These primer combinations could produce 379 fragments,including 265 polymorphic bands,an average of 10.8 bands and 7.6 polymorphic bands per primer combination.The numberofbandsusing each primercombination ranged from 7 to 14 with a percentage ofpolymorphic bands (PPB)spanning from 33.3 to 100.0%.

Notably,most of the primer combinations consisted of the securable forward or reverse primers,e.g.Me1,Me10, Me11,Em1,and Em19.Based on these primer sets,we also found thatthere were 29,25,44,and 33 SRAP bands specific to c707,c718,c763,and c779 respectively.The genetic distances(GS)among genotypes(clones)could be calculated according to the matrix of SRAP bands scored as present(1)or absent(0),and the methods as described by Neiand Li(1979),and Ferrioletal.(2003),indicating a moderate distance(0.58–0.61)among c707,c718,c763, and c779.This resultagreed with previous supposition that these clones were substantially differentin genomes albeit of the same in provenance.

Conclusion

Our work presented a possible method for applying the SRAP technique on Chinese fir and the genomic bias of this species on different SRAP primer nucleotides.The optimal primer sets we obtained would facilitate the next SRAP analysis assays,and be usefulin the marker-assisted breeding schemes of Chinese fir.

Table 2 The optimal SRAP primer combinations and their reference parameters in Chinese fir

AcknowledgmentsThis research was supported by the National Natural Sciences Foundation of China(No.31200506)and the Special Fund for Forest Scientific Research in the Public Welfare(No. 201404127).

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29 October 2013/Accepted:3 April 2014/Published online:24 January 2015

Huiquan Zheng and Hongjing Duan contributed equally to this work.

The online version is available athttp://www.springerlink.com.

Guangdong Academy of Forestry,Guangzhou 510520, The People’s Republic of China

e-mail:hudehuo@sinogaf.cn

National Engineering Laboratory for Tree Breeding,Beijing Forestry University,Beijing 100083,The People’s Republic of China