SSR genetic linkage map construction of pea (Pisum sativum L.) based on Chinese native varieties

Xuelian Sun,Tao Yang,Junjie Hao,Xiaoyan Zhang,Rebea For,Junye Jiang,Fang Wang,Jianping Guan,Xuxiao Zong,*

aInstitute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing,100081,China

bNational Key Facility for Crop Gene Resources and Genetic Improvement,Chinese Academy of Agricultural Sciences,Beijing,100081,China

cQingdao Academy of Agricultural Sciences,Qingdao 266100,China

dDepartment of Agriculture and Food Systems,Melbourne School of Land and Environment,The University of Melbourne,Melbourne,Victoria 3010,Australia

1.Introduction

Field pea (Pisum sativum L.) is the fourth largest legume crop globally,with 97 and 87 countries growing dry pea and green pea,respectively,in 2011 [1].China,where pea has been cultivated for more than 2000 years,has remained the largest global green pea and third largest dry pea producer over the last decade.The crop plays an important role in sustainable agricultural systems[2].

Although progress has been made by conventional breeding for agronomically desirable traits such as seed shape,size and other quality traits[3],the large(～4 × 109bp)and somewhat complex genome structure [4] of pea has imposed limitations.However,the use of molecular approaches provides the necessary tools for accurate and rapid selection of more complex quantitatively inherited traits,such as disease resistance,tolerance to abiotic stresses,and yield.At least 16 genetic maps have been constructed with different kinds of markers,including morphological markers,isozymes,RFLP,RAPD,SSR,EST-based,PCR-based,and markers from highthroughput parallel genotyping [5–20].These maps were not based on Chinese germplasm,which is very different from that in other areas.

Past molecular assessment of the Chinese pea population structure,and its comparison with the global pea core collection,has clearly shown the genetic uniqueness of the species both within China as a whole and among the pea growing regions of China.This uniqueness is reflected not only by a diverse allelic variation at the SSR loci assessed but also by many examples of non-transferability of flanking primers (null alleles) [21].To develop a reliable and robust genetic map of elite and unique Chinese breeding germplasm,a novel set of SSR markers is required.

The aims of this study were to 1)isolate and characterize a novel set of Chinese pea-derived SSR loci and 2) construct a dense genomic map for subsequent use in marker-assisted breeding.

2.Materials and methods

2.1.Germplasm and population development

The female parent G0003973 (winter hardy) was crossed to the male parent G0005527 (cold sensitive).The dry seed color of G0003973 was olivine and that of G0005527 was green.The segregating F2population comprised 190 individuals.Both F1and F2populations were grown in a protected field at Qingdao Academy of Agricultural Sciences,Qingdao,Shandong,China.

2.2.SSR markers developed

A total of 6287 SSR markers were developed from flanking primer sequences isolated from 12 accessions (G0005527,G0004462,G0003462,G000145,G000391,G0005389,G0005669,G0004847,G0005039,G0005763,G0002915,and X9002) at the Chinese Academy of Agricultural Sciences,Beijing,China via the magnetic beads enrichment method following Yang et al.[22].Genomic DNA was sheared into 500 to 800 bp fragments.The probes containing p(GA)10,p(AC)10,p(AAT)8,p(AAC)8,p(AAG)8,p(ATGT)6,p(GATA)6,and p(AAAT)6were hybridized with the genomic DNA fragments.Then magnetic beads were used to isolate the positive DNA fragments with selected motifs,followed by washing and elution.The positive DNA fragments were constructed as a library for 454 sequencing with GS-FLX Titanium reagents at Beijing Autolab Biotechnology Co.,Ltd(China).

2.3.Existing marker transfer

A total of 2166 SSR loci were assessed for transferability to the Chinese germplasm,comprising 953 EST-SSRs developed from faba bean(Vicia faba L.),pea(Pisum sativum L.),grass pea(Lathyrus sativus L.)or lupin(Lupinus albus)and retrieved from NCBI EST databases [23,24],115 pea SSR sequences sourced from Gong et al.[25]and Kwon et al.[26],and 906 pea and 192 faba bean SSR sequences that we developed using the transcriptome sequencing data of Kaur et al.[27].

2.4.Marker optimization

Genomic DNA was extracted from young leaves of field-grown plants by the improved CTAB method of Liu et al.[28].PCR amplification using flanking SSR loci sequences was performed in 10 μL reaction volumes containing 50 ng genomic DNA,1 μL of 10×buffer,0.2 μL of dNTP(10 mmol L-1each),1 μL of each primer(2 μmol L-1),0.4 U Taq DNA polymerase.PCR reagents were supplied by Dingguo Changsheng Biotechnology Ltd.,Beijing,China.Amplifications were performed in an EDC-810 Heijinggang Thermal Cycler (Beijing,China),using the following program:an initial denaturation at 95 °C for 5 min,followed by 35 cycles of denaturation at 95 °C for 30 s,annealing at an appropriate temperature specific to the primer pair for 45 s,and an extension at 72 °C for 45 s,and a final elongation at 72 °C for 10 min.The PCR products were separated on 8% non-denaturing polyacrylamide gel electrophoresed under 280 V and 50 W and visualized by 0.1%silver nitrate staining.

2.5.Linkage map construction

Chi-squared analysis (P = 0.05) was applied to test the distorted segregation of the markers against the expected Mendelian segregation ratio by QTL ICIMapping V3.2 software [29].The SSR marker states were encoded according to Map Manager QTXb 20[30],whereby the male parent allele was encoded as“A”and the female parent allele as“B”.For the F2population,the same male allele was encoded as “A” and the same female allele as “B”,“H” was recorded when a locus was heterozygous,and“-”when there was a missing or null allele.The linkage map was constructed using the Kosambi function(P = 0.0001)in Map Manager QTXb 20,with marker distances in centiMorgans (cM),and presented using JoinMap 4.0[31].

3.Results

3.1.SSR marker screening

Of the total of 8453 SSRs developed,4342 yielded amplification products.From these SSRs we selected 815 pairs of primers for polymorphism screening.The polymorphism ratios of G0003973 × G0005527 were 15.8%for the magnetic bead enrichment method,26.0% from pea EST-SSR markers deposited in the NCBI EST database,68.3% from faba bean EST-SSR markers developed by Ma et al.[23],26.2% from grass pea EST-SSR markers developed by Sun et al.[24],27.7% from lupin EST-SSR markers developed by screening the NCBI EST database,34.0% and 6.8% by designing primers from Kaur's pea and faba bean transcription sequencing data[27],respectively,and 76.3%from Gu et al.[32].Among the 815 SSR markers,567 pairs of markers were eliminated owing to indistinct bands,missing bands,or absence of target bands.Finally,248 pairs of SSR markers were subjected to χ2testing for linkage map construction.

Fig.1-Genetic linkage map of the G0003973 × G0005527 F2 population.

3.2.Genetic map construction

Of 248 polymorphic markers,50(34 genomic SSRs and 16 ESTSSRs) showed significant segregation distortion (P = 0.05)including 23 biased toward the female parent,9 biased toward the male parent,and 18 biased toward the heterozygote.These distorted markers were excluded from linkage map construction.

After application of the Kosambi function in Map Manager QTXb 20 (P = 0.0001),41 markers could not be placed in any linkage group.As a result,the map based on F2genotyping data contained 157 SSR markers,including 52 genomic and 93 EST-SSR markers from pea,8 EST-SSRs from grass pea,and 4 EST-SSR-derived markers from faba bean(Table S1).The map contained 11 linkage groups with an average genetic distance of 9.7 cM between neighboring markers and covered 1518 cM(Kosambi) (Fig.1).Each linkage group contained from 5 to 31 markers,with a length ranging from 12.8 to 335.1 cM.

Thirteen anchor markers were used in an attempt to reference our linkage groups to published consensus maps.However,only AF016458 (LG I),PSAD147 (LG I),PsAS2 (LG I),PD23 (LG II),and PSAB60 (LG VII) were finally used as anchor loci (Table 1).

4.Discussion

Although diploid pea has 14 chromosomes,many genetic linkage maps including the one constructed in this study contain more than seven linkage groups [7,33,34].This result is most likely due to the large genome size and the insufficient number of markers for complete coverage.This deficiency leads to gaps too large for statistical linkage between markers that may in fact be linked.Increasing the number of loci and using a larger mapping population will likely improve map resolution further.

Although the map in this study represents a largely novel genome background,it can be aligned with existing maps produced using non-Chinese material via a set of shared anchor markers [20,26,32,35].PEACPLHPPS and PS11824 were common markers between this study and a previous study[26],but could not be anchored on a specific chromosome.Other markers,AF016458 (LG I),PSAD147 (LG I),PsAS2 (LG I),PD23 (LG II),and PSAB60 (LG VII) were used as anchor loci on our linkage map.These are more important markers than the others because they are bridges between our map and those from the pea research community.

The linkage map reported here is the first map constructed purely with SSR markers and based on the Chinese pea germplasm,with conserved order with RIL-derived maps[35].This map may facilitate marker-assisted breeding of pea in the future.

Table 1-The distribution of anchor SSR markers on the linkage groups in previous linkage maps.

This study was supported by the International Cooperation projects (2010DFB33340 and 2010DFR30620),and the National Key Technology R&D Program of China from the Ministry of Science and Technology of China (2013BAD01B03-18),the National Natural Science Foundation of China (31371695),and also supported by the Agricultural Science and Technology Innovation Program(ASTIP)in CAAS.

Supplementary material

Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.cj.2014.03.004.Table S1-SSR markers used to construct a linkage map of pea.

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