Abstract

Linkage maps have become important tools for genetic studies. With the aim of evaluating the SRAP (sequence-related amplified polymorphism) technique for linkage mapping in Pisum sativum L., a F₂ mapping population derived from an initial cross between cvs. DDR11 and Zav25 were generated. A total of 25 SRAP primer combinations were evaluated in 45 F₂ plants and both parental lines, generating 208 polymorphic bands/markers. The markers were analyzed by the chi-square goodness-of-fit test to check the expected Mendelian segregation ratio. The resulting linkage map consists of 112 genetic markers distributed in 7 linkage groups (LGs), covering a total of 528.8 cM. The length of the LGs ranged from 47.6 to 144.3 cM (mean 75.54 cM), with 9 to 34 markers. The linkage map developed in this study indicates that the SRAP marker system could be applied to mapping studies of pea.

Key words:
Pea; plant breeding; linkage map; F₂ population; molecular markers

INTRODUCTION

Pea (Pisum sativum L.) is an autogamous, annual cool-season legume originated from areas in the Middle East, in the East of the Caucasus, Iran and Afghanistan, and West of the Mediterranean basin (Smýkal et al. 2011Smýkal P, Kenicer G, Flavell AJ, Corander J, Kosterin O, Redden RJ, Ford R, Coyne CJ, Maxted N, Ambrose MJ and Ellis NTH (2011) Phylogeny, phylogeography and genetic diversity of the Pisum genus. Plant Genetic Resources: Characterization and Utilization 9: 4-18.). Its genome is organized in seven chromosome pairs (2n = 2x = 14), and the haploid size estimated at 4.45 Gb (Smýkal et al. 2012Smýkal P, Aubert G, Burstin J, Coyne CJ, Ellis NTH, Flavell AJ, Ford R, Hýbl M, Macas J, Neumann P, McPhee KE, Redden RJ, Rubiales D, Weller JL and Warkentin TD (2012) Pea (Pisum sativum L.) in the genomic era. Agronomy 2: 74-115.). Peas were an important source of animal and human food for many centuries. The species is rich in protein, slowly digestible starch, soluble sugars, fiber, minerals, and vitamins (Dahl et al. 2012Dahl W, Foster L and Tyler R (2012) Review of the health benefits of peas (Pisum sativum L.) British Journal of Nutrition 108: 3-10. ). The global dry pea production averages 10 million tonnes a year. Argentina is one of the top exporting countries, eighth in the global ranking between 2008 and 2011 (FAO 2012FAO (2012) The Statistics Division of the FAO. Food and Agriculture Organization of the United Nations. Available at <Available at http://faostat.fao.org/site/567/default.aspx#ancor. > Accessed on June 6, 2014.
http://faostat.fao.org/site/567/default.... ). The rising world population will require increased crop production. Moreover, some researchers suggest that the current rate of increase in crop yields will not be enough to meet this demand (Tester and Langridge 2010Tester M and Langridge P (2010) Breeding technologies to increase crop production in a changing world. Science 327: 818-822.). Therefore, plant breeding programs are needed to further raise crop yields. In this context, linkage mapping will be useful to maximize the success probability. Genetic linkage maps are powerful tools for genetic research and breeding of plants. The linkage maps are the first step in: 1) the analysis of qualitative and quantitative traits; 2) the introgression of desirable genes and quantitative trait loci (QTLs); 3) positional or map-based cloning of genes responsible for economically important traits (Semagn et al. 2006Semagn K, Bjornstad A and Ndjiondjop MN (2006) Principles, requirements and prospects of genetic mapping in plants. Africal Journal of Biotechnology 5: 2569-2587.). Different kinds of markers, such as simple sequence repeats (SSR; Loridon et al. 2005Loridon K, McPhee K, Morin J, Dubreuil P, Pilet-Nayel ML, Aubert G, Rameau C, Baranger A, Coyne C, Lejeune-He`naut I and Burstin J (2005) Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.). Theoretical and Applied Genetics 111: 1022-1031.), single nucleotide polymorphisms (SNP; Deulvot et al. 2010Deulvot C, Charrel H, Marty A, Jacquin F, Donnadieu C, Lejeune-Hénaut I, Burstin J and Aubert G (2010) Highly-multiplexed SNP genotyping for genetic mapping and germplasm diversity studies in pea. BMC Genomics 11: 468-478.), inter simple sequence repeats (ISSR; Mishra et al. 2009Mishra RK, Kumar A, Chaudhary S and Kumar S (2009) Mapping of the multifoliate pinna (mfp) leaf-blade morphology mutation in grain pea Pisum sativum. Journal of Genetics 88: 227-232.), and sequence tagged sites (STS; Barilli et al. 2010Barilli E, Satovic Z, Rubiales D and Torres AM (2010) Mapping of quantitative trait loci controlling partial resistance against rust incited by Uromyces pisi (Pers.) Wint. in a Pisum fulvum L. intraspecific cross. Euphytica 175: 151-159.) have been used to develop moderate density linkage maps in pea. Several markers were common in different maps corresponding to different crosses; this allowed an integration of these maps (Loridon et al. 2005Loridon K, McPhee K, Morin J, Dubreuil P, Pilet-Nayel ML, Aubert G, Rameau C, Baranger A, Coyne C, Lejeune-He`naut I and Burstin J (2005) Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.). Theoretical and Applied Genetics 111: 1022-1031., Aubert et al. 2006Aubert G, Morin J, Jacquin F, Loridon K, Quillet MC, Petit A, Rameau C, Lejeune-Hénaut I, Huguet T and Burstin J (2006) Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula. Theoretical and Applied Genetics 112: 1024-1041.), as well as the development of consensus linkage maps for the species (Weeden et al. 1998Weeden NF, Ellis THN, Timmerman-Vaughan GM, Swiecicki WK, Rozov SM and Berdnikov VA (1998) A consensus linkage map for Pisum sativum. Journal of Heredity 83: 123-129., Bordat et al. 2011Bordat A, Savois V, Nicolas M, Salse J, Chauveau A, Burgeois M, Potier J, Houtin H, Rond C, Murat F, Marget P, Aubert G and Burstin J (2011) Translational genomics in legumes allowed Placing in silico 5460 unigenes on the pea functional map and identified candidate genes in Pisum sativum L. G3: Genes, Genomes, Genetics 1: 93-103. ).

In this study, we proposed the use of the sequence-related amplified polymorphism (SRAP) technique (Li and Quiros 2001Li G and Quiros CF (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics 103: 455-461.) to generate a number of markers distributed across all pea chromosomes. Since its development, SRAP has been employed in a wide range of plant species for genetic diversity estimation (Cravero et al. 2007Cravero VP, Martin EA and Cointry EL (2007) Genetic diversity in Cynara Cardunculus determined by sequence-related amplified polymorphism markers. Journal of the American Society for Horticultural Science 132: 208-212., Espósito et al. 2007Espósito MA, Martin EA, Cravero VP and Cointry EL (2007) Characterization of pea accessions by SRAP's markers. Scientia Horticulturae 113: 329-335., Aneja et al. 2012Aneja B, Yadav NR, Chawla V and Yadav RC (2012) Sequence-related amplified polymorphism (SRAP) molecular marker system and its applications in crop improvement. Molecular Breeding 30: 1635-1648. ), gene tagging (Martin et al. 2008Martin E, Cravero V, Espósito M, López Anido F, Milanesi L and Cointry E (2008) Identification of markers linked to agronomic traits in globe artichoke. Australian Journal of Crop Science 1: 43-46., Zhang et al. 2010Zhang W, He H, Guan Y, Du H, Yuan L, Li Z, Yao D, Pan J and Cai R (2010) Identification and mapping of molecular markers linked to the tuberculate fruit gene in the cucumber (Cucumis sativus L.) Theoretical and Applied Genetics 120: 645-654. ), and map construction (Lin et al. 2005Lin Z, He D, Zhang X, Nie Y, Guo X and Feng C (2005) Linkage map construction and mapping QTL for cotton fibre quality using SRAP, SSR and RAPD. Plant Breeding 124: 180-187. , Sun et al. 2007Sun Z, Wang Z, Tu J, Zhang J, Yu F, McVetty P and Li G (2007) An ultradense genetic recombination map for Brassica napus, consisting of 13551 SRAP markers. Theoretical and Applied Genetics 114: 1305-1317., Wang et al. 2008Wang J, Yao J and Li W (2008) Construction of a molecular map for melon (Cucumis melo L.) based on SRAP. Frontiers of Agriculture in China 2: 451-455., Martin et al. 2013Martin E, Cravero V, Portis E, Scaglione D, Acquaviva E and Cointry E (2013) New genetic maps for globe artichoke and wild cardoon and their alignment with an SSR-based consensus map. Molecular Breeding 32: 177-187 ). The aim of the current study was to evaluate the usefulness of SRAP markers in the development of a genetic linkage map of Pisum sativum L.

MATERIAL AND METHODS

Plant material

The F₂ mapping population was derived from an initial cross between the cvs. DDR11 and Zav25. The latter is an experimental line obtained from the IICAR-CONICET breeding program (Espósito et al. 2007Espósito MA, Martin EA, Cravero VP and Cointry EL (2007) Characterization of pea accessions by SRAP's markers. Scientia Horticulturae 113: 329-335.). For most yield-related traits, such as number of pods and seeds per plot, the values of 'DDR11' are lower than those of 'Zav25'.

Both parents and 45 plants of the F₂ population were sown in an experimental field of Universidad Nacional de Rosario (lat 33° 1' S, long 60° 53' W) in the winter of 2012, in a completely randomized design (inter-row spacing 70 cm, plant spacing 10 cm).

DNA extraction

The genomic DNA of each F₂ plant and both parents was isolated from fresh leaves by the CTAB method described by Doyle and Doyle (1990Doyle JJ and Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12: 149-151.), with the following modifications: after DNA precipitation, the samples were stored at -20 °C for 30 min, the two washes of the final step were performed with ethanol 70%, and the resulting pellet was resuspended in distilled water.

After RNAase-treatment, each DNA sample was quantified using agarose gel electrophoresis (1% w/v) and comparison of band intensity with the standard λ DNA (76 ng μL^-1). The hybrid origin of each plant was checked using two microsatellite markers SSR: PSMPSAA135 and PSMPSAA205 (Tar'an et al. 2005Tar'an B, Zhang C, Warkentin T, Tullu A and Vandenberg A (2005) Genetic diversity among varieties and wild species accessions of pea (Pisum sativum L.) based on molecular markers, and morphological and physiological characters. Genome 48: 257-272.), which were contrasting in both parental lines.

SRAP genotypic analysis

The F₂ population was genotyped using 25 SRAP primer combinations generated from five forward and five reverse primers developed by Li and Quiros (2001Li G and Quiros CF (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics 103: 455-461.) (Table 1). The primers are 17-18 bases long and have a core sequence, which includes 10-11 non-specific bases at the 5´end and sequence CCGG in the forward and AATT in the reverse primer. The core sequence is followed by three selective nucleotides at the 3´ end of each primer. The primers were selected based on the results of Espósito et al. (2007Espósito MA, Martin EA, Cravero VP and Cointry EL (2007) Characterization of pea accessions by SRAP's markers. Scientia Horticulturae 113: 329-335.) in a characterization of pea accessions with this type of molecular markers.

Polymerase chain reactions were carried out in a final volume of 20 μL containing 15 ng genomic DNA, 0.2 mM dNTPs, 1.5 mM MgCl₂, 0.5 μM of each primer, 1X Taq buffer (Invitrogen, California, USA), and 1 U of Taq recombinant polymerase (Invitrogen). Samples were subjected to the following thermal profile: 5 min denaturing at 94 °C and five cycles of three steps: 1 min denaturing at 94 °C, 1 min annealing at 35 °C, and 1 min elongation at 72 °C; for the following 35 cycles, annealing temperature was elevated to 50 °C with a final elongation step of 10 min at 72 °C.

The resulting amplicons were separated on 6% (w/v) denaturing polyacrylamide gels and then visualized by silver staining (Bassam et al. 1991Bassam BJ, Caetanoanolles G and Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry 196: 80-83.). The SRAP fragments were treated as dominant markers. Each marker was labeled according to the primer combination used for its generation plus the estimated amplicon size.

Linkage analysis and linkage map construction

Linkage analyses were performed using JoinMap v4 (van Ooijen 2006van Ooijen JW (2006) Software for the calculation of genetic linkage maps in experimental populations. Demo Version. Kyazma B.V., Wageningen. Available at <https://www.kyazma.nl/index.php/JoinMap/Evaluate/> Accessed in June 2015.
https://www.kyazma.nl/index.php/JoinMap/... ). Each segregating marker was tested for deviations from the expected 3:1 segregation ratio using Chi-square tests. Markers with Mendelian segregation ( χ 2 ≤ χ α=0.1 2 ) or with minor distortion ( χ 𝛼=0.1 2 < χ 2 ≤ χ 𝛼=0.01 2 ) were used for the construction of the linkage map. Markers with highly distorted segregation ( χ 2 > χ 𝛼=0.01 2 ) were included in a second step of mapping only when their presence did not affect the local marker order. Linkage groups (LGs) were established at a minimum LOD (logarithm of odds) value of 3.0. The marker order for each LG was determined at LOD = 1.0, REC = 0.40 and Jump = 5. Recombination values were converted to genetic distances using the Kosambi (1994) mapping function. Linkage groups were numbered sequentially according to their length in cM. Linkage maps were drawn using Map Chart 2.2 software (Voorrips 2002Voorrips RE (2002) MapChart: Software for the graphical presentation of linkage maps and QTLs. The Journal of Heredity 93: 77-78.).

RESULTS AND DISCUSSION

Since the development of molecular marker techniques, the number of marker loci identified on genetic maps is increasing at a high rate. The SRAP marker system designed by Li and Quiros (2001Li G and Quiros CF (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics 103: 455-461.) is a simple and efficient technique. It has several advantages over other molecular markers, namely its simplicity and reasonable throughput rate. It also allows easy isolation of bands for sequencing and, most importantly, it targets ORFs (open reading frames). Elsewhere, SRAPs were established as a powerful tool for construction of genetic linkage maps, e.g., of Brassica (Li and Quiros 2001Li G and Quiros CF (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics 103: 455-461., Sun et al. 2007Sun Z, Wang Z, Tu J, Zhang J, Yu F, McVetty P and Li G (2007) An ultradense genetic recombination map for Brassica napus, consisting of 13551 SRAP markers. Theoretical and Applied Genetics 114: 1305-1317.), Gossypium (Lin et al. 2005Lin Z, He D, Zhang X, Nie Y, Guo X and Feng C (2005) Linkage map construction and mapping QTL for cotton fibre quality using SRAP, SSR and RAPD. Plant Breeding 124: 180-187. , Yu et al. 2007Yu J, Yu S, Lu C, Wang W, Fan S, Song M, Lin Z, Zhang J and Zhang X (2007) High-density linkage map of cultivated allotetraploid cotton based on SSR, TRAP, SRAP and AFLP markers. Journal of Integrative Plant Biology 49: 716-724.), Cucumis melo L. (Wang et al. 2008Wang J, Yao J and Li W (2008) Construction of a molecular map for melon (Cucumis melo L.) based on SRAP. Frontiers of Agriculture in China 2: 451-455.), and more recently of Cynara cardunculus (Martin et al. 2013Martin E, Cravero V, Portis E, Scaglione D, Acquaviva E and Cointry E (2013) New genetic maps for globe artichoke and wild cardoon and their alignment with an SSR-based consensus map. Molecular Breeding 32: 177-187 ). Our study presents the first application of SRAP markers for the construction of a linkage map of P. sativum L.

The parental lines to develop the mapping population ('DDR11' and 'Zav25') were selected based on observations of Espósito et al. (2007Espósito MA, Martin EA, Cravero VP and Cointry EL (2007) Characterization of pea accessions by SRAP's markers. Scientia Horticulturae 113: 329-335.), who reported that these lines are divergent at the morphological (Euclidean distance = 0.47) and molecular levels (Dice distance = 0.66) and that they were grouped separately by hierarchical cluster analysis.

A total of 25 SRAP primer combinations were used of which 23 pairs were amplified. Most combinations produced clear bands without overlapping, but in some cases the scoring of the markers was somewhat cumbersome, because of the high number of bands and their different intensities, and the presence of minor bands in some plants. The number of fragments amplified by each primer combination ranged from 3 (Me1-Em1; Me1-Em4) to 24 (Me3-Em2), with an average of 9.96. A total of 208 polymorphic bands (PB) were generated. The most polymorphic primer combination was Me2-Em3, with 18 PB (Table 2).

The markers proved efficient for genetic studies in pea, producing an average of 8.32 PB/primer combination. This value is similar to those of the other species, where 3 to 14 bands per primer combination were reported (Li and Quiros 2001Li G and Quiros CF (2001) Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics 103: 455-461., Lin et al. 2003Lin Z, Zhang X, Nie Y, He D and Wu M (2003) Construction of a genetic linkage map for cotton based on SRAP. Chinese Science Bulletin 48: 2064-2068., Sun et al. 2007Sun Z, Wang Z, Tu J, Zhang J, Yu F, McVetty P and Li G (2007) An ultradense genetic recombination map for Brassica napus, consisting of 13551 SRAP markers. Theoretical and Applied Genetics 114: 1305-1317., Yu et al. 2007Yu J, Yu S, Lu C, Wang W, Fan S, Song M, Lin Z, Zhang J and Zhang X (2007) High-density linkage map of cultivated allotetraploid cotton based on SSR, TRAP, SRAP and AFLP markers. Journal of Integrative Plant Biology 49: 716-724., Wang et al. 2008Wang J, Yao J and Li W (2008) Construction of a molecular map for melon (Cucumis melo L.) based on SRAP. Frontiers of Agriculture in China 2: 451-455., Martin et al. 2013Martin E, Cravero V, Portis E, Scaglione D, Acquaviva E and Cointry E (2013) New genetic maps for globe artichoke and wild cardoon and their alignment with an SSR-based consensus map. Molecular Breeding 32: 177-187 ). Since one primer combination may detect a high number of polymorphic loci, this technique can be used to construct ultra-dense genetic maps. Furthermore, SRAP markers can be combined with next-generation techniques to enhance their capacity and effectiveness. Li et al. (2011Li W, Zhang, J, Mou Y, Geng J, McVetty P, Hu S and Li G (2011) Integration of Solexa sequences on an ultradense genetic map in Brassica rapa L. BMC Genomics 12: 249-263) combined SRAP with Illumina/Solexa sequencing to directly integrate genetic loci in the B. rapa genetic map based on paired-end Solexa sequencing. Results of the SRAP technique obtained in this way may prove invaluable for QTL analysis and map-based cloning.

Chi-square analysis of the 208 loci revealed that 116 loci (~ 55.8%) were consistent with the expected Mendelian 3:1 segregation ratio ( χ 2 ≤ χ 𝛼=0.1 2 ), the distortion of 37 loci (~ 17.8%) was minor ( χ 𝛼=0.1 2 < χ 2 ≤ χ 𝛼=0.01 2 ) and 55 loci (~ 26.4%) were highly distorted ( χ 2 > χ 𝛼=0.01 2 ).

Initially, the 153 markers with Mendelian or slightly distorted segregation were used for the construction of linkage groups (LG), using a minimum LOD value of 3.0 and the mapping parameters Rec = 0.40, LOD = 1.0, and Jump = 5. Under these conditions, an initial framework map with 62 loci was constructed. Then the framework order was fixed and a second round of mapping performed, including markers with distorted segregation. These markers were only included in the final map if their presence did not alter the surrounding marker order in a given linkage group. By this step, we incorporated 29 Mendelian markers and 21 distorted markers in the previously established linkage groups. This strategy ensures the accuracy and a high coverage of the final map. Similar strategies were successfully used for linkage mapping in different species, e g., of olive (Khadari et al. 2010Khadari B, El Aabidine AZ, Grout C, Sadok IB, Doligez A, Moutier N, Santoni S and Costes E (2010) A genetic linkage map of olive based on amplified fragment length polymorphism, intersimple sequence repeat and simple sequence repeat markers. Journal of the American Society for Horticultural Science 135: 548-555.), globe artichoke and cardoon (Martin et al. 2013Martin E, Cravero V, Portis E, Scaglione D, Acquaviva E and Cointry E (2013) New genetic maps for globe artichoke and wild cardoon and their alignment with an SSR-based consensus map. Molecular Breeding 32: 177-187 ), lentil (Verma et al. 2015Verma P, Goyal R, Chahota RK, Sharma TR, Abdin MZ and Bhatia S (2015) Construction of a genetic linkage map and identification of QTLs for seed weight and seed size traits in lentil (Lens culinaris Medik.). PLoS ONE 10.1:1-17.), poplar (Zhou et al. 2015Zhou W, Tang Z, Hou J, Hu N and Yin T (2015) Genetic map construction and detection of genetic loci underlying segregation distortion in an intraspecific cross of Populus deltoides. PLoS ONE 10 . 11-13), and wheat (Li et al. 2015Li C, Bai G, Chao S and Wang Z (2015) A high-density SNP and SSR consensus map reveals segregation distortion regions in wheat. . BioMed Research International 2015: 13 Article ID 830618, 10 pages.).

The resulting map comprises 112 loci distributed over seven linkage groups (LGs), which is equal to the haploid number of chromosomes in the pea genome (Figure 1). The overall map length was 528.8 cM and the mean inter-marker distance 4.72 cM. The LG length varied from 47.6 cM to 144.3 cM. The number of markers included in each LG ranged from 9 to 34 (Table 3). A total of 21 distorted markers (19%) were included in the genetic map.

Although several linkage maps for pea have been developed using different kinds of markers, this is the first linkage map of this species constructed with SRAP markers. The length of our map (528.8 cM) is shorter than that of previous ones generated with other molecular markers, which covered 1430, 1458, and 1283 cM, respectively (Loridon et al. 2005Loridon K, McPhee K, Morin J, Dubreuil P, Pilet-Nayel ML, Aubert G, Rameau C, Baranger A, Coyne C, Lejeune-He`naut I and Burstin J (2005) Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.). Theoretical and Applied Genetics 111: 1022-1031., Aubert et al. 2006Aubert G, Morin J, Jacquin F, Loridon K, Quillet MC, Petit A, Rameau C, Lejeune-Hénaut I, Huguet T and Burstin J (2006) Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula. Theoretical and Applied Genetics 112: 1024-1041., Barilli et al. 2010Barilli E, Satovic Z, Rubiales D and Torres AM (2010) Mapping of quantitative trait loci controlling partial resistance against rust incited by Uromyces pisi (Pers.) Wint. in a Pisum fulvum L. intraspecific cross. Euphytica 175: 151-159.). All these genetic maps were constructed using mapping populations with different sizes, derived from different crosses. Both, the size of the mapping population and their origin affect the marker coverage and map length because an increasing divergence between parents generates a greater number of possible recombinations and the possibility of finding recombinant plants is higher when populations are large. Then, the small size of our F₂ population (45 plants) could be the cause of the smaller size of the map obtained in this study (Ferreira et al. 2006Ferreira A, Flores da Silva M, Silva LC and Cruz CD (2006) Estimating the effects of population size and type on the accuracy of genetic maps. Genetics and Molecular Biology 29: 187-192. ). To enhance accuracy and reduce the statistical error, a great number of plants should be evaluated. On the other hand, the large number of unlinked markers with Mendelian or slightly distorted segregation (62) reflects the need to enrich this map with additional markers to cover the entire genome.

CONCLUSIONS

The linkage map generated in this study provided basic information for assistance of future molecular marker application in the local breeding program of Pisum sativum L. Since pea has no reference genome, molecular markers that do not require sequence information must be evaluated. In this context, sequence-related amplified polymorphism (SRAP) represents an efficient tool for genetic analysis of pea even though the proposed linkage map was only partly saturated. For the first time, SRAP markers were applied in this study to develop a linkage map of pea. Moreover, since these markers target coding regions of the genome, they can potentially identify markers with inherent biological significance. Additional markers are required to expand the coverage of this map for QTL analysis. The segregating population used to develop this linkage map is currently being phenotyped for yield-related traits to detect QTLs associated to this character.

ACKNOWLEDGEMENTS

This research was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and Fondo para la Investigación Científica y Tecnológica (FONCyT, Argentina).

REFERENCES

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