Simple Sequence Repeats as Reference Points in Cucumis Mapping

Y. Danin-Poleg, N. Reis, G. Tzuri, and N. Katzir

Department of Vegetable Crops, Agricultural Research Organization, Newe Ya'ar Research Center, P.O.B. 1021, Ramat Yishay 30095, Israel

Abstract. Mapping projects of melon (Cucumis melo) and cucumber (C. sativus) that are currently being conducted at different laboratories are all directed at mapping economically important traits. The present study examines the hypothesis that Simple Sequence Repeats (SSR, also termed microsatellite) are useful markers for mapping and for map merging purposes. Thirty polymorphic SSRs, representing 27 loci, were tested for polymorphism between the parental lines of four melon mapping populations. Eighteen SSRs were polymorphic between 'Védrantais' and 'Songwhan Charmi', the parental lines of the first melon map. Three other populations shared 12, 13, and 17 SSR loci with the above map. Nine SSR loci were common to all four biparental populations, enabling the merging of five linkage groups in all melon maps. In addition, 12 of the 30 SSRs tested were polymorphic between GY14 and PI 183967, the parental lines of a cucumber mapping population, sharing six loci with the melon map described above. Our results indicate that SSRs are most useful points of reference for map merging in melon, as well as for comparative mapping of melon and cucumber.

This research is supported by grant no. 255-0436-96 from the Chief Scientist of the Israel Ministry of Agriculture, and by grant no. IS-2708-96 from the US-Israel Binational Agricultural Research and Development (BARD) Fund. Contribution from the Agricultural Research Organization, Newe Ya'ar Research Center, Israel, No. 132/98. E-mail: geneweya@netvision.net.il.

 

Siimple sequence repeats (SSR, also termed Microsatellite) consist of a variable number of tandem repeats, of 1 to 5 nucleotides, forming highly informative, locus-specific genetic markers. SSRs are abundant and evenly dispersed throughout the human, other mammalian and plant genomes. They can be analyzed efficiently by the polymerase chain reaction (PCR), using primers specific to their flanking regions. Variation in PCR product length is a function of the number of SSR units. SSRs were recommended as standard markers to be used in the preparation of highly saturated genetic maps of any eukaryote genome (Beckmann and Soller, 1990). They were applied for mapping in a large number of plant species including Arabidopsis, soybean, maize and rice (Bell and Ecker, 1994; Akkaya et al., 1995; Senior and Heun, 1993; Wu and Tanksley, 1993, respectively). The high level of polymorphism associated with SSR in melon (Cucumis melo) and cucumber (C. sativus) was demonstrated in a previous study, as was the cross homology of SSR

between the two species (Katzir et al. 1995, 1996; Danin-Poleg et al., 1996). In addition, the application of Cucumis SSR markers for mapping has been demonstrated (Katzir et al., 1996, 1997).

The existing map of melon (Baudracco-Arnas and Pitrat, 1996) was recently expanded by the integration of 18 SSRs, five RFLP and two RAPD markers. This yielded a genetic map of 13 linkage groups with a total length of 1747 cM (LOD 3) (Danin-Poleg et al., unpublished data). The previous map included 110 loci in 14 linkage group with a total length of 1390 cM (LOD 5). Thus, the addition of these markers increased the defined map length by almost 26%. The new map covers 54% to 77% of the melon genome according to the estimate of between 2276 cM and 3250 cM (Baudracco-Arnas and Pitrat, 1996). The average interval between two loci was 15.5 cM, compared with the previous 17.7 cM. Pitrat (1994) summarized the information in melon, based on classical genetics. This provided 13 linkage groups of 28 traits, including several economically important genes. Only four of these linkage groups are currently related to the molecular map (Baudracco-Arnas and Pitrat, 1996). Additional mapping projects will enable the integration of the information of the two maps.

 

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Recently, an AFLP-based map was constructed for melon, using 197 AFLPs, six RAPDs, one SSR and one disease resistance marker. The map covers 1942 cM with 14 major and six minor linkage groups (Wang et al., 1997). Further mapping projects are being currently conducted (e.g., Katzir et al., 1996; Oliver et al., 1998) with the aim of saturating the melon map and integrating additional agriculturally and economically important traits. At present, the number of markers scored for each of these maps is insufficient. There are no common markers scored among the different populations, to be used as anchor points for map merging.

This study describes the potential of SSR as anchor markers for merging the different melon maps. In addition, their potential role in comparative mapping with cucumber is discussed. The integration of available maps within melon, and between melon and cucumber, will accelerate mapping of both crops, and provide information on their genome organization and evolutionary relationships.

Material and methods

Plant material. The parental lines of four melon and one cucumber mapping populations were tested for SSR polymorphism. The melon genotypes were 'Védrantais' and 'Songwhan Charmi' (PI 161375, Baudracco-Arnas and Pitrat, 1996), obtained from M. Pitrat, INRA, Montfavet, France; PI 414723 and 'Dulce' (Danin-Poleg et al., 1997); MR-1 and 'Ananas Yoqne'am' (Wang et al., 1997), obtained from R. A. Dean (Clemson Univ., Clemson, S.C.); and 'Piel de Sapo'. The cucumber genotypes were GY14 and PI 183967 (Kennard et al., 1994), obtained from J.E. Staub (USDA­ARS, Univ. Wisconsin, Madison, Wis.).

ssr markers. The 30 SSR markers used in this study are presented in Table 1. Twenty-five SSRs were developed from a DNA library of 'Noy Yizre'el', one SSR was derived from a cucumber cDNA library (obtained from R. Perl-Treves, Bar Ilan U., Ramat Gan, Israel) and four were derived from EMBL database sequences (as described in Katzir et al. 1996).

Microsatellite loci amplification. Microsatellite analysis consisted of the following steps. a)

PCR reaction mixtures for separation of SSR on a sequencing gel (radiolabeled) contained: 60 ng of plant genomic DNA, 1 mm of Mg2+, 8 to 10 pmol of 3' and 5' primers, 166 mm of dATP, dTTP, dGTP, 2 mm of dCTP, 0.1 mL of 3000 Ci/mmol [a-33P] dCTP, 1x Taq Buffer, 1 unit of Taq DNA polymerase (Advanced Biotechnologies, U.K.), in a total volume of 15 mL. The amplification program was as follows: 30 s at 94 oC, 30 s at 51 oC, and 60 s at 72 oC for 34 cycles on a thermocycler (PTC-100 MJ Research Inc.). PCR products (3 mL/lane) were separated on a DNA sequencing gel, containing 6% polyacrylamide, 8 m urea and 1x TBE, at 60 W constant power for 1.5 to 2.5 h. M13 DNA sequence was used as size marker. After drying, the gels were exposed to a Kodak MR film (Eastman Kodak). b) PCR reaction mixtures for separation on an agarose gel (nonradiolabeled) contained: 30 ng of plant genomic DNA, 2 mm of Mg2+, 10 to 15 pmole of 3' and 5' primers, 0.1 mm of each of the dNTPs, 1x Taq Buffer, 0.5 unit of Taq DNA polymerase (AB, UK), in a total volume of 25 mL. The PCR conditions were the same as above but for 44 cycles. PCR products produced by this amplification were separated by electrophoresis on 2.5% agarose (Techcomp, Hong Kong) gel and stained with ethidium bromide.

Results and discussion

Thirty melon and cucumber SSR markers were developed and evaluated for length polymorphism in melon (Table 1; Katzir et al., 1996; Danin-Poleg et al., 1996). Twenty-five of the SSRs were developed through screening of the melon genomic library and one from screening of a cucumber cDNA library. In addition, one melon SSR and three cucumber SSRs were developed from database sequences. The 30 SSRs had their origin in 27 loci, as three were from a second repeat, found at a distance of less than 1500 bp from the first SSR (Table 1, CMTA170a and CMCT170b; CMTA134a and CMCT134b; CMCT160a and CMTC160a+b). Amplification products of SSR loci were separated on either a sequencing or an agarose gel, according to the length difference between the alleles of the parental genotypes (Figs. 1 and 2). Size difference of >6 bp enabled the detection on agarose gel (Fig. 1b), while a smaller difference

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required the high resolution of a sequencing gel (Fig. 2).

Eighteen of the 30 SSR markers detected polymorphism between 'Védrantais' and PI 161375, the parental lines of the mapping population that had been used to develop the first melon map (Baudracco-Arnas and Pitrat, 1996). The 18 SSRs were integrated into this linkage map by segregation analysis with 117 markers, scored earlier for this population (Katzir et al., 1997; Danin-Poleg et al., unpublished data). Fifteen of the 18 SSR loci were located on nine distinct linkage groups. Of the 15 SSRs mapped (Table 1): five

were located on each of five different linkage groups (5, 6, B, G, and J), three pairs of different SSRs were located on each of three linkage groups (A, C, and F+I), and four SSRs were located on another linkage group (E). These results suggested that the microsatellite loci were randomly distributed throughout the melon genome.

An additional melon map, based on AFLP, has recently been published (Wang et al. 1997). This map consists of 204 markers (197 AFLPs, six RAPD markers and one microsatellite). These were scored as dominant markers in a backcross population consist

Table 1. Polymorphic simple sequence repeat(SSR) markers in five Cucumis mapping populations.

Mapping populations

Melon

Piel de MR-1 Cucumber

Védrantais PI414723 Sapo x GY14

SSR Chromosomal x x x Ananas x

designation locationz PI 161375 Dulce PI 161375 Yoqne'am PI183967

From genomic library melon

CMTC13 ­ + + + ­

CMAG59 E + + + + ­

CMGA127 ­ + ­ +

CMGA128 J + + + ­

CMGA15 F + + + ­ ­

CMGA104 nl + + + +

CMCT44 ­ + ­ ­ +

CMACC146 E + + + ­ ­

CMTC47 5 + + + + +

CMTC123 B + ­ + +

CMAT141 E + + + + ­

CMCTT144 E + + + + +

CMCCA145 G + ­ + + +

CMGA/AC108 ­ ­ + ­

CMTAA/GA166 ­ ­ ­ + +

CMTC168 C + + + +

CMTA170a ­ + + +

CMCT170b ­ + + +

CMGA172 A + ­ + + +

CMGA165 ­ ­ + + ­

CMTA134a + + + +

CMCT134b A + + + +

CMCT160a ­ + ­ ­ +

CMTC160a+b 6 + + + ­ +

CMCT505 nl + + + + ­

From cucumber cDNA library

CSCTTT15 ­ ­ ­ ­ +

From database melon

CMAT35 nl + ­ + ­

From database cucumber

CSGA057 ­ + + + +

CSAT425 F + + + + +

CSCCT571 C + ­ ­ ­

zChromosomal location of SSRs in the map constructed by Baudracco-Arnas and Pitrat (1996); +/­ = polymorphic/not polymorphic between the parental lines of each mapping population; nl = not located yet.

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Figure 1. PCR amplification products from the parental lines of the four melon mapping populations using CMCT134b. Separation was performed on (a) a sequencing gel and on (b) an agarose gel. Lanes are as follows: 1 Védrantais, 2 PI161375, 3 PI 414723, 4 'Dulce', 5 MR-1, 6 'Ananas Yoqne'am', 7 'Piel de Sapo,' 8 PI 161375 , M Size marker was 100-bp DNA ladder (Biolabs, New England). Inverted u-lines denote the biparental lines of each of the four populations.

ing of 66 progenies. The markers were assigned to 20 linkage groups covering 1942 cM (Wang et al., 1997). Other mapping projects that are currently being conducted at different laboratories are all directed at mapping economically important traits. These include traits described by Pitrat (1994).

Map merging is essential to obtain a single detailed map of melon. The findings that SSRs are evenly dispersed throughout the melon genome and are codominant, highly polymorphic PCR markers, establish their future role in mapping and map merging. The thirty SSRs tested here detected polymorphism in a sample of 13 melon varieties, having between two and six alleles (Katzir et al., 1996, Danin-Poleg unpublished results). They are thus likely to be polymorphic and informative in many biparental populations (as predicted by Akkaya et al., 1995, for soybeans). The 30 SSRs were tested for polymorphism between the parental lines of three other melon mapping populations and one cucumber population (Table 1). Eighteen loci were polymorphic between MR-1 and 'Ananas Yoqne'am', the parental lines of the Wang et al. (1997) population. Eighteen loci were polymorphic between PI 414723 and Dulce, the parental lines of a mapping population planned for viral resistance traits (Danin-Poleg et al., 1997). Twenty-two loci were polymorphic between PI 161375 and 'Piel de Sapo', the parental lines used by Oliver et al. (1998). These mapping populations

shared 12, 13, and 17 SSR loci, respectively (Table 1), with the expanded map of Baudracco-Arnas and Pitrat (1996). Nine SSR loci were common to all four biparental populations tested, enabling the merging of five linkage groups in all melon maps (Table 1, Figures 1 and 2). These SSRs are expected to be polymorphic in additional populations that are currently being used by other groups (Perl-Treves and Kyle; Staub and McCreight, personal communication). Moreover, 12 of the tested SSRs were polymorphic between GY14 and PI183967, the parental lines of a cucumber mapping population (Kennard et al., 1994), sharing six loci with the melon map described above (Table 1, Fig. 2). It is concluded that SSRs are most useful points of reference for map merging in melon, as well as for comparative mapping between melon and cucumber.

Figure 2. PCR amplification products from the parental lines of the five Cucumis mapping populations, using CMCTT144. Separation was performed on a sequencing gel. Lanes are C. melo, 1 'Védrantais', 2 PI161375, 3 PI414723, 4 'Dulce', 5 MR-1, 6 'Ananas Yoqne'am', 7 'Piel de Sapo,' 8 PI 161375; C. sativus, 9 GY-14, 10 PI 183967. Inverted u-lines denote biparental lines of each of the five populations.

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