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Development and characterization of simple sequence repeat (SSR) markers for an apomictic species Allium senescens

Ki-Won Oh1, Chan-Sik Jung1, Hyeun-Kyeung Kim1, Myung-Hee Lee1, Chung-Berm Park1, Byung-Joo Kim2 and Duck-Yong Suh1

1 Yeongnam Agricultural Research Institute, NICS, RDA, Milyang, 627-130, Korea , www.nyaes.go.kr Email ohkw1004@rda.go.kr
2
Research Management Bureau, RDA, Suwon, 441-707, Korea, www.rda.go.kr Email kimbj@rda.go.kr

Abstract

Enrichment methods were optimised in order to isolate large numbers of simple sequence repeat (SSR) markers for Allium senescens, with the aim of developing a comprehensive set of loci for apomixis and developing useful markers for genetic studies of Allium species. Four libraries were constructed showing greater than 20 % enrichment for a variety of SSR-motif types. Sequence characterisation of 796 clones identified 263 SSR-Containing closes. Truncation of flanking sequences limited potential primer design to 100 clones. The enrichment efficiency of six cutter restriction enzyme Sau3AI was higher than that of four cutter restriction enzyme MseI (48% vs. 22%). Developed primers were characterized and the size of amplified fragment was same as expected.

Media summary

We report an enrichment methods of simple sequence repeat in Allium senescens.

Key Words

SSR, Enrichment, Allium senescens,

Introduction

The genus Allium includes several important vegetable and ornamental species, and is an economically important crop. Allium senescens (2n = 6x = 48) is an apomictic species that diplospory, parthenogenesis, and pseudogamy occur (Kim et al. 1999). Molecular marker systems provide the means for genetic mapping of apomixis and marker assisted selection (MAS) of key agronomic target traits in Allium species. Molecular marker system based on SSLP (Fischer and Bachmann 2000) and Expressed sequence markers (McCallum et al. 2001) have been developed for onion (Allium cepa L.) and have been used to germplasm and genetic analysis and intra- and interspecific relatedness. However, molecular markers developed in onion were not adaptive in A. senescens in our experiment. Due to the likely genetic complexity of mapping heterozygous population, the ideal marker system for genetic map construction is simple sequence repeat polymorphism (SSRP). SSR markers are highly reproducible, genetically co-dominant and multiallelic. SSRs are suitable for framework mapping as they map to the same location in different crosses, and are amenable to automation for high-throughput genotyping (Rafalksi et al. 1996). Here we describe the optimisation of SSR enrichment conditions for A. senescens.

Methods

Oligonucleotides

Synthetic biotin labelled oligonucleotides were constructed having the following nucleotide sequences which are complementary to consensus microsatellite repeat sequences : Mixture : (CAT)10, (CAA)10, (AGA)10, (CTT)10, (ACT)10 ; Mixture : (CT)15, (CA)15, (AGC)10, (GAC)10, (CTG)10.

Plant material

Allium senescens (2n = 6x = 48) was used to construct enrichment and characteriazation

Construction of SSR-Enriched libraries and sequencing

Genomic DNA of YCA2 (A. senescens) was isolated using Qiagen DNeasy Plant Mini Kit. Two micro gram of genomic DNA was digested with MseI and Sau3AI, following size selection between 400 and 1000 base pare on the 1.5% agarose gel electrophoration. Size selected DNA was enriched with biotin labelled oligonucleotide mixture I and mixture II. SSR-enriched fragments were cloned using the PCR cloning kit from Qiagen. DNA from recombinant colonies was extracted using the QIAprep kit from Qiagen and sequenced.

Results

The efficiency of microsatellite enrichment was higher in oligo mixture than that of oligo mixture (48% vs. 22%). Among ten different kinds of repeatitive oligos, two sequence repeat (CT)15 and (CA)15, was frequently observed. From microsatellite-enriched library, 796 clones were sequenced and 303 clones inclued repeatitive sequence. From 303 sequences, 100 sets of primer were designed and characterized.

Table 1. SSR discovery in different libraries from A. senescens.

Restriction enzyme

Oligo Mixture

Number of
sequenced close

Number of
microsatellites

Primer
designed

MseⅠ

230

48

32

219

48

21

Sau3AⅠ

25

12

10

322

155

37

Fig 1. Sequence difference from three different enriched clones in A. senescens.

References

Kim BJ, Kwon YC, Kwack YH, Lim MS, and Park EH (1999). The mode of seed formation in Allium senescens and two Korean Allium species. Plant Breeding 118, 435-438.

Fischer D and Bachmann K (2000). Onion microsatellites for germplasm analysis and their use in assessing intra- and interspecific relatedness within the subgenus Rhizirideum. Theoretical and Applied Genetics 101, 153-164.

McCallum J, Leite D, Pither-Joyee M and Havey MJ (2001). Expressed sequence markers for genetic analysis of bulb onion (Allium cepa L.). Theoretical and Applied Genetics 103, 979-991.

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