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Isolation of differentially expressed sex genes in garden asparagus using suppression subtractive hybridization

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Abstract

Garden asparagus (Asparagus officinalis L.) is a dioecious species whose male and female flowers are found in separate unisexual individuals. A region called the M-locus, located on a pair of homomorphic sex chromosomes, controls sexual dimorphism in asparagus. To date, no sex determining gene has been isolated from asparagus. To identify more genes involved in flower development in asparagus, subtractive hybridization library of male flowers in asparagus was constructed by suppression subtraction hybridization. A total of 107 expressed sequence tags (ESTs) were identified. BLASTX analysis showed that the library contained several genes that could be related to flower development. The expression patterns of seven selected genes believed to be involved in the development of asparagus male flower were further analyzed by semi-quantitative or real-time reverse-transcription polymerase chain reaction (RT-PCR). Results showed that AOEST 4-5, AOEST 12-40, and AOEST 13-38 were strongly expressed in the male flower stage, whereas no transcript level of AOEST 13-38 was detected in the female flower stage. The expression levels of AOEST 13-87, AOEST 13-92, AOEST 13-40, and AOEST 18-87 in the male flower stage were also higher than those in the female flower stage, although these transcripts were also expressed in other tissues. The identified genes can provide a strong starting point for further studies on the underlying molecular differences between the male and female flowers of asparagus.

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Abbreviations

SSH:

Suppression subtraction hybridization

ESTs:

Expressed sequence tags

RT-PCR:

Reverse-transcription polymerase chain reaction

DIG:

Digoxigenin

RTs:

Reverse transcriptions

GO:

Gene ontology

SqPCR:

Semi-quantitative RT-PCR

qRCR:

Quantitative PCR

PCD:

Programmed cell death

Tdr:

Tapetum degeneration retardation

MFB:

Male flower bud

MF:

Male flower

MP:

Male phylloclade

MS:

Male stem

MR:

Male root

FFB:

Female flower bud

FF:

Female flower

FP:

Female phylloclade

FS:

Female stem

FR:

Female root

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Acknowledgments

The research was supported by grants from the National Natural Science Foundation of China (31000165, 31300202) and Program for Innovative Research Team (in Science and Technology) in University of Henan Province (15IRTSTHN020).

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Correspondence to Chuan-liang Deng.

Electronic supplementary material

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10265_2015_735_MOESM1_ESM.tif

Fig. S1 PCR analysis of adaptor ligation efficiency. Lane 1: PCR products using Tester 1-1(Adaptor 1-ligated) as the template and the SLactin 3′Primer and PCR Primer 1. Lane 2: PCR products using Tester (male flower buds cDNA) 1-1(Adaptor 1-ligated) as the template, and the SLactin 3′ and 5′ Primers. Lane 3: PCR products using Tester (male flower buds cDNA) 1-2(Adaptor 2R-ligated) as the template, and the SLactin 3′ Primer and PCR Primer 1. Lane 4: PCR products using Tester (male flower buds cDNA) 1-2(Adaptor 2R-ligated) as the template, and the SLactin 3′ and 5′ Primers. Lane M: 400, 500, 600 bp (TIFF 646 kb)

10265_2015_735_MOESM2_ESM.tif

Fig. S2 Analysis of subtraction efficiency by polymerase chain reaction (PCR). PCR was performed on the unsubtracted (lanes 1–4) or subtracted (lanes 5–8) secondary PCR product using the SLactin 5′ and 3′ primers. Lanes 1 and 5: 30 cycles; lanes 2 and 6: 25 cycles; lanes 3 and 7: 20 cycles; lanes 4 and 8: 15 cycles. Lane M: 300, 400, 500 bp (TIFF 888 kb)

Fig. S3 PCR analyses of selected clones from SSH libraries. Lane M: 100 bp ladder (TIFF 1979 kb)

Table S1 The primer sequences used in this study (DOC 41 kb)

Table S2 BLASTx analysis for sequenced differentially expressed cDNA clones (DOC 76 kb)

10265_2015_735_MOESM6_ESM.doc

Table S3 tBLASTx analysis for part of sequenced differentially expressed cDNA clones from male subtracted cDNA library (DOC 77 kb)

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Deng, Cl., Wang, Nn., Li, Sf. et al. Isolation of differentially expressed sex genes in garden asparagus using suppression subtractive hybridization. J Plant Res 128, 829–838 (2015). https://doi.org/10.1007/s10265-015-0735-6

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  • DOI: https://doi.org/10.1007/s10265-015-0735-6

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