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

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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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Suppression subtraction hybridization


Expressed sequence tags


Reverse-transcription polymerase chain reaction




Reverse transcriptions


Gene ontology


Semi-quantitative RT-PCR


Quantitative PCR


Programmed cell death


Tapetum degeneration retardation


Male flower bud


Male flower


Male phylloclade


Male stem


Male root


Female flower bud


Female flower


Female phylloclade


Female stem


Female root


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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.

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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)


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)


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).

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