Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (22): 4266-4276.doi: 10.3864/j.issn.0578-1752.2017.22.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Molecular Cloning, Expression Analysis and Development of Functional Markers for SiARGOS1 Gene in Foxtail Millet

WANG ZhiLan1, DU XiaoFen1,WANG Jun1, YANG HuiQing1, WANG XingChun2, GUO ErHu1, WANG YuWen1, YUAN Feng1, TIAN Gang1, LIU Xin1, WANG QiuLan1, LI HuiXia1, ZHANG LinYi1, PENG ShuZhong1   

  1. 1Millet Research Institute, Shanxi Academy of Agricultural Sciences/Shanxi Key Laboratory of Genetic Resources and Breeding in Minor Crops, Changzhi 046011, Shanxi; 2 College of Life Science, Shanxi Agricultural University, Taigu 030800, Shanxi
  • Received:2017-04-17 Online:2017-11-16 Published:2017-11-16

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Abstract: 【Objective】The homologous genes of Arabidopsis ARGOS (auxin-regulated gene involved in organ size) family were isolated from foxtail millet, which were induced expression by hormone and participate in the regulation of plant organ size. Bioinformatics, including alignment of amino acid sequences and promoter cis acting elements, and the expression pattern in different tissues and plant hormones were analyzed. Functional markers were developed from sequences analysis of gene encoding region and promoter, which will play an important role in genetic improvement of yield traits, accelerate breeding process and increase yield of foxtail millet.【Method】The number of ARGOS family members and the sequence of the ARGOS family in foxtail millet were analyzed through BLAST of conserved domain with the existing ARGOS in other plants. The encoding region and promoter sequences of SiARGOS1 gene, one of the family members of ARGOSin foxtail millet, were obtained with homologous cloning method. The promoter cis acting elements of SiARGOS1 was analyzed by the bioinformatics analysis. Expression patterns in different tissues and plant hormone of SiARGOS1 were analyzed using real-time PCR. Functional markers were developed from sequence analysis of SNP and insertion/deletion in gene encoding region and promoter. Functional markers of elite alleles of yield related traits were tested using data analysis of significant differences between genotypes associated with the yield traits such as panicle weight, grain weight and thousand-grain weight in 85 cultivars. 【Result】A total of six ARGOS family members were found to have a conserved OSR (Organ Size Related) domain consisting of two transmembrane helical structures and a highly conserved proline-rich motif in foxtail millet. the open reading frame (ORF) and the promoter of SiARGOS1, one of the family members homologous to Arabidopsis AtARGOS were cloned. Sequence analysis showed that the ORF of SiARGOS1 on chromosome 8 encoding a putative protein composed of 113 amino acids was 342 bp in length, without intron. The promoter contains a variety of regulation related components including auxin, ethylene and other plant hormone with 2 109 bp in length. SiARGOS1 is expressed in all organs of foxtail millet, at high levels in root, whereas at lowest level in panicle. The gene was insensitive to indole-3-acetic acid (IAA), but was up-regulated by ethephon (ETH). On the basis of the alignment of gene SiARGOS1 genomic DNA sequence, CAPS-AccⅡmarker was developed on the missense polymorphism site (C/G→Ala/Gly) at the 150 bp of the encoding region, two SSR markers, AP-1 and AP-2, were developed at -1652--1651(TA)2/3 and -1165--1163(TCA)1/2 of promoter, respectively. No significant association was found among the yield associated traits such as panicle weight, grain weight and thousand-grain weight with CAPS-AccⅡ and AP-1 in 85 cultivars. However, AP-2 proved to be highly correlated with panicle weight and grain weight in 85 cultivars, in addition to thousand-grain weight. The average values of panicle weight for two genotypes were 15.52 g and 20.61 g, respectively, while the ones of grain weight for two genotypes were 12.24 g and 16.74 g, respectively. 【Conclusion】Six ARGOS family members were found in foxtail millet , all of which had conserved OSR domain. The open reading frame (ORF) of SiARGOS1, homologous to Arabidopsis thaliana AtARGOS, is 342 bp in length without intron. The SSR marker AP-2, which was developed in the promoter region of thegene, could be used as functional marker for the selection of superior allelic variation in yield traits such as panicle weight and panicle weight of foxtail millet varieties.

Key words: foxtail millet, SiARGOS1;promoter, expression analysis, functional marker

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