Identification Of Genes Required For Barley Spike Development

To identify the genes required for spike development,forwards and reserve genetics are performed in different barley cultivars including Golden Promise and Hua30(Chinese domestication cultivar).Using the combined methods of genetics,molecular and bioinformatics,I aim to identify key genes required for barley spike development,and obtain the following results:1.Some reported inflorescences architecture genes in rice and maize were selected as references to find homologs in barley.The functions of these barley candidates genes were investigated using CRISPR-Cas9 genome-editing system.Firstly,I established barley transformation system using the barley homologs of rice cold1 and CSA genes.And the mutants of 12 barley homologous genes related to inflorescence development of rice and maize were created.Among these lines,we identified the mutants of barley homologs of Cold1 and qSW5.Interestingly,no abnormal phenotype was detected in cold1-1 spikes.But the height of cold1-1 mutant seemed shorter compared with the WT when it grows in the paddy field during the Shanghai winter season.After the treatment of 5?-8? in the greenhouse for 2 weeks seedling,the height of the mutant appeared shorter than the wild type,suggesting its role in regulating plant height in a temperature dependent manner,and the function of this gene was preliminarily analyzed.2.To understand the role of auxin in regulating barley spike development,using in silico method,36 Auxin/IAA genes were identified from the barley genome,which were distributed on 7 chromosomes,and 9 HvIAAs predominantly located on chromosome 5.18 Aux/IAA proteins contain four conserved domains The expressions of HvIAA16,19 and 20 were tissue-specifics,specifically expressed during the grain development stage and tillering stage,respectively.The qRT-PCR results showed that HvIAA3,7,8 and 18 were highly expressed in DR(Double ridge),LP(Lemma primordium),SP(Stamen primordium),AP(Awn primordium)and WA(White anther),and HvIAA24 was only highly expressed in the WA stage.These genes may be involved in the development of barley spike,pro viding strong evidence for reverse genetics gene knockout.After obtaining transgenic seedlings,verify the functions of these genes on the spike development.At the same time,it was tested whether the Aux/IAA genes responded to exogenous auxin.The results shown that the transcription levels of HvIAAs in C2,4,5 and 6 groups were up-regulated 4h after NAA treatment.Particularly,the expression of HvIAA30 was up-regulated over 10-fold at 4h,and the expression of HvIAA2 and HvIAA30 was more than 2-fold at 2h and 4h.It shows that genes such as HvIAA2 and HvIAA30 are responsive to exogenous auxin treatment,which provides a reference for further research on the initial response factors of auxin.3.To identify the genes regulating barley spike development,Co60 ?-rays were used to induce Hua30 seeds to obtain mutants with spike phenotype.After 3 generations of self-pollination,mutants with defects of spike were obtained.Backcrossed with Hua30 and crossed with Gold Promise,to establish F2 generation segregated population.At the same time,prepare the materials for development the InDel molecular markers system.In the meantime,the InDel molecular markers system was established for early mapping of mutants.Among them,there is still a large gap in the InDel molecular marker,which needs further supplementation.A total of 88 InDel molecular markers with significant differences were obtained for subsequent map-based cloning experiments of mutants,especially mutants related to spikes.