| Finger millet,Eleusine coracana(L.)Gaertn.,is an annual crop of the genus Eleusine of Gramineae.It is the fourth most important millet across the world,next to sorghum,pearl millet and foxtail millet,with annual planting area of 4.5 million hectares in the word.The finger millet is one of the special dry grain crops and served as medicine,food,feeding stuff in China.Most of the research about finger millet in China were keeping on yield and cultivation improvement.Few efforts were put into systematic survey and in-depth research on diversity of germplasm resources,quality and drought tolerance mechanism.In order to screen germplasm resources with strong drought tolerance and great genetic diversity for genetic breeding,we analyzed the phenotypic traits,quality,genetic diversity and drought tolerance response of the 69 core millet germplasm resources in Hunan province were,which could promote the development of millet industry in China.The main results of this thesis are including:1.Evalution on phenotypic variation of finger millet germplasm resourcesThe phenotypic diversity of 69 core finger millet germplasm resources were evaluated.The results showed that 69 core germplasm resources were mainly distributed in the middle and low altitude areas of 200-600 m.The diversity analysis based on 19 phenotypic traits revealed a higher genetic diversity in this collection.The correlation analysis and principal component analysis suggested that the cumulative contribution rate of 7 main features including tiller number,gross weight,leaf color,1000 grain weight,glutinousness,whole growth period was reached 75.53%.Based on the clustering analysis,the 69 finger millets could be clustered into two groups.The first group included 44 germplasms,which have smaller biomass,height and low yield,while the second group have larger biomass,plant height and higher yield.We selected 12 germplasms as the core finger millet resources,which could be summarized into four groups based on the characteristics: long spike(EC23,EC36,EC64),fodder type with large biomass(EC5,EC13,EC19,EC33),grain and fodder type with high yield and biomass(EC25,EC50,EC53)and glutinous type(EC16,EC46).2.Evalution on quality diversity of finger millet germplasm resourcesThe magnesium(Mg),calcium(Ca),potassium(K),iron(Fe),VB2,VB1,starch,total sugar,protein,dietary fiber and total amino acid of 69 core finger millet germplasm resources were comprehensively evaluated.The results showed that VB2(38.51 %),calcium(24.40 %)and total amino acid(30.33 %)had high variation among different resources,and the genetic diversity index of iron(2.03)and calcium(2.01)were higher than other indicators.Principal component analysis divided 11 nutritional indicators into three principal components,with a cumulative contribution rate of 58.85 %.The comprehensive ranking of nutritional value was conducted by membership function method and similarity priority ratio.The top five varieties with nutritional value were selected,including EC39,EC50,EC34,EC62 and EC64.3.Comparison of Finger Millet by ITS Sequences and Cluster Analysis of EleusineIn order to establish a method for rapid identification of finger millet(Eleusine coracana)and determine the genetic relationship of finger millet,the ribosomal DNA transcribed spacer(ITS)sequences of 69 core finger millet germplasm resources were sequenced and compared,and the phylogenetic tree of Eleusine genus was constructed.It was found that there were single base mutants in the ITS sequences of the three finger millet populations,including EC25,EC53,and EC59,and there was no difference in other populations.There are 3 to 43 bases varied in ITS within Eleusine Gaertn species.The phylogenetic tree shows that the Eleusine Gaertn species are divided into 2 categories,including 4 subgroups,and the finger millet was found to be clustered with Eleusine kigeziensis.These results have shown that the ITS sequences are highly conserved in finger millet,which indicates that the ITS sequence cannot be used as an adequate marker gene for discrimination of the different finger millet population.4.Evaluation of drought resistance of Finger milletWe evaluated the drought tolerance of 69 core finger millet germplasm resources using membership function method and the results showed that EC64 was drought sensitive and EC38 was more drought resistant in the drought experiment at seedling stage.Principal component analysis showed that the main information of 10 physiological and biochemical indexes was concentrated in the first two principal components,and the cumulative contribution rate was 86.60%.Among them,catalase,glutathione reductase,superoxide dismutase,peroxidase,hydroxyl radical inhibition ability,soluble sugar and protein,proline and MDA contribute the most,which can be used as the main index to measure drought tolerance and variety screening.The comprehensive evaluation of the screened materials was carried out by the membership function method,and the evaluation results were consistent with the screening results,which could be used to establish the drought tolerance evaluation system.5.Integration of transcriptomic and proteomic analyses for Finger Millet in response to drought stressIn order to further explore the genetic mechanism of drought tolerance of Finger millet,in this study,transcriptomic(RNA-seq)and proteomic(i TRAQ)technologies were combined to investigate the finger millet samples(C38)treated with drought at different stages.A total of 80,602 differentially expressed genes(DEGs)and 3,009 differentially expressed proteins(DEPs)were identified in the transcriptomic and proteomic levels,respectively.An integrated analysis,which combined transcriptome and proteome data,revealed the presence of 1,305 DEPs were matched with the corresponding DEGs(named associated DEGs-DEPs)when comparing the control to samples which were treated with 19 days of drought(N1-N2 comparison group),1,093 DEGs-DEPs between control and samples which underwent rehydration treatment for 36 hours(N1-N3 comparison group)and607 DEGs-DEPs between samples which were treated with drought for 19 days and samples which underwent rehydration treatment for 36 hours(N2-N3 comparison group).Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis identified80 DEGs-DEPs in the N1-N2 comparison group,49 DEGs-DEPs in the N1-N3 comparison group,and 59 DEGs-DEPs in the N2-N3 comparison group,which were associated with drought stress.The DEGs-DEPs which were drought tolerance-related were enriched in hydrolase activity,glycosyl bond formation,oxidoreductaseactivity,carbohydrate binding and biosynthesis of unsaturated fatty acids.Co-expression network analysis revealed two candidate DEGs-DEPs which were found to be centrally involved in drought stress response.These results suggested that the coordination of the DEGs-DEPs was essential to the enhanced drought tolerance response in the finger millet.The finger millet germplasm resources with high yield,good quality and high drought tolerance ability were screened.The genetic relationship of these resources from different populations was clarified and the phylogenetic tree was constructed of Eleusine,and the drought tolerance response mechanism of finger millet was clarified.It is helpful for people to understand the evolution process of finger millet,which provides a basis for the safe protection and effective utilization of finger millet germplasm resources,and takes an important step for drought resistance breeding of finger millet in China. |
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