| Rapeseed(Brassica napus L.)is most important oil crops and high quality protein feed crops in China,its planting area and yield accounted for one third of the world.Nowadays how to improve the yield and breed fine varieties is an important subject in face of rapeseed.Seed as one of the most important means of production of agricultural production,its good or bad quality directly determine the germination rate and seedling growth.However the seed aging,deterioration and degradation frequently happened in actual production because of the improper and difficult storage lead to the waste of production and the loss of seed resource.Thus it is of great significance to research the mechanism of rapeseed aging,breed aging resistant varieties for rapeseed production in China.In this study the RIL population derived from GH06 and P174 was used the experimental material and QTL analysis was performed by the combination of a SNP genetic linkage map which based on the RIL population and the germination index after artificial aging.We had screened the extreme materials with the RIL population germination index,measured its physiological indicators,fatty acid and hormone levels and found related candidate genes coupled with the RNA-seq data to laying foundation of seed aging explanation and excellent rapeseed selection.Our main results are as follows:1.The germination index of the seeds of RIL population in 2015 and 2016(40?,90%)was calculated after 15 days,and the germination index was calculated..The results showed that the seed germination index was range from 1.46 to 47.58 after aging treatment in 2015,the mean was 23.92.Seed germination index in 2016 was range from 3.24 to 98,the average was 57.31,which was twice as many as in 2015 indicated that the seed vitality was quickly lost in the process of storage causing largedifference of seed quality between the two years.From the overall distribution,the seed germination index in 2015 shows obvious normal distribution and which in 2016 shows skewed normal distribution.This implied that the seed aging was controlled by minor effect polygene and the population was suitable for QTL analysis.We have investigated seed germination index after aging in 15,16 years,13 QTL were detected based on SNP genetic linkage map.Six QTL were identified in 2015 which mapped on chromosome A02,A03,C02,C05 and C08.Seven QTL were identified in 2016 which mapped on chromosome A03,A06,A09 and C08.The q2016AGI-C08-2 and q2015AGI-C08 were overlapped on the chromosomes C08 that confidence interval was 36.8 to 38.4.And there is no found for other overlapping QTL in two years.2.Extreme phenotypic materials were screened according to the seed germination condition about the 2015 year RIL population after aging,and germination index was calculated after the high temperature and high humidity,repeated three times,it is found that rape seed germination index decreased after aging,but the two kinds of materials were compared that the sensitive materials had low germination index both before and after the treatment,and the difference was more obvious artificial processing,it indicated that the artificial aging makes the structure change inside the seeds,affecting the normal germination.The results of physiological index of extreme phenotype material after the artificial aging showed that the seed relative conductivity and MDA content are increased gradually,and the content of soluble sugar and soluble protein are reduced,and the proline content are increased,the activities of CAT and POD are decreased,the activity of SOD is increased first and then decreased along the seeds aging.Among them,the changes of the relative conductivity,the content of proline and CAT were more obvious,and the changes of other physiological indexes were smaller.From the two kinds of different materials,the relative conductivity of resistant materials is lower than the sensitive material before the aging,after treatment the result is opposite;the content of proline in the sensitive material is higher before and after the treatment;and the activity of CAT in resistant materials is high both before and after aging,no significant difference about other indexes between the two materials.After that,the seed germination index after aging and physiological index are analyzed.The results showed that the seed germination index showed significant negative correlation with relative conductivity,malondialdehyde and proline,the correlation coefficients were-0.666,-0.636 and-0.822,and it was positively correlated with other indicators,which had extremelysignificant positive correlation with CAT,the correlation coefficient was 0.736.3.The results showed that the content of fatty acids(linoleic acid,linolenic acid,oleic acid and erucic acid)in the aging of rape seeds did not change obviously during the artificial aging of seeds,which may be due to the change of fatty acid content in the process of storage,and the aging treatment had little effect on it.From the different materials,the content of erucic acid in resistant materials was much higher than the sensitive materials,oleic acid and linoleic acid content was low,linolenic acid content and sensitive materials were little difference,which indicated that the difference of fatty acid content and composition would affect the seed vigor,hinder its normal germination,and led to significantly difference of its germination index.The content of hormone of aging after extreme material mixing was determined by liquid mass spectrometry.It was found that the content of abscisic acid increased with the high temperature and high humidity treatment,the content of sensitive materials decreased and the content of gibberellin increased in two different types of materials.On the whole,the content of ABA and GA3 did not change obviously before and after seed aging,especially the low GA3 content.However,the GA3/ABA ratio of rapeseed seeds increased,so the endogenous hormone imbalance could be one of the reasons for the biochemical reaction or the deterioration of rape seeds in the process of seed aging.4.The extreme materials(R0,R15,S0 and S15)after artificial aging of the B.napus seed was subjected to transcriptional sequencing.In gene differential expression analysis,R15 Vs R0 yielded 2843 DEGs,S15 Vs S0 obtained 1086 DEGs,and the up-regulated gene was significantly more than down-regulated.The GO enrichment and KEGG pathway of differently expressed genes were analyzed in the genome of the B.napus genome as the reference genome.We compared the R15 Vs R0 and S15 Vs S0 to the top 10 significantly enriched GO entries and the KEGG pathway.The results showed that the two groups were enriched to the same GO function annotation and KEGG pathway,such as reactive oxygen,protein folding stool,but the number of genes involved in which is very different,indicating that the degree of response is different.Then 461 genes up-regulated in R0 / R15 and S0 / S15 were analyzed and sorted.Among them,62 important genes related to seed aging were found,encoding some proteins,kinases and transcription factors respectively.In this study,the gene positions near the SNP marker in the transcriptome analysis were compared with the previous QTL loci of the RIL germination index to find thedifferentially expressed genes in the QTL interval.Fifteen genes were found in A02,A03,A09,C02,C05 and C09 chromosomes,three of which are related to protein ubiquitination,and genes that encode hydrolases and heat shock proteins,which may be associated with seed aging and are worthy of further study. |
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