| Cowpea is a relatively common vegetable among legumes.It is commonly known as beans,an annual herb of the Papilionaceae family,and has strong adaptability.It is planted more in China,India,and Southeast Asia and has higher economic benefits.In recent years,due to the impact of human factors such as changes in environmental conditions and continuous cropping obstacles,the pests and diseases in the cowpea production process have increased,making the yield and quality of cowpea difficult to meet the needs of vegetable production.Cowpea is a butterfly-shaped corolla,heterosis is difficult to reflect in the F1generation.Polyploids obtained through chromosome doubling in breeding generally have many excellent characteristics such as large fruits,strong resistance,and good nutritional quality.Using current scientific and technological means to induce the existing diploid cowpea to obtain tetraploid cowpea,and to explore the physiological and molecular mechanism of tetraploid cowpea is of great significance to the future production of tetraploid cowpea and the production of polyploid species.In this experiment,common diploid cowpea（HN-56）was used as the material,and three different methods of colchicine solution were used to induce and compare different induction methods through the three methods of soaking method of dew seed,stem tip drip method and in vitro induction method.And the advantages and disadvantages between different induced concentrations.The physiological and biochemical indexes of the obtained tetraploid cowpea material were analyzed,their gene expression profile and DNA methylation were analyzed.The diploid cowpea was used as a control for comparison,and the main differences between the two were explored.The research of the body provides the data foundation and theoretical basis.The main results were as follows:1.Different induction methods could induced tetraploid cowpea,but the three methods had their own advantages and disadvantages.The induction rate of the tetraploid with the white seed soaking method at 0.15%concentration of colchicine for 1 h was 23.33%.This method was simple to operate and had a short treatment cycle,but the induction rate of tetraploid cowpea was relatively low.The tip-point drip method had the highest tetraploid induction rate of 36.67%after treatment with 0.15%concentration of colchicine for 4 times.This method was more complicated.The drip-tip was time-consuming and labor-intensive,the treatment cycle was longer than the soak method of exposed seeds.But the induction rate of tetraploid cowpea was relatively high.In vitro induction method under the treatment of0.004%concentration of colchicine,the tetraploid bud induction rate was the highest,reaching 40.91%.This method was the most cumbersome operation.First,the optimal medium for seed germination and the optimal medium for adventitious bud induction must be selected.The selected adventitious bud induction medium was added with colchicine for induction,the treatment period was long,and the conditions for inoculating explants were strict,but the tetraploid induction rate was the highest in this method.2.There was difference between the phenotype and physiology of tetraploid cowpea and diploid cowpea.Compared with diploid,the leaf,stem thickness,petals,and pods of tetraploid plants all showed hugeness.The stomatal density of the leaves decreased by44.92%,the guard cells increased,and the number of chloroplasts in the guard cells increased by 67.17%.3.There was difference in the photosynthetic characteristics of tetraploid and diploid cowpea.Compared with diploid,the chlorophyll content in the tetraploid cowpea leaves increased by 35.18%,soluble sugar content increased by 28.09%,and soluble protein content increased by 33.33%.The chlorophyll fluorescence parameters of the maximum fluorescence（Fm）,photochemical quenching coefficient（q P）,photochemical effect（Fv/Fm）daily change trends were similar,but the initial fluorescence（Fo）,non-photochemical quenching coefficient（NPQ）and effective photochemical quantum yield（Y（Ⅱ））were quite different.The tetraploid Fo was less than diploid,q P,NPQ,Fv/Fm,Y（Ⅱ）were all higher than diploid.tetraploid cowpea plants showed excellent photosynthetic performance,and had better environmental adaptability.The diurnal and tetraploid plants photosynthesis parameters of photosynthesis rate（Pn）,transpiration rate（Tr）,stomatal conductance（Gs）had a large daily variation trend,while the intercellular CO2 concentration（Ci）The daily trend was similar.The overall levels of Pn,Tr,and Gs in tetraploid plants were higher than diploid,the maximum net photosynthetic rate was higher than diploid,and the light compensation point was lower than diploid,indicating the light energy utilization rate of tetraploid cowpea plants.The photosynthetic yields of both were higher than diploid.4.Perform transcriptome sequencing on tetraploid and diploid cowpea,compare the gene expression of them,and refer to databases such as Gene Ontology and KEGG Pathway for functional classification and enrichment analysis of differentially expressed genes.The results showed that there were 2678 genes differentially expressed in tetraploid and diploid cowpea.Among them,421 genes were expressed in tetraploid higher than diploid,and 2257genes were expressed lower than diploid.Gene Ontology annotation analysis showed that these differentially expressed genes were mainly involved in the related functions of cowpea metabolism and cell components.KEGG Pathway analysis showed that most of the differentially expressed genes were significantly enriched in 6 pathways including phenylpropane biosynthesis,starch sucrose metabolism and circadian rhythm.After screening,there were 8 significantly differentially expressed genes related to phenylpropane biosynthesis,6 significantly differentially expressed genes related to starch sucrose metabolism,and 4 significantly differentially expressed genes related to circadian rhythm,among these 18 genes It can be used as an important candidate gene for diploid and tetraploid cowpea to produce differences in phenotype and photosynthesis.5.Performed genome-wide methylation analysis on tetraploid and diploid cowpea,and compared the DNA methylation characteristics of them and the DNA methylation differences between ploidy.The results showed that the average methylation level of CHH type on each chromosome of tetraploid was higher than diploid.The methylation levels of different types of tetraploid on the element were higher than diploid.The methylation sites were mainly distributed in the repeat region,followed by 2000bp upstream of the transcription start site,2000bp downstream of the transcription start site and Cp G islands,And less distributed in the CDS region and m RNA region.In the promoter region,the number of CHH type differential methylation was the largest,and in the body region,the number of CG type differential methylation was the largest.GO enrichment and KEGG Pathway enrichment of differential methylation genes in the promoter region and the gene body region revealed that the differential methylation genes between ploidy were mainly enriched in gene pathways related to cowpea growth,development,metabolism,etc.Enriched in genetic pathways related to genetic information,signal transduction,etc. |
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