| Cucurbita moschata Duchesne belongs to the annual herbaceous plant of the Cucurbitaceae.C.moschata,Cucurbita maxima and Cucurbita pepo are the three main cultivars of the genus Cucurbita,those which are not only provide fruit products for the fresh food-used market but also provide raw materials for the seed-used market.The hull-less-seeded Cucurbita moschata are very popular in the Chinese market for its unusual trait which can eliminate the process of hull removal prior to use.Although the demand for hull-less-seeded C.moschata eagerly,low yield restricted the development.How to make good use of this excellent gene is an urgent issue facing us.To this end,relevant research has been carried out and the results are as follows:1.The 153 group constructed from hull-seeded and hull-less-seeded pumpkin was used to investigate the variability,correlation and principal components.The genetic diversity of 153 pumpkin materials was abundant,among which the coefficient of variation of seed weight of per fruit,number of seeds per fruit and weight of per fruit.The variation coefficient of all three is more than 30%,which indicates that the genetic basis of these three traits is very rich and with the potential for developing excellent C.moschata varieties.In hull seeded C.moschata,the average of seed traits such as seed width,seed length,seed thickness,and 1000-seed weight are significantly larger than those of hull-less seeded C.moschata,and the difference between the two materials is not significant in other fruit traits.By analyzing the main components of fruit characters and seed traits,it is found that the top 15 materials of fruit traits have the characteristics of high weight of per fruit,thick pulp and high soluble solids matter content.The top 15 materials of seed traits have the excellent characteristics of good number of seeds per fruit,high weight 1000-seed and seed weight of per,big and thick seeds.There are four identical materials in the top 15 fruit traits and seed traits,of which there are three hull seeded C.moschata and one hull-less seeded C.moschata.These four materials can be gradually used in fruit and seed breeding.2.Using a scanning electronic microscope,we investigated the anatomical structure of the seed testa in two C.moschata inbred lines with hulled and hull-less seeds.We found that the seed coat of hulled seeds possessed a typical five-layer structure including,from the outside to inside,the epidermis(E),hypodermis(H),sclerenchyma(S),aerenchyma(A)and the chlorenchyma(C).Indeed,SEM observations on the hulled and hull-less seeds showed that its seed testa had all five layers by 10 DPA,and was slightly lignified.The seed testa of hulled seeds enhancement of lignification at 20 DPA,heavily lignified at 50 DPA.Instead,in the seed coat of hull-less seeds,after 15 days post anthesis(DPA),the five layers exhibited successive degradation,and were devoid of vascular bundle and lipid droplets.At 50 DPA,significant differences were observed in the sclerenchyma(S)and hypodermis(H)layers between hulled and hull-less seeds.In the seed coat of hulled seeds,the S-layer intercellular became ossified,and the stratified cell wall became thicker,denser,and lignified;the H-layer formed thick walled cells,and the small intercellular space was compact.On the contrary,in the seed testa of hull-less seeds,the cells lumens in the S-layer were hollow and the stratified cell wall was extremely thin;the thick-walled cells of H-layer were collapsed.3.We measured the lignin content at 10,20,30,40 and 50 days after post pollination and corresponding activities for four enzymes involved in lignin biosynthesis,the enzymes were lignin polymerase,phenylalanine ammonia-lyase(PAL),cinnamic acid-4-hydroxylase(C4H),4-coumarate-coenzyme A ligase(4CL)and cinnamyl alcohol dehydrogenase(CAD).The results showed that the lignin content in the testa of hull-less seeds also showed continued decrease over time,which was 0.22 mg/g at 50 DPA,on the contrary,the lignin content continued to increase in that of hulled seeds,which was 64.22 mg/g at 50 DPA,suggesting lignin plays a critical role in the formation of seed testa.The activities and contents of five enzymes in the hull-seeded were always higher than those in the hull-less-seeded.In the seed coat of hull-seeded lines,their activities continued increasing,which was 243.45(PAL),16.76(C4H),63.37(4CL)and 89.37 U/g·h(CAD)respectively at 50 DPA.In contrast,activities of PAL,C4 H,and CAD enzyme decreased gradually in the seed coat of hull-less lines from 20 DPA(4CL activity from 15 DPA),which was 46.15(PAL),2.84(C4H),7.33(4CL)and 12.40 U/g·h(CAD)respectively at 50 DPA.The enzymatic activities for four enzymes in seed coat of hulled seeds were positively correlated with lignin content.These data strongly suggested that lacking lignin accumulation and associated lignin biosynthesis activities are the cause of lack of seed coat developed in the hull-less seeds.4.Based on C.moschata F2 generation and two parents(hull-less-seeded and hull-seeded),we developed a high-throughput molecular marker using specific-length amplified fragment sequencing(SLAF-seq)technology.Results showed that reads totaled 33.05 M,the number of polymorphisms of SLAF tags reached 6816(4.85%).Correlation analysis revealed that the associated number of SLAF tags totaled 34,with 55 associated single nucleotide polymorphism loci.Compared with the SLAF tags in the cucumber genome,nine tags were mapped to the genomes.In the KEGG metabolic pathway aligned with the cucumber gene,Csa6M108530.1 is related to ovule development,and the bead of the ovule contains a large amount of cytoplasm,which may be closely related to seed coat development.Further research will provide theoretical and practical basis for molecular assisted breeding of hull-less-seeded C.moschata.In summary,the collapse or lack of hull-less seeded C.moschata is due to the reduced activity of four lignin biosynthesis enzymes: PAL,C4 H,4CL,and CAD.Four enzymes are directly involved in the synthesis of seed lignin.these data strongly suggested that lacking lignin accumulation and associated lignin biosynthesis activities are the cause of lack of seed coat developed in the hull-less seeds.The decrease in lignin deposition not only caused the collapse of hull-less seeded C.moschata seed coat,but also significantly changed seed traits such as seed width,seed length and seed thickness.The Csa6M108530.1 is related to ovule(seed coat)development.Further research on the gene Csa6M108530.1 and screening of other candidate genes,combined with lignin metabolism pathway research,is expected to further understand the developmental mechanism of hull-less-seeded C.moschata. |
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