Transcription Analysis Of Summer Squash In Response To Cold Stress And The Development Of SSR Molecular Marker

The cultivated area of Summer squash is one of the largest vegetable in the protection production in our country, it has important economic value and social value. While the chilling damages is one of the most serious nature disasters during the process of cultivation. Currently, research about stress response mechanism under low temperature on summer squash has not been reported. This experiment analyzed the expressed difference of genes under low temperature between cold hardiness and sensitive to low temperature on summer squash through high-throughput sequencing technologies, in order to provided theoretical foundation in the molecular biology breeding on the hardiness on summer squash.In this study, the experimental material were three squash strain SS1, SS2, SS3 provided by our summer squash, and the Dongyu (French varieties) was served as control. We measured the physiological and biochemical indexes on summer squash under artificial low-temperature and comprehensive evaluated the hardiness ability using the method of subordinate function between three kinds of subject material and control. In order to reveal the molecular mechanism of cold stress, we conducted the transcriptome sequencing on cold hardiness and sensitive summer squash filtrated using Illumina Hi-SeqTM 2500 sequencing platform, analyzed and compared the difference of different expressed genes in order to understand the cold response mechanism on summer squash. Meanwhile, combined the SSR molecular marker technology, we developed the SSR molecular of summer squash and provided a certain basis for authenticate the ties o consanguinity between different summer squash kinds(strains). The results are as follows:1) After low temperature treatment, the percentage of conductance, soluble sugar content, MDA content, praline content and POD activity showed a rise trend, while SOD activity showed a decline trend.2) Analysising six measures of hardiness physiology using the method of subordinate function, the cold hardiness ability of 4 summer squash from strong to weak was:CK>SS1>SS2 >SS3.3) Using the Illumina HiSeqTM2500 sequencing technologies for the transcriptome sequencing on cold resistance and cold sensitive summer squash screened. We received 112,392,423 clean reads, and total bases were 22,701,006,115 bp. After assembled, we acceped 61,493 Unigenes. Conducted bioinformatics annotation on Unigenes with NR, SwissProt, GO, COG, KEGG database, we accepted 28,493 results of Unigenes. Summer squash Unigenes library was constructed. Analysised structure of the Unigenes library constructed, we acceped 61,208 ORF and overall length was 24,986,310 bp.4) Plant cold hardiness was the number of characters controled by multiple effect genes. Comparative analysised the transcriptome between the cold hardiness and cold sensitive summer squash in cryogenic treatment group and control group, the results showed that the cold hardiness summer squash had more differentially expressed genes than cold sensitive summer squash, and the pencentage of up-regulated DEGs was higher than cold sensitive. There were 612 DEGs found both cold hardiness and sensitive summer squash, and they constructed the common cold resistance mechanism. There were 430 DEGs only detected in the cold hardiness summer squash, and the main enrichment pathways was photosynthesis pathways ko00195 after analysised by KEGG database. There were 268 DEGs only detected in the cold sensitive summer squash, and the main enrichment pathways was photosynthesis pathways ko04075 after analysised by KEGG database.5) The experiment received 5302 SSR molecular marker combining with SSR molecular technology. Among this, there were 1973 single-base repeat,1173 double-bases,1508 three-bases repeat,92 four-bases repeat,30 five-bases repeat and 15 six-bases repeat. To verify the feasibility of SSR loci,98 pairs of primers were designed and to using for PCR amplification with DNA selected from 24 summer squash varieties(lines), and ultimately we selected 26 peimers which were polymorphism. This can be used for analysising genetic evolution of different summer squash varieties(lines).This experiment conducted the transcriptome of summer squash using high-throughput sequencing technologies and comparated the different cold response mechanism between the cold hardiness and cold sensitive summer squash and provided a certain basis for summer squash researching.