Factors Affecting The Seed Hardness And The Transcriptome Analysis Of Soft-seed Pomegranate

Pomegranate(Punica granatum L.)belongs to Punicaceae, Punica L. It has been valued for its social,ecological, economic, aesthetic value and more lately for its health benefits. Especially, ‘Tunisia soft-seed’, which has the qualities of soft seeds, big arils and easily swallowed, is popular among person.However, the molecular mechanisms of the hard-seed and soft-seed’s formation not clear. In order to know how the factors affecting the seed-hardness of ‘Tunisia soft-seed’, the experiment chose five factors to study the affecting mechanism. Then took the seeds of different development stage of ‘Tunisia soft-seed’ and ‘Sanbai’ as the materials, using Illumina Hi Seq 2500 high-throughput sequencing to find the deferentially expressed genes, and then validated some related genes by q RT-PCR. The main results are as follows.1. The results showed that: at 20 days after blooming(DAB), the testa and embryo of the two cultivars were not developed. The arils appearance of the two varieties was similar. Both of them were white and the size of the two varieties had little difference. At DAB 40, testa was developed, the embryo hadn’t developed yet. The arils size of ‘Yudazi’ was larger than ‘Tunisia soft-seed’. The arils color of ‘Tunisia soft-seed’ was pink, and ‘Yudazi’ was white. At DAB 60, the testa and embryo were both developed. At DAB 120, the appearance and size of the two varieties were obviously different. Color of the arils of Tunisia was bright red, and the color of ‘Yudazi’ is lighter than ‘Tunisia soft-seed’. The seed size of‘Yudazi’ was larger than ‘Tunisia soft-seed’. The two pieces of the leaf are intertwined with each other.The development process of the seed of the pomegranate: first the testa developed, and then the embryo developed. Selected the 8 a, 10 a, 12 a, 15 a ‘Tunisia soft-seed’ as the materials to measure the seed hardness and fruit quality. As the age of the pomegranate tree increased, the seed hardness of the fruit decreased. Most of the fruit quality improved with the increase of age. The results found that quality of fruits in east, south, west and north four directions were without significant difference, but the seed size and hardness had differences: south < east < north < west. The quality of fruit and seed hardness at upper, middle and lower three levels were significantly different, with the quality of fruits: upper <middle < lower; seed hardness and lignin content: middle < upper < lower.The seed hardness of self pollination and hybrid combinations fruits were soft. ‘Yudazi’ and ‘Sanbai’ as the pollinated variety had the higher fruit setting rate. ‘Heizitian’ and ‘Shandazi’ as the pollinated variety had the better fruit quality; ‘Tunisia soft-seed’ as the pollinated variety had the smaller seed hardness; at the same time, they had the smaller lignin content. The hardness of seed was positively correlated with lignin content; the greater of the seed hardness, the higher of the lignin content was in testa. Bagged ‘Tunisia soft-seed’at DAB 40, then keep bagging for 20 d, 40 d, 60 d and 80 d respectively. The results showed that, compared with the control group, there was no significant difference in the hardness of the seed. However, bagging time had some effects on fruit size, Thousand grain weight, and soluble solid content and so on. The effect of the application of different fertilizers on the quality of pomegranate fruit was significant. Different fertilizer application compared with the control could increase the fruit weight, seed size and fruit quality. The application of phosphate fertilizer could significantly reduce the seed hardness of pomegranate.2. Took the seeds of DAB 30, 60 and 120 of ‘Tunisia soft-seed’(soft-seed, seed hardness < 3.67kg/cm2)and ‘Sanbai’(hard-seed, seed hardness > 4.2kg/cm2) as the materials to extracted total RNA, then reverse transcription. Used the new generation of high-throughput sequencing technology, Illumina Hiseq 2500 to initially revealed the genes expression and transcription information of two varieties at DAB 30, 60, 120, to enrich the pomegranate molecular databases. 35.1G of the data was obtained by sequencing, and the 280881106 original reads were obtained by using the Trinity platform to carry out the de novo assembly of the transcription group. In this study, we have assembled a total of 132287 transcripts and 105743 representative Unigenes sequences, and 76806 complete protein coding sequences were obtained. The seed hardness of two pomegranate cultivars was related to lignin biosynthesis and metabolism, including some genes of laccase and peroxidase, WRKY, MYB, NAC and other transcription factors. In the hard-seed pomegranate, lignin related genes, cellulose synthesis related genes, MYB transcription factor expression had high levels; flavonoid related genes,programmed cell death related genes expression had higher levels in soft-seed pomegranate.