| Potato is one of the world’s most important crops together with rice, wheat and corn. Due to the long-term vegetative propagation, the degeneration caused by viral disease of the seed potato is the key factor to cause low yield and benefit of potatoes and it is also the most important constraint of the potato production in China. The production of virus-free seed potato is the main way to solve this problem. The traditional virus-free seed potato is to plant the potato in vitro plantlets to produce the minitubers, and then to get the commercial tubers in an isolated field. However, the cost of this method is very high and the survival rate is low. So it is difficult to meet the requirement at present. In vitro tuber of potato is a kind of minituber induced in tissue culture conditions which is virus-free, easily storage and also has high survival rate. So, it has got widespread attention and using in these years.Most of the previous reports mainly focused on the affects of different culture conditions on in vitro tuberization, such as photoperiod, temperature and the level of sucrose in the media, but the genetic basis of this process is far away from being fully understood. In addition, because of the genetic complexity of autotetraploid potato, the present genetic researches of potato are mostly based on the populations developed by double haploid potato or the relative diploid Solanum brevidens. In other words, the genetic research progress of tetraploid potato is very slow. Reports of in vitro tuberization in the tetraploid potato is also very rare.In this research, we used the tetraploid potato population MT I as our research material, which is built in our lab and shows segregation on in vitro tuberizaton. We made further identification and analysis of their characteristics of in vitro tuberizaton. Then we utilized the BSA-based RNA-seq analysis to compare the transcription of individuals with significant different %IVT(the percent of in vitro tuberizaton plantlets), trying to develop new markers associated with in vitro tuberizaton. So that we could add the new SNP markers to the existing genetic map to narrow the previous QTL regions. Meanwhile, we also hope to explore more useful information about the genetic basis of potato tuberization.The main results are as follows:1. According to the phenotype of in vitro tuberization of the tetraploid potato population MT I(209 individuals) under two photoperiods(16 h/d and 8 h/d), the initiation of in vitro tuberization of most genotypes have begun after 30 days cultivating. The initiation of tuberization under short daylight(8h/d, SD) condition was earlier than long daylight(16h/d, LD), and the %IVT and single in vitro tuber weight under SD were also higher than that of LD. For population MT I, the segregation ratio between tuberized and nontuberized genotypes under SD is 201:8. And the segregation ratio between tuberized and nontuberized genotypes under LD is 109:100, which is close to 1:1, and the 109 offsprings also tuberized under SD.2. In our study, we utilized BSA-based RNA-seq analysis, and constructed extreme pools twice. By comparing the differences of transcriptomes of groups with significant different %IVT, we identified the significant differences of SNP loci, and selected 47 SNPs located in the area of QTL MT05 to develop primers. By means of high-resolution melting curve screening and genotyping, we ultimately got 13 polymorphic SNP markers. Only 7 SNP markers finally located on the chromosome V of parents respectively in previous linkage map. And 5 of them belong to the male parent while 3 of them belong to the female parent. One belongs to both male and female parent. The other 6 SNP makers were not mapped on parents’ linkage map.3. The result of the marker-trait association analysis showed that 3 and 12 markers were significantly(P<0.01) associated with the %IVT under LD and SD photoperiod; 6 and 10 markers were significantly(P<0.01) associated with the single in vitro tuber weight under LD and SD photoperiod; 4 and 10 markers were significantly(P<0.01) associated with the initial tuberization time under LD and SD photoperiod. And two markers were significantly associated with all three phenotypes; 5 markers were significantly associated with both %IVT and the single in vitro tuber weight; 3 markers were significantly associated with both %IVT and the initial tuberization time; 2 markers were significantly associated with both the single in vitro tuber weight and the initial tuberization time. The related markers mainly distributed on the parents’ I, II, V, VI, IX, XI chromosomes.4. We utilized the IM routine of TetraploidMap for all linkage groups identified, and 10 QTL were identified using the phenotype data. Four QTLs were identified for %IVT under SD, including MT0502 detected on chromosome V of E108(explained 10.665 % of the variation for % IVT), MT0913 detected on chromosome IX of E108(7.364%), mt0502 detected on chromosome V of E20(17.662%), and mt0203 detected on chromosome II of E20(7.389%). For the single in vitro tuber weight, 2 QTLs were identified using the phenotype data under LD, there are TW1113 detected on chromosome XI of E108(6.413%), and tw0224 detected on chromosome II of E20(9.599%). Also 2 QTLs were identified using the phenotype data under SD, there are TW0502 detected on chromosome V of E108(15.245%), tw0502 detected on chromosome V of E20(9.058%). For the initial tuberization time, only 2 QTLs detected under SD, there are FT0934 and ft0502 which were detected on chromosome IX and V of E108 and E20 respectively, and explained 9.020% and 11.578% of the variation for the initial tuberization time under SD.Our study had made more detailed phenotype identification on the tetraploid potato population MT I. And we had developed some new SNP markers through the utilization of BSA-based RNA-seq analysis to compare the differences between transcriptions of individuals with significant different %IVT. After the encryption of genetic linkage map, we detected 10 QTLs associated with 3 phenotypes in total. Compared to the previous QTL mapping results, we found that the existence of QTLs on chromosome V of parents associated with %IVT under SD are stable. Although there are some deviation on peak and confidence interval between two results, but the trends of QTL effect are consistent. This result strengthens the possibility of genes associated with in vitro tuberizaton in this area. In addition, we found that there are coincident points of the QTLs related to above three kinds of phenotypes. It reflects the close correlation between these three phenotypes according to the genetic basis, and provides some new ideas for further exploration about the genetic mechanism of in vitro tuberizaton. |
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