| Nelumbo, an ancient aquatic genus, comprises only two extant species, N. nucifera (Asian sacred lotus) and N. lutea (American lotus). These two species disjunctly distributed in East Asia, North Australia, and North America. The sacred lotus has a unique geographical distribution pattern, such as temperate zone and tropic zone distributions. In each climate zone, there distributed a distinct ecotype (temperate N. nucifera and tropic N. nucifera). Tropical N. nucifera grows without significant pauses during the four seasons, and its underground stem will not become into lotus, mainly distribute in tropical Southeast Asia (Thailand, India, Indonesia and Malaysia or other countries) and northern Australia. On the contrary, temperate N. nucifera has obvious annual growth and development cycle, the underground stem eventually swelling into lotus and dormancy. Its distribution area is mainly in temperate and boreal countries, such as China, Japan, South Korea and Russia. In order to reveal how the unique geographical distribution patterns of the two ecotypes of N. nucifera come into being, in this study, we use genome re-sequencing method and sample almost all the distribution range of this species to study the adaptive genetic differentiation between the two ecotypes. Our main results are as follows:(1) By resequencing and genome mapping, in total, we found 16,727,549 SNPs. The number of SNPs which located in the gene region was 320,547, and the percentage of missense mutation was up to 62.875%, meaning about 201,545. At the same time, there were about 7937 nonsense mutations and 111,065 synonymous mutations. The ratio of missense and synonymous mutation is about 1.8147, which indicated that the positive selection may have occurred during the evolution of the two ecotypes of N. nucifera.(2) Based on the phylogenetic analysis of the SNP loci, we found that found that there was significant genetic differentiation between Tropical N. nucifera and Temperate N. nucifera. The results also suggested that in different environment with different temperature, the selection pressure between the two ecotypes were different.(3) From the analyses of the three dimensional principal components of 37 samples of Tropical N. nucifera and Temperate N. nucifera, we found that the Tropical N. nucifera could cluster together and the Temperate N. nucifera also could cluster together, indicating that during the long evolution process, the environment differences, such as the temperature, sunshine time and light intensity and so on may have promoted the genetic differentiation between the two ecotypes. From the effectively distinguish of the top three principal components in Tropical N. nucifera and Temperate N. nucifera, we conclued that these two ecotypes of N. nucifera might have been affected by at least three kind of factors during their long-term adaptive evolution process.(4) Based on the population structure analysis of 37 N. nucifera samples, we also found that there was a unique genetic group for each ecotype. However, in the geographic zone between temperate and tropical region, the genetic structure of Tropical N. nucifera and Temperate N. nucifera were not obvious, which may be caused by the lower selection pressure in this transitional zone and the relatively high gene flow between these plants.(5) From the multiple tests of selection using Pi value in Fst, Tajiama D and Z test, we found that a total of 349 genes were under selection. More than 50% of those genes are categroried into catalytic activity and binding protein genes from our Go enrichment analyses. These genes are mostly belong to oxidoreduction, transfer, hydrolase pathways, which have been showed playing important roles in macromolecule synthesis. In addition, some of the genes were belong to energy metabolism pathway. Therefore, our results indicated that the energy metabolism might have greatly contributed to the genetic differentiation between the Tropic N. nucifera and Template N. nucifera. |
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