| Summary: | 博士 === 國立臺灣大學 === 農藝學研究所 === 107 === Botanists have been fascinated by the genetic mechanism of heterostyly since Darwin’s theory of evolution. It was believed that the genes controlling self-incompatibility and floral morphology were linked tightly, so-called S-locus. According to the classical evolutionary studies, when a plant evolved from outcrossing to selfing, it was necessary to lose self-incompatibility and then adjusted the positions of male and female floral organs through the rare recombination within the S-locus. However, new evidence suggested that homostyly resulted from hemizygote rather than the rare recombination. In agriculture, studying the genetic mechanism of self-incompatibility and heterostyly can understand the changes of crop genomes under the selection forces during domestication processes. Additionally, it can accelerate the production of hybrid seeds or ensure the pollination to increase yield.
Solanum pimpinellifolium is a wild tomato originated from the coastal region of Peru and Ecuador. It serves as an important germplasm in tomato breeding programs because it displays many resistant traits and can freely cross to cultivated tomatoes. Previous studies classified this species as complete or near complete allogamy, complete autogamy and intermediate type based on its mating system. In addition, allogamous accessions displayed higher genetic diversity and more exsertion of stigma than autogamous ones. Because S. pimpinellifolium contains the variations of outcrossing rate and floral morphology within its own species, it could be an ideal material to study the genetic mechanism of self-incompatibility and heterostyly.
Nowadays, molecular markers have been applied to crop breeding extensively. Accompanying by the cost down of next generation sequencing, the development of genome-wide high-density markers for germplasm becomes essential in breeding programs. In this research, we performed the PstI-digested associated DNA sequencing for 99 accessions of S. pimpinellifolium, resulting in 24,330 SNPs. The coverage extended to 12,790 genes, and a total of 7,383 genes were targeted directly by 16,365 SNPs. Besides, the sequencing regions and the annotated genes presented similar distributions through each chromosome. This suggested that PstI-digested associated DNA sequencing was an appropriate strategy to investigate candidate genes. This collection was divided into three subpopulations of single-ancestral genome and four subpopulations of mix-ancestral genome by ADMIXTURE. Principle component analysis, pairwise Fst and AMOVA all supported the subpopulations, implying this set of high-density markers was capable to estimate the subpopulations stably. Moreover, the overall LD decay was within 18 Kb, suggesting a fine resolution in genome-wide association study even to a single-gene level. However, to achieve such fine resolution, at least 50,000 markers were required.
Three candidate loci controlling stamen length were identified via the mixed linear model in genome-wide association study of 98 S. pimpinellifolium accessions, but all three loci presented high false discovery rate. Since the power and false positive rate of genome-wide association study depend on the sample size of a studying population, we suggest two approaches to increase sample size. One is to increasing samples in each subpopulation evenly. This approach can potentially make rare alleles to common alleles by increasing the allele frequency. The other is to sampling more individuals in the northern Peru because the accessions in the northern Peru present more genetic diversity. This approach can also increase both rare alleles and common alleles.
On the other hand, following the previous studies, stamen2.2 and stamen2.3 were located in the downstream interval next to style2.1. We performed a RNA sequencing experiment of M82 and TA3178. TA3178 is an introgression line of M82 and contains a segment of Solanum pennellii near style2.1. We identified this introgression region by comparing the difference of SNPs between these two lines. Afterwards, following the previous work in our team, we screened 18 candidate genes from marker cLED19A24 to CT9 by comparing the fold change and cDNA polymorphism between M82 and TA3178. This result suggested that Solyc02g087960.2, Solyc02g087970.1 and Solyc02g088070.2 should be the candidates of stamen2.2 and stamen2.3.
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