Genetic Analysis And Differential Genes Correlated With Heterostyly In Eggplant(Solanum Melongena L.)

Heterostyly is a unique form of polymorphism and herkogamy in flowers, which is widely distributed among angiosperm plants, including eggplant（Solanum melongena L.）.Generally, L-morph patterns owned stronger fruit setting capability and wider habitat suitability, while S-morph patterns owned lower fruit setting capability. Although lots of reports have been published on the difference in fruit setting capability and adaptability to environment of light, temperature, etc., between S-morph and L-morph flowers, the molecular regulation mechanism of eggplant heterostyly development and variety improvement is still unknown. In southern China, the main planting is distylous flower eggplant. Due to the lower fruit setting capability and unfavorable facility for cultivation, the improvement of style in eggplant was necessary in practice. In this study, we studied the growth development, the fruit setting capability, inheritance law and molecular regulation mechanism. The main results are as follows:1. We divide the period of flower from budding to blooming into 6 stages, Visible flower bud stage, Flower bud enlargement stage, Flower revealing corolla stage, Flower corolla enlargement stage, Soft bud stage and Flowering stage, respectively. In the view of the bud length, the S-morph shows no difference with the L-morph in pistil length between 0 10 mm length, but the growth of S-morph pistil ground to a halt when it enters into Flower bud enlargement stage（about 10 mm）. During the flower development, style length of L-morph flower shows a linear relationship with flower bud. The style length shows extremely significant correlation with the height of the ovary and bud length, and significant correlation with the pedicel, but show less significant correlation with ovary diameter and anther length. Through fluorescence microscopic observation, we found that the style and pollen of L-morph flower have reproductive activity, however, only the pollen of S-morph flower has reproductive activity. The egg cells, central cell, antipodal cells and synergids of in only longistylous flower eggplant and Hetero-Distylous flower eggplant embryo sac are presented as cellular structure integrity by the optical microscopic.2. The fruit rate of S-morph flower selfing is only about 5%, but the fruit rate of L-morph flower selfing is above about 94%. The hybrid test suggests the fruit rate of S-morph style was significantly less than L-morph style.3. We use only longistylous flower eggplant and Hetero-Distylous flower eggplant to crossbreed, to find that all of the F1 generation are only longistylous flower eggplant. The specific value of only longistylous flower eggplant and distylous individuals among F2 generations of B3-3×56 and 01×119 are 12.14:1 and 11.85:11, respectively. The separation specific value of only longistylous flower eggplant and distylous individuals in the combined BC1 population are 3.64:1 and 2.56:1, respectively. After the chi square test, only longistylous flower eggplant shows the dominant inheritance compared with Hetero-Distylous, which may be the overlap effect of two gene pairs. The Shapiro-Wilk test of F2 generation shows style length is quantitative genetic character controlled by multigene heredity, and main effect genes may exist.4. High-throughput miRNA sequencing, microarray and degradome sequencing are used to identify the pistil miRNA expression and target genes of Hetero-Distylous flowers in same individual. 55 identified known miRNAs of eggplant and 35 miRNAs having the same sequences with the known miRNAs in the miRBase are identified using miRNA sequencing.Among the 594 novel miRNAs, majority of the miRNA families are identified to be highly conserved compared with other plants. Among the 686 miRNAs on the microarray chip, 10 are demonstrated to be differentially expressed, only two of which are significantly down-regulated in L-morph pistil development. Moreover, the expression profiles of the eggplant miRNAs obtained through the microarray assays are validated by quantitative real-time polymerase chain reaction（qRT-PCR）. We also predict and annotate the target genes for the differentially expressed miRNAs.5. The expression profile of pistil miRNAs in S-morph of B3-3 and L-morph of Thousand Finger eggplant are identified by microarray. Among the 686 miRNAs on the microarray chip, 35 are identified as differentially expressed miRNAs, among which 9 are down-regulated miRNAs in L-morph and 26 up-regulated. PC-5p-1408812₁ and PC-5p-3215765₁ are the most significantly down-regulated miRNAs, and PC-3p-1358₄05and PC-5p-132923₇ are the most significantly up-regulated. Furthermore, the expression patterns obtained from the microarray assays are evaluated by qRT-PCR. We also conduct target gene prediction of differentially expressed miRNAs, and annotate GO terms for the putative targets. Among the target genes of differentially expressed miRNAs, many genes encode TFs, such as basic Helix-Loop-Helix（bHLHs）, MYBs and AP2, some important enzymes or functional proteins, including triphosphate hydrolases superfamily protein,protein kinase, phosphatase, and ATPase, which may play important roles in regulation the heterostyly development in eggplant.6. We perform the iTRAQ proteomic analysis of eggplant pistils for L-morph and S-phorm of flowers in same individual. A total of 300 differential expressed proteins are identified. During flower development period, we discover that there are 26 down-regulated proteins and 23 up-regulated proteins in S-morph compared with L-morph. Among down-regulation proteins, there are maximum expression difference in methionine sulfoxide reductase A3（MSRA3） and putative DNA-damage-repair/toleration protein-like. And among up-regulated proteins, there are maximum expression difference in methionine sulfoxide reductase a（MSRA） and sarcosine oxidase（SOX）. The differential expressed proteins are mainly involved in energy storage, environmental stress response and pathogens infection resistance. During flower mature period, 97 proteins are up-regulated and 157 proteins are down-regulated. Among down-regulated proteins, there are maximum expression difference in leucine-rich repeat protein and sexual organ expressed protein. LAT52 and ntp201 are two top differentially up-regulated genes. The differential expressed proteins mainly participate in biosynthetic process, small molecule transport and metabolic pathway. In addition, during flower mature period, the expression level of alcohol NADP+ oxidoreductase, alcohol dehydrogenase, cysteine proteinase and peroxidase are up-regulated in S-morph, and those genes are involved in programmed cell death, which are related with S-morph fruit setting incapability. Our qRT-PCR result is not in complete conformity with iTRAQ result, probably due to post-transcriptional regulation of flower morphology.Our study on cell morphological features and differential expression of miRNAs and during heterostyly development in eggplant will provide important information foruncovering the heterostyly development model and molecular mechanism of angiosperm plant, and give important reference value for the research on basic theory and agricultural improvement, breeding, etc.