Morphology And Transcriptome Analysis Of Brassica Napus-Orychophragmus Violaceus Addition Lines With Female Sterility And Red Petals

Orychophragmus violaceus （L.） O.E.Schulz, （2n=24） belonging to Brassicaceae family keeps some specific phenotypic traits （such as purple color of petals）, which should be studied for their genetics and development by production of the addition lines carrying its individual chromosomes in Brassica napus background. In this study, two addition lines with the novel female sterility or lightly red petals were used and investigated for the anatomy of the gynoecium abortion and the transcriptomic analysis via RNA-Seq, to reveal the developmental stage of gynoecium abortion and genetic control, and to unravel the anthocyanin synthesis pathway and genetic regulation. The main results were described as follows:1. Comparative anatomy of gynoecium development in female sterilityIn comparison with the normal B. napus, the gynoecia of female sterile addition line （S1） were completely aborted, slow-growing and much shorter from early development stage （flower buds ～2 mm）, which produced no seeds. After artificial pollination, few pollen grains could adhere and germinate on stigmas of S1, and the pollen tubes grew spirally over the surface of papilla cells and hardly penetrated into stigmas. SEM observation showed that papilla cells on S1 stigmas were short, small and not fingerlike as normal ones, but globular. DIC observation of cleared ovules showed that the ovules in S1 initiated only one short inner integument primordium which underwent no further development and the outer integument could not initiate. The female gametophyte development was blocked after the tetrad stage but before megagametogenesis initiation, without the formation of functional megaspore.2. Transcriptome analysis of female sterile addition lineAfter RNA-Seq,7,502,922 （99.14%） and 6,990,973 （99.03%） clean reads were obtained in S1 and the recipient B. napus （H3）, respectively. Using Brassica₉5k_unigene as the reference genome, a total of 28,065 and 27,653 unigenes were identified to be transcribed in S1 and H3, respectively. Further comparison of the transcript abundance between S1 and H3 revealed that 4540 unigenes showed more than two fold expression differences. GO and pathway enrichment analysis of the Differentially Expressed Genes （DEGs） showed that a number of important genes and metabolism pathways were involved in the development of gynoecium, embryo sac, ovule, integuments as well as the interactions between pollen and pistil. DEGs for the ovule development were detected to function in the metabolism pathways regulating brassinosteroid （BR） biosynthesis, adaxial/abaxial axis specification, auxin transport and signaling. A model was proposed to show the possible roles and interactions of these pathways for the sterile gynoecium development in S1 plant.3. Transcriptome analysis of lightly-red-petals addition lineIn the lightly-red-petal disomic addition line （4-10） with one pair of the O. violaceus chromosomes, red pigment emerged on the petals with different extent under yellow background. After mRNA sequencing of 4-10, B. napus （H3）, natural plants of O. violaceus with purple petals （Zihua） and white petals （Baihua）, a total of 14G data and 115 million high-quality clean reads were obtained. For each of the four samples, approximately 30 million reads were obtained and 89% of which were clean reads. The clean reads of each sample were aligned to the genome of Brassica rapa, and for 4-10 and H3, approximately 60% of the clean reads matched to either a unique or multiple genomic positions,31956 and 30979 genes were obtained, respectively. Differently, only 10% of the clean reads of Zihua and Baihua matched to a unique or multiple genomic positions,30009 and 30275 genes were obtained, respectively. Using "p-value ≤ 0.01" and "fold change （FC）≥ 2 or ≤ 0.5" as DEG standard,9917 and 3818 genes exhibited different expressions between "4-10 vs H3" group and "Zihua vs Baihua" group, respectively. Gene functional classification analysis of the two groups of DEGs showed similarly that the genes related with photosynthesis and light reaction were down-regulated in both 4-10 and Zihua, but the protein translation ability was increased. Analysis of the expression of genes involved in anthocyanin synthesis revealed that most of structural genes for the anthocyanin synthesis were up-regulated in both 4-10 and Zihua, especially the last step key gene ANS. Only one regulatory gene, GL3, showed different expression in Zihua and Baihua group, moreover, its RPKM value was down-regulated to 0 in Baihua. However, there were 10 regulatory DEGs in 4-10 and H3 group, containing 8 positive regulators and 2 negative. PAP2 was one positive regulator and significantly up-regulated in 4-10. Reads assembling and sequence alignment showed that the PAP2 in 4-10 was from the transcript of O. violaceus gene.4. Over-expression vector construction of PAP2 and ANS from 4-10 and O. violaceus, and the genetic transformation of Arabidopsis thaliana and B. napusAccording to the assembly seguences and genome information of Brassica rapa, PAP2 and ANS were cloned from 4-10 and O. violaceus. Sequences alignment showed that the sequence of PAP2 in 4-10 was completely consistant with that in O. violaceus. Two copies of ANS were obtained from 4-10, one of which corresponded to one copy of B. rapa and another to one copy of B. oleracea. Two copies of ANS were also obtained from O. violaceus. Three over-expression vector, PAP2-7, ANS-12 and AZKR-7, were constructed, corresponding to PAP2 from 4-10, one copy of ANS from 4-10 and one copy from O. violaceus, respectively. Except AZKR-7, A. thaliana transgenetic plants of both PAP2-7 and ANS-12 showed different levels of purple pigments in most of the organs, including the petals. At present, only some regenerated seedlings of transgenetic B. rapa were obtained, and PAP2-7 were found to have some obvious purple leaves on the transgenetic seedlings.