Analysis Of EST Sequence And Expression Profile Of Wheat Introgression Cultivar SR3

Genomic variation is a fundamental driving resource for species evolution, and is always going on during the whole period of evolution. Wheat is an allopolyploid, and during its formation the hybridity and polyploidy played a strong genomic shock, which induced a high frequency of genomic variation and made a diploidizaton of the allopolyploid. Using the asymmetric somatic hybridization method, we have released the bread wheat cultivar Shanrong No.3 (SR3), a somatic hybrid introgression line obtained from hybrids of common wheat Jinan 177 (JN177) as a recipient with Thinopyrum ponticum as a donor. During asymmetric somatic hybridization, transitory co-existing of biparent genomes in one nucleolus, large-scale extinction of donor chromatins and introgression of a few donor chromatins may occur. These bio-events are of a specific diploidization, which may also play a strong genomic shock and induce genomic variation. However, the characteristics and mechanism of asymmetric somatic hybridization induced genomic variation as well as its difference from allopolyploidization induced one has not been well documented.Biomarker and functional gene analysis indicated that genetic and epigenetic variation had been taken place in the genome of SR3, but the extent and characteristics of this variation is still unclear. In this work, we proposed to construct cDNA libraries of SR3 and its parent JN177, and perform a large-scale EST sequencing, EST functional annotation and cDNA microarray hybridization, and then outline the effect of asymmetric somatic hybridization on the structure and expression of SR3’s genome as well as its relationship with the high salt tolerance capacity of SR3, with the aim to provide evidence for elucidate the mechanism of asymmetric somatic hybridization induced genomic variation.1. Construction of cDNA libraries of SR3 and JN177 Based on the object of this work, four cDNA libraries were constructed with two methodologies:SR3 salt-stressed root and leaf mixed library and SR3 drought-stressed root and leaf mixed library were constructed with the Stratagene method; SR3 salt- and drought-stressed root mixed library and JN177 salt- and drought-stressed root mixed library were constructed by the Gateway method. The result showed that the capacities of these four libraries were all more than 10’, and the average lengths of inserted sequences were all larger than 1.3kb, showing that these high-quality libraries can be used for large-scale sequencing.2. Large-scale sequencing and annotation of cDNA libraryWith the 5’-sequecing method, we obtained 18192 SR3 and 9770 JN177 5’-ESTs with high-qualities, and their total lengths were 11678320 bp and 7131983 bp, respectively. These sequences were assembled into 9634 SR3 and 7107 JN177 unigenes using the CAP3 software, of which contigs were 2097 and 1207, and sigletons were 7537 and 5900, respectively. These unigenes were subjected to local-BLAST against the public wheat EST database, and 476 SR3 and 594 JN177 unigenes had no identified alignments, which were new wheat ESTs or exogenous sequence of Thinopyrum ponticum. GO annotation and classification were performed with the Blast2GO software, and high abundance of stress and pathogen resistance associated ESTs were found in SR3. The codon bias of SR3 and JN177 ESTs were analyzed using the codonw software, and the result showed that the GC contents of third nucleotide of codon of SR3 and JN177 ESTs were 67.4% and 69.7%, respectively, significantly higher than those of whole sequences, and that the GC content of SR3 ESTs were significantly lower than that of JN 177.3. Polymorphism analysis of SR3 and JN177 ESTsIn order to comprehensive analysis the sequence polymorphism among wheat cultivars and that induced by asymmetric somatic hybridization, three alignments were carried out:JN177 ESTs vs public wheat EST database (JN177-Ta), SR3 ESTs vs public wheat EST database (SR3-Ta), SR3 ESTs vs JN177 ESTs (SR3-JN177). Then the aligned sequences with identity more than 96% were selected for polymorphism analysis, of which SR3-JN177 had 2581, SR3-Ta had 7284, JN177-Ta had 5072.In comparison with Ta, the frequencies of single nucleotide polymorphisms (SNPs) and 1-22nt insertions/deletions (InDels) were 9.02 SNPs/kb EST and 3.81 InDels/kb EST in JN177 ESTs, and 9.09 SNPs/kb EST and 4.29 InDels/kb EST in SR3 ESTs. In comparison with JN177, the frequencies of SNPs and InDels were up to 11.8 SNPs/kb EST and 5.44 InDels/kb EST in SR3 ESTs. These results indicated that asymmetric somatic hybridization can induce high frequency of genomic variation, and the polymorphism of SR3-Ta were not the plus of polymorphism of JN177-Ta to new-occurred one during asymmetric somatic hybridization, which suggested that there may have a reversible variation mechanism, and InDels has a lower reversible potential.The results indicated that the frequencies of different types of SNPs and InDels were unequal. In comparison with JN177-Ta and SR3-Ta, the proportion of Aâ†'T and Tâ†'A in total SNPs were lower in SR3-JN177, whereas the proportion of Câ†'T was remarkably higher (16.6%:14.8%). The substitution of Câ†'T is majorly achieved through deamination of methylated cytimidine, demonstrating the shift of methylation in genome during asymmetric somatic hybridization. The proportions of different types of SNPs between JN177-Ta and SR3-Ta were also inequable, of which Gâ†'A shared 17% in SR3-Ta and 14% in JN177-Ta, suggesting the difference of reversion among SNPs. One-nucleotide insertions contributed to 62% of InDels in SR3-JN177,74.6% in JN177-Ta, and 76.4% in SR3-Ta, showing the role of inequilibrium between insertion and deletion in low reversible potential of InDels, and the higher equilibrium between insertion and deletion during somatic hybridization than natural and allopolyploidized evolution. The higher frequencies of large (>22bp) deletions than those of insertions in JN177-Ta and SR3-Ta indicated that diploidization is an on-going evolutionary event in allopolyploidy wheat. However, the higher frequency of larger insertions than that of deletions implied the obvious distinction between diploidization mechanisms in asymmetric somatic hybrids and allopolyploids.The analysis showed that the GC content of InDel flanking sequences had specificity. In JN177-Ta and SR3-Ta, the GC contents of the left flanking nucleotide of insertions were very high whereas those of the right one were very low, but the result of deletion was quite opposite. In SR3-JN177, the GC contents of the left flanking nucleotide of insertion and deletion were both high whereas those of the right ones were both low. This revealed that InDels are not random bio-event and GC content may be the identification label of InDel sites, and that the identification mechanisms may be somewhat distinct from each other in asymmetric somatic hybrids and allopolyploids. Interestingly, we found the "3N" rule of 6-22nt InDels, that the frequencies of 6,9,12, 15,18 and 21nt InDels as well as the GC contents of their flanking sequences were all higher than those of InDel with adjacent sizes. Also a correlationship between InDel frequency and GC content of flanking sequences was found, and the correlationship in SR3-JN177 was the highest.Besides, repeated, slippage and translocated sequences as well as inverted repeats were found in SR3-JN177, showing diverse patterns of genomic variation were taken place during asymmetric somatic hybridization.3. cDNA microarry analysisIn order to reveal the effect of high frequency of genomic variation on transcripteome and the molecular basis of SR3’s stress tolerance, we performed a cDNA microarry analysis to compare the difference of transcriptome between roots of SR3 and JN177 seelings as well as the difference of their response to salt and drought stress. In this work, a customized 60-mer cDNA microarray with 15172 probes designed from public wheat EST assembled sequences and part of our cDNA library sequences was used to cover the exogenous sequences from Thinopyrum ponticum.Under the control, there had 508 probes with differential expressions between SR3 and JN177, including 241 with high and 267 with low transcription levels in SR3. GO term ’response to stress’ was significantly enriched in highly transcribed probes, including cold-regulated protein, heat-shock protein and dehydrins. Besides, the expression of phosphate pathway, ascorbate and aldarate metabolism and some JA and GA biosynthesis and response associated probes was enhanced, but carbon and energy metabolism associated probes were restricted.Under 0.5h NaCl treatment, up-regulated probes in SR3 and JN177 were 122 and 87, and down-regulated were 101 and 218, of which 48 and 75 were shared by two cultivars. Under 0.5h PEG treatment, up-regulated probes were 215 and 210, down-regulated were 81 and 143, and 71 and 29 were overlapped, respectively. Under 24h NaCl treatment, up- and down-regulated probes were increased to 1451/1556 and 1579/1507 in SR3 and JN177, and 950 and 978 were found in both of cultivars. Interestingly, except for four probes, all other un-co-responsive probes showed cultivar-specific responsive pattern, that is those responded to stress in one cultivar and kept constant in the other. Bio-process enrichment showed that unsaturated fatty acids and lipids synthesis and alpha-linolenic acid metabolism were obviously promoted in SR3 under 0.5h PEG and NaCl treatment; Ascorbate and aldarate, glycolysis/gluconeogenesis, unsaturated fatty acids and flavoniods synthesis were markedly accelerated in SR3 under 24h NaCl treatment. The enhancement of these stress-responsive bio-processes partially contributed to the systematic improvement of G protein-phospholipid-ABA/DREB signaling pathways and hormone synthesis and regulatory network.The alignment of ESTs with cDNA microarry probes extracted 3471 and 1618 probe-matched ESTs in SR3 and JN177, respectively, and correlationship between EST abundances and matched probes’signals was not found. In order to address the effect of variation on expression, we selected 61 SR3 ESTs that were homologous between SR3 and JN177 and had matched probes. Of them,17 had matched probes with higher abundance in SR3, all of which had SNPs, and 15 of which had InDels; 42 had matched probes with lower abundance in SR3,39 of which had SNPs and 27 of which had InDels.According to EST polymorphisms and probe annotations, we found the difference in gene expression between SR3 and JN177 was partially resulted from following genetic and epigenetic factors:1. high frequency of genomic variation such as SNPs and InDels; 2. the introgression of exogenous sequences from Thinopyrum ponticum; 3. change in transcriptional activity of retrotranspons and transpons; 4. chromatin remodeling resulted from the change in expression of histone variants and histone modification genes; 5. the alternation of DNA methylation patterns.Taken together, the results of large-scale sequencing of SR3 and JN177 cDNA libraries, comparison of sequence and cDNA microarry analysis indicated that high frequency of genomic variation and gene expression were taken place during asymmetric somatic hybridization, which changed the stress tolerance capacity and development potential of SR3; the mechanisms of genomic variation during asymmetric somatic hybridization, allopolyploidization and natural evolution were largely distinct. This work provided important information for understanding the mechanisms of asymmetric somatic hybridization and salt tolerance in plants.