| Pre-harvest sprouting (PHS) was a universal disasters, which seriously influence yield and quality. PHS in wheat is controlled by quantity trait locus and these QTLs for PHS were dispersed on wheat all 21 chromosomes. QTLs on group 3 and 4 homologous chromosomes have been identified frequently. In additional, PHS was influenced by many biological factors including dormancy, ABA sensitivity, late maturity amylase, grain color etc. Several genes involving in these pathway have been isolated. However, the relationship between regulated locus of genes and PHS reisitance QTL have been seldom reported. Therefore, detection more locus for PHS resitance and identification of its relationship with regulated locus for functional genes will be valuable to marker assistant breeding and effectively use of QTLs. In present study, a high-generation recombinant inbred lines (F10,171 lines) derived from PHS resiatant synthetic wheat SHW-L1 (synthesized from strong dormancy Aegilops tauschii AS60 and local Sichuan landrace Triticum turgidum AS2255) and PHS susceptible wheat Chuanmai32 were used for construction of high density genetic map though wheat 660K SNP assay. Then using this genetic map to detect PHS-resistance QTLs and evaluation of QTL effect in SHW-L1 breeding progeny. Meanwhile, several genes{TaAmyl encoded high PI amylase, TaVpl?TaSdr4? TaAIP2 and TaSnrk2.6 invloved in ABA signal) were carried out for expression pattern analysis and eQTL mapping, which was in order to analysis overlapped eQTLs with PHS-resistance QTLs and detect candidate genes. The main studies and results were described as follows:1) Construction of high density genetic map in wheat RILs though AxiomTM wheat 660K Arrays. The genetic map contained 121222 markers with total length of 17889.62cM. The average length of each marker was 0.148cM with corresponding 143kb each markers (wheat genome size referenced IWGSC database). Moreover, QTL mapping for PHS resistance though high density genetic map was processed in ten independent environments at 2010-2015. Nine QTLs were detected in at least two independent environments and two QTLs (qPHS.sicau-1B and qPHS.sicau-3D.2) were repeatly detected at 8 environments. Meanwhile, QTL qGC.sicau-3D associated with grain color was overlapped with qPHS.sicau-3D.2, which was close to grain color related transcriptional factor TamyblO-D. Another new major QTL qPHS.sicau-1B was independent to grain color.2) Large number of breeding lines were generated from hybridization by SHW-L1 and other Sichuan cultivars. Of them, new wheat line Shumai580 (Liu? Zhang selected, passed Yunnan area test)showed same PHS resistance level with SHW-L1 and decreased 30% germination ratio compared its bread wheat parents. Genetic background of Shumai580 showed that two QTLs (qPHS.sicau-3D.1 and qPHS.sicau-3D.2) derived from Aegilops tauschii were successfully introgressed and another major QTL qPHS.sicau-1B was probably introgressed (under fine-mapping).3) TaAmyl gene controlled grain germination rate, which was belonged to barley AMY2-1 subgroup. Six SNPs were detected in TaAmyl nucleotide sequences of SHW-L1 and Chuanmai32. In additional, the expression level at late stage of seed development (DPA25 and DPA30) in Chuanmai32 was significantly higher than that in SHW-L1. One significant eQTL eqamyl.sicau-DPA30 for both TaAmyl at DPA25 and DPA30 was overlapped with major PHS resistance QTL qPHS.sicau-1B, indicated that PHS resistance improvement by qPHS.sicau-1B was probably though decreasing TaAmyl gene expression at later stage of seed development.4) The expression level of TaVpl gene in SHW-L1 was consistently higher than that in Chuanmai32 during seed development. Meanwhile, eQTL result for TaVpl showed that one of its eQTL eqABA.sicau-1B.1 was overlapped with major QTL qPHS.sicau-1B and TaAmyl eQTL eqamy1-DPA30, revealed the expression variation of TaVpl probably participate in qPHS.sicau-1B mediated PHS resistance improvement.5) Further analysis of significant eQTL eqABA.sicau-1D for TaVpl at DPA15 and DPA20, which was orthologe to 2.8Mb interval of rice chromosome 5 and hence 4 candidate genes were found based on gene annotation (TaABIl, TaGAox20, TaAkinlO and TaRav1). Advanced expression pattern analysis showed that TaRavl gene expression pattern was consistant to that of TaVpl, revealed that TaRavl as a trans-regulator in acting TaVpl gene expression. In additional, several eQTLs controlled TaRavl gene expression at DPA15 and DPA20 were overlapped with other eQTLs for TaVpl, TaSdr4, TaAIP2, TaSnrk2.6 and TaAmyl, indicated that they were probably regulated by common upstream genes.6) Five SNPs located at -234bp,-91bp,+18bp,+379bp and +584bp in TaRav1D were detected in SHW-L1 and Chuanmai32. Among them, two SNPs located at +379bp and +584bp could change the varation of amino acid. TaRavl-D sequences in 52 hexaploid wheat were consistently with that in Chuanamai32. Others, sequences of AetRavl in Ae. tauschii showed rich gene variation and its haplotypes was correlated with the classification of Ae. tauschii. Among them, one type of Ae. tauschii ssp. strangulata was consistant with that in bread wheat. |
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