Saturation Mapping Of FHB Resistance Gene Fhb7 Derived From Thinopyrum Ponticum And Its Marker Assisted Selection

Wheat is one of the most important crops in the world. Wheat Fusarium head blight, also called wheat scab, predominantly caused by the fungus Fusarium graminearum Schwabe[telomorph, Gibberella zeae(Schw.) Petch] is one of the major diseases of wheat in many areas of the world. It causes significant yield losses as well as reduced grain quality and functionality due to contamination with mycotoxins, such as deoxynivalenol(DON) and nivalenol(NIV). No single strategy has proven effective in mitigating the effects of FHB,but one promising avenue is the development of more resistant wheat cultivars, limiting both fungal growth and accumulation of mycotoxins. Sources of FHB resistance used in current wheat breeding programs can be traced to very few parents, which include Sumai 3 and its derivatives, Wangshuibai and Wuhan 1. Development of only one or a few sources of resistance over large crop production areas poses vulnerability to resistance breakdown and disease epidemics. It is rare to find wheat cultivars with high levels of resistance to FHB,and no source of immunity has been identified. Therefore, discovery, development and characterization of new resistance sources will provide breeders with a wider choice of germplasm.The objectives of the present study were to(1) produce 7el1-, 7el2-, 7Ee-, and 7Eichromosome-specific COS markers to supplement meiotic chromosome pairing and GISH data for revealing genetic relationships among the 4 above-mentioned Thinopyrum chromosomes;(2) saturate this FHB resistance gene region using SSR, DArT and conserved markers;(3) develop and characterize small segment translocation lines carrying Fhb7 by reducing the amount of Th. ponticum chromatin using ph1b-induced homoeologous chromosome pairing; and(4) pyramid Fhb7 with Fhb1 by marker-assisted selection. The results were described in detail as follows.(1) The results indicated that four Thinopyrum chromosome 7E paired with each other in wheat, indicating that these chromosomes were closely related. The frequency of chromosome pairing was different in different combination of Thinopyrum chromosome 7E.Out of them, chromosome 7Ee with 7el1, 7el2 and 7Ei, and 7el2 with 7Ei had the lower meiotic chromosome pairing rate. However, 7el1 paired quite well with both 7el2 and 7Ei with meiotic pairing rates of 71.64 and 85.96%, respectively.(2) Four wheat-Thinopyrum 7E substitution lines, 7el1(7D), 7el2(7D), 7Ei(7D), and7Ee(7D), were utilized for GISH study. The alien chromosomes in the wheat-Thinopyrum substitution lines 7el1(7D), 7el2(7D), 7Ei(7D), and 7Ee(7D) were identified by using P.strigosa genomic DNA as the probe and Triticum aestivum genomic DNA as the block. The results showed that strong hybridization signals were distributed at the centromeres and the terminal regions of 7el2, indicating that 7el2 belonged to the Js genome. When using T.elongatum genomic DNA or P. strigosa genomic DNA as the probe and T. aestivum genomic DNA as the block, the hybridization signals on 7Ee were similar to each other.However, strong hybridization signals were only observed on the terminal regions of 7el1 and 7Ei, showing that both of them belonged to the J genome.(3)Based on the data given in online supplementary table 1, cluster analysis on the 4Thinopyrum 7E chromosomes was performed to generate dendrograms constructed by the COS markers and the frequency of meiotic pairing, respectively. The genetic relationships of the 4 chromosomes estimated by the COS markers were in agreement with the results revealed by the frequency of chromosome pairing. The results indicated that 7el1 and 7Ei were the most closely related. 7el2 was relatively distant from the 7el1–7Eicomplex. While7 Ee was distant from 7el1, 7el2 and 7Ei.(4)This is the first report showing that 7el1 and 7Ei may be similar, which could be explained by the similar chromosome signal distribution revealed by GISH as well as UPGMA analysis revealed by both molecular marker and the highest frequency of meiotic pairing.(5)Based on the corresponding EST sequences of these EST-STS markers and blast analysis, the orthologous genomic regions were identified in rice, Brachypodium and sorghum. The Thinopyrum genomic region containing Fhb7 was presumably orthologous to rice chromosome 6, Brachypodium chromosome 1 and sorghum chromosome 10.(6) The predicted genes of rice, Brachypodium and sorghum in the corresponding orthologous region of Thinopyrum chromosome 7el were used to develop molecular markers linked to FHB resistance gene Fhb7. The putative orthologous or non-orthologous genes between Os06g51330 and Os06g51570 in rice, Bradi1g29130 and Bradi1g29510 in Brachypodium and Sb10g031110 and Sb10g031310 in sorghum were selected to develop markers to narrow the collinearity regions among Thinopyrum, rice, Brachypodium and sorghum. The coding sequences(CDS) of these genes were used as queries to search fororthologous wheat, barley and wheatgrass ESTs. After elimination of unlinked markers and other small linkage groups, the high density genetic map contained 167 markers covering158.97 cM, with an average density of one marker per 0.95 cM. Of the six conserved markers, XsdauK60 and XsdauK130 were assigned to deletion bin 7BL10-0.78-1.0, and XsdauK13 and XsdauK66 were assigned to deletion bin 7DL10-0.82-1.0. These markers permitted mapping of Fhb7 within a 1.7 cM interval flanked by molecular markers XsdauK66 and Xcfa2240. Thus, Fhb7 was most closely linked to Os06g51490 and Os06g51500 in rice representing a 18.8 kb genomic region and corresponding to a 37.1 kb genomic region in Brachypodium and a 27.0 kb genomic region in sorghum.(7) Of the 6 polymorphic markers mapped in the refined Fhblop linkage map in Thinopyrum, 4 conserved molecular marker can be used to identify orthologous genes in the corresponding genomic regions of rice, Brachypodium and sorghum. The one Thinopyrum orthologue XsdauK144 is Bradi1g29441 in Brachypodium, but its orthologue is not present in rice and sorghum in the corresponding genomic regions. In addition, the other Thinopyrum orthologue XsdauK130 is Bradi1g29320 in Brachypodium and Sb10g031180 in sorghum, but its orthologue is not present in rice.(8) Gene inversion, which is one of driving forces for genome evolution and play important role in adaption to stress and environmental changes for species, is very common in the grass family. In this study, one segmental inversion(Bradi1g29250-Bradi1g29300)was found in Brachypodium compared with the corresponding genomic regions of Fhblop locus in rice and sorghum. Our high density genetic map of the Fhblop revealed that the Thinopyrum genomic region(XsdauK60-XsdauK71) was syntenic to the orthologous genomic regions of rice(Os06g51490-Os06g51510) and sorghum(Sb10g031240-Sb10g031265), but complete inversion was observed in the corresponding region of Brachypodium(Bradi1g29250-Bradi1g29300). Brachypodium is phylogenetically closer to wheat relative species than rice and sorghum and could be more useful for Thinopyrum marker development and comparative genomic research.(9)To identify introgression lines carrying Fhb7 on smaller alien segments, 208 BC1F2 individuals were genotyped for five markers Xcfa2240, Xgwm333, Xswes130, XsdauK66 and Xmag1759; Lines 338-29 and 338-63 carrying much smaller translocated Thinopyrum segments were presumed to have the structure T7 DS.7DL-7el2 L. In addition, six molecular markers(Xbarc76, Xwmc273, XBM137749, XCJ664083, XsdauK352 and Xpsp3003)between Xmag1759 and Xcfa2240 were selected. Our results indicated the 7elL-7DLtranslocation might be between Xwmc273 and XBM137748. The two lines with putatively shortened segments and homozygous for the Thinopyrum Xcfa2240 and XsdauK66 alleles were evaluated for FHB response using the parental lines and the resistant and susceptible varieties as controls. Plants were classified as resistant or susceptible. FHB response differences represented by NDS between the parental lines KS24-2 and CS ph1 b were significant at P = 0.01. The two new lines with terminal 7el2 L fragments, designated as SDAU1881 and SDAU1886, were resistant, with mean NDS of 1.4 and 1.5, respectively,similar to KS24-2.(10) Based on FHB responses, GISH and molecular maker analysis, the shortened Thinopyrum chromatin segments in SDAU1881 and SDAU1886 were located in the distal region of chromosome 7DL. The sizes of the Thinopyrum chromatin segments in the entire chromosomes were 16.1%(25 cells) for SDAU1881 and 17.3%(30 cells) for SDAU1886 compared to 49.0%(20 cells) for line KS24-2. Therefore, about 65% of the Thinopyrum chromatin proximal to Fhb7 was removed in the two new translocation lines after ph1 binduced homoeologous recombination.(11) The three co-dominant markers XsdauK352, Xcfa2240 and XsdauK66, were selected and validated with 35 wheat accessions. The co-dominant marker XsdauK352 derived from the scaffold76033 amplified two fragments of approximately 210 and 483 bp in KS24-2, 7el2(7D), SDAU1881 and SDAU1886, and amplified one 490 bp fragment in Thatcher and CS ph1 b. SSR marker Xcfa2240 amplified two fragments of 240 and 280 bp in KS24-2, 7el2(7D), SDAU1881 and SDAU1886, and two fragments of 245 and 280 bp in Thatcher and CS ph1 b. The conserved marker XsdauK66, derived from rice gene Os06g51500, amplified three fragments of approximately 358, 360 and 420 bp in KS24-2,7el2(7D), SDAU1881 and SDAU1886, and amplified one 360 bp fragment in Thatcher and CS ph1 b.(12)To pyramid Fhb7 and Fhb1, a population totaling 300 F2 plants was generated from crosses of the new wheat-Thinopyrum derivatives and cv. Ning 7840. Four lines homozygous for markers Xcfa2240 and XsdauK66 flanking Fhb7, and Xgwm493 and Xgwm533 flanking Fhb1 were selected. The FHB responses indicated that four F2:3 lines were resistant to FHB with mean NDS ranging from 1.3 to 1.6 and not significantly different from KS24-2 or Ning 7840. The four pyramided lines putatively homozygous for both Fhb7 and Fhb1 were designated as SDAU1902, SDAU1903, SDAU1904 and SDAU1906.(13) To confirm the FHB resistance conferred by Fhb7 in different common wheat background, Fhb7 was introgressed into wheat cv. Jimai 22, Shannong 22, Taishan 23, Jinan17, Liangxing 99 and Shannong 14 by marker-assisted selection. A lot of lines with Fhb7 were obtained and then evaluated for FHB response in the greenhouse. The results indicated that lines with Fhb7 had lower NDS than its parents, meaning that Fhb7 can express in different wheat background. Compared to lines carrying Fhb7 in CS background, the lines carrying Fhb7 in different elite cultivar background were agronomically more attractive to breeders.