| Genus Thinopyrum, a relative species to Triticum, as third gene pool for wheat conferring characteristics of cold resistance, drought resistance, lodging resistance, barley yellow dwarf virus resistance and rust immunity, has been considered as valuable resource in wheat genetic improvement. Thinopyrum intermedium (2n=42, genome symbol EeEeEbEbStSt or JJJsJsStSt) and Thinopyrum elongatum (2n=14, genome symbol EeEe) have been widely used in wheat improvement due to their traits of immunity or resistance to many wheat diseases. To date, many of valuable genes in these two species, including Bdv2, Lr19, Sr26, Cmc2 and Wsm1 have been successfully transferred into common wheat by chromosome engineering approach. Therefore, a series of addition lines, substitution lines and translocation lines are available for both genetic improvement and wheat breeding. However, so far molecular markers for Thinopyrum chromatin detection are lack, its necessary to develop more molecular markers and create new germplasm of wheat-Thinopyrum, in order to meet the demand of disease resistance, high production and quality.In this study, our goal is to transfer excellent genes from Thinopyrum to common wheat. Therefore, we developed molecular markers on E chromosomes (arms) and St chromosomes (arms) and identified newly synthesized Thinopyrum-wheat introgression lines. Molecular markers that we developed in this study can be used to rapidly identify E chromosomes (arms) and St chromosomes (arms) in Thinopyrum-wheat introgression lines. New Thinopyrum-wheat amphidipliods and substitution lines with strip rust and powdery mildew resistance that we characterized could provide valuable genes for wheat improvement. Detailed results in this research are listed below.1. Development and application of Thinopyrum E chromosomes (arms) and St chromosomes (arms) markers: (1) A total of 258 pairs of EST-SSR primers located on homologous group 1 to 7, 46 pairs of PLUG primers and 30 pairs of STS primers tested in Chinese Spring (CS), Th. elongatum and a set of CS-Th. elongatum addition lines, results showed 37 pairs of EST-SSR primers, 5 pairs of PLUG primers and 1 pair of STS primer can be amplified polymorphism bands in both CS and Th. elongatum. Polymorphic bands amplified by those primers located on single chromosome (arm) of Th. elongatum, which could be employed as molecular markers to trace single chromosome (arm) of Th. elongatum in common wheat genetic background. (2) By using EST-SSR primers and PLUG primers from homologous group 1 to 7 amplified in CS, Pseudoroegneria spicata and Th. intermedium. Polymorphic bands were obtained in both Ps. spicata and Th. intermedium, CS as control by 46 pairs of EST-SSR primers and 39 pairs of PLUG primers, which indicated those polymorphic bands were from St chromosomes. The primers that could amplify specific bands in St chromosomes were further validated in more species containing St chromosomes, St chromosome substitution lines and their progeny. Results showed polymorphic bands appeared in St chromosome containing species, which indicated the corresponding primers could be used as molecular markers to trace St chromosomes (arms) in common wheat background. (3) Molecular marker results also suggested that the genetic distance between genomes in Th. intermedium and D genome is shorter than genome A and B genomes. Meanwhile, frequent rearrangement was observed in E chromosomes of Th. intermedium in its evolutionary process.2. Molecular and cytological characterization and disease resistance identification of newly partial amphiploid of Wheat-Th. intermedium ssp. trichophorum. (1) C-banding result of Wheat-Th. intermedium ssp. trichophorum partial amphiploids showed that there were few bands on Th. intermedium ssp. trichophorum chromosomes, which is difficult to be used for chromosome identification. GISH analysis of Wheat-Th. intermedium ssp. trichophorum partial amphiploids revealed that genome of 1520-1, 1508-2-1, Y69, Y70 and L451-3-1 consisted of 42W+8J+6St (W indicates wheat chromosomes), 42W+6St+4Js+2J, 40W+4St+4J+2Js+2St/D, 42W+4St+4Js+2St/Js, and 40W+8St/Js+4St+2T (T indicates telomere), respectively. (2) E and St chromosome specific molecular markers from homologous group 1 to 7 that developed in this study, were employed to identify which homologous group the alien chromosomes belong to in Wheat-Th. intermedium ssp. trichophorum partial amphiploids. Our preliminary results suggested that in 1520-1, J chromosomes belonged to homologous group 4, 5, 6, 7 and St chromosomes belonged to homologous group 5, 6 and 7; in 1508-2-1, J chromosomes belonged to homologous 4 and St chromosomes belonged to homologous group 3, 6 and 7, and Js chromosomes belonged to homologous group 5 and 6; in Y69, St chromosomes belonged to homologous group 2 and 4, and J chromosomes belonged to homologous 3 and 7, and Js chromosomes belonged to homologous group 5, as well as St/D translocation chromosome belonged to homologous group 5; in Y70, St chromosomes belonged to homologous group 5 and 7, and Js chromosomes belonged to homologous group 4 and 6, and St/Js translocation chromosome belonged to homologous group 3; while in L451-3-1, there were many Js/St translocation chromosomes appeared by molecular markers. (3) Disease resistance tests including strip rust, powdery mildew and fusarium head blight showed that 1520-1, 1508-2-1, Y70 and L451-3-1 were highly resistant to stem rust; L451-3-1 was also highly resistant to powdery mildew; 1520-1, 1508-2-1, Y70, Y69 and L451-3-1 were highly resistant to fusarium head blight. Meanwhile, the transferring disease resistances from those partial amphiploids into wheat are in process.3. Characterization of wheat-Thinopyrum substitution lines: (1) 1St (1D) substitution line AS1677 was identified from wheat-Th. intermedium ssp. trichophorum offsprings by the combination of methods including molecular markers, seed protein SDS-PAGE analysis, sequential Giemsa C-banding and FISH, strip rust and powdery mildew resistance tests. AS1677 was highly resistant to strip rust and powdery mildew in both seedling and adult stage. AS1677 has a pair of St chromosomes demonstrated by sequential C-banding and GISH. PCR-based molecular marker analysis among AS1677, Th. intermedium ssp. trichophorum, Ps. spicata, Th. intermedium and CS as control, showed a pair of St chromosomes in AS1677 belonged to homologous group 1, designated as 1St. Both gliadin and glutelin analysis showed the lack of a protein band which is encoded by gene located on 1D, while FISH with pAS1, a D genome specific probe, revealed the lack of 1D chromosomes. Therefore, AS1677 is a new 1St (1D) wheat-Thinopyrum substitution line with high disease resistance. (2) 6Js (6B) substitution line X005 was identified from wheat-Th. ponticum by the combination of molecular marker, sequential Giemsa C-banding and GISH, strip rust resistance test. X005 was highly resistant to strip rust. Sequential Giemsa C-banding and GISH analysis on both 7430 and X005 revealed that they contained a pair of Js chromosomes instead of 6B chromosomes, which was also demonstrated by PCR-based molecular marker confirmation. PCR amplification were performed on X005, 7430, Th. intermedium and CS as control with newly developed molecular markers in this study. Result indicated that Js chromosomes in X005 were originated from 7430, which belonged to homologous group 6, designated as 6Js. Therefore X005 is 6Js (6B) wheat-Thinopyrum substitution line with high disease resistance.4. Characterization and genetic analysis of wheat-Th. intermedium introgression lines: F1 hybrids were obtained from crosses between 1St (1D) substitution line with powdery mildew resistance and MianYang11 with powdery mildew susceptive, and then F1 selfed to get F2 and F3 population. The powdery mildew resistance among 500 individual F3 plants was segregated, which showed that resistant strains and susceptible strains were 296 and 104 respectively, the segregation ratio is about 3:1. Therefore, we assume that the resistance of powdery mildew is from 1St chromosome controlled by a dominant gene in Th. intermedium ssp. trichophorum. Cytological observations on both F2 and F3 plants suggested that the number of chromosomes in F2 was unstable, much variable and they were abundant of telosomic, while the number of chromosomes in F3 was also variable and they had monotelosomic, ditelosomic, short chromosome (fragment) and isochromosome. These results indicated that chromosomes in F2 and F3 experienced breakage, deletion and translocation. Alien chromosomes detection of F3 population was performed by SSR, EST-SSR, STS and PLUG molecular markers. Results revealed that there were 3 types of alien chromosomes existing in F3 population, including 1StS/W (W indicates wheat chromosomes), 1StL/W translocation, 1StS and 1StL telosomic, 1StS fragment and 1StL fragment introgression. Then, the GISH analysis confirmed that the translocation line 1St/1D existing. Moreover, analysis of molecular markers, gliadin and glutelin indicated that deletion happened on the terminal of D chromosomes, which provided theoretical bases for screening small fragment translocation and deletion in wheat. To date, accurate identification of these wheat-Th. intermedium translocation lines and development of powdery mildew newly resistant gene markers are in process.In summary, series of new E and St chromosome- (arm-) specific molecular markers have been developed, which could be used for tracing Thinopyrum chromosomes. Furthermore, frequent chromosome rearrangement in Thinopyrum was revealed by molecular markers analysis. Therefore Thinopyrum as a valuable gene pool could provide excellent genes for continuous wheat chromosome engineering breeding due to its frequent rearrangement characteristic. 5 new wheat-Th. intermedium ssp. trichophorum partial amphiploids were identified and they displayed highly resistant to strip rust, powdery mildew and fusarium head blight respectly. 2 new substitution lines AS1677 and X005 were identified from 52 wheat-Th. intermedium ssp. trichophorum offsprings and 45 wheat-Th. ponticum offsprings. AS1677 is highly resistant to strip rust and powdery mildew, and X005 is highly resistant to strip rust. 2 translocation lines with powdery mildew resistance, 1StS/W (W indicates wheat chromosomse) and 1StL/W, were roughly identified from hybrids of wheat-Th. intermedium ssp. trichophorum substitution line 1St (1D) and MianYang11. Therefore, molecular markers that developed in this study could be used for assisting wheat breeding, and new disease resistant materials identified could be used as valuable intermediate gene pool for wheat breeding in the future, and they possess the important application value. |
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