Identification Of Tritielytrigia Germplasm, The Chromosomal Location And SSR Molecular Marker Of Its Resistant Gene

Elytrigia elongata is a perennial partial auto-allo-decaploid species (2n=10x=70, previously designated with genomes StStStStEeEeEbEbExEx), that is a potentially useful source of a number of genes for wheat improvement, including rust resistance, powdery mildew resistances, low-temperature and drought tolerance, high protein, an increased number of florets per spike. Tritielytrigia Shannong87074, Shannongl9, Shannong20 and Shannong122 all are novel germplasm created by the hybridization between decaploid Elytrigia elongata and common wheat. Shannong87074 is a progeny from hybrid cross ofElytrigia elongata and common wheat Lumai5 and Jinanl 3, the ten lines Shannong87074-513,-519,-526,-541,-551, -554,-555,-557,-565,576 all are derived from Tritielytrigia Shannong87074; Shannongl9, Shannong20 and Shannong122 all are octaploid Tritielytrigia types. The inoculation assessment, cytological analysis, biochemistry assay, simple sequence repeat(SSR) molecular marker technique, and genomic in situ hybridization(GISH) were employed in the identification of Tritielytrigia germplasm; The specific repeat sequence of decaploid Elytrigia elongata amplified by SSR was retrieved and cloned, and was prepared as the specific probe used in the GISH. The results are as following:1. The cytological identification indicated that Shannong87074-519 was a alien disomic addition line, with 44 chromosomes in root tip cells and basic chromosomal configuration of PMC MI 2n=22â…¡, Shannong87074-513,-526,-541,-551,-554,-555,-557,-565,576 all were alien disomic substitution lines, with 42 chromosomes in root tip cells and basic chromosomal configuration of PMC MI 2n=21â…¡, and the three Tritielytrigia interspecies Shannongl 9,Shannong20 and Shannong 122 all were novel octaploid Tritiielytrigia containing a genome of Elytrigia elongata. The morphological characterization and disease resistance evaluation showed that they appeared well agronomic characteristic, powdery mildew resistance or yellow rust resistance, more florets per spike. They will play an important role in the wheat genetic improvement.2. The GISH identification of the addition line Shannong87074-519 with root tip cells was conducted by using Ps.strigosa(2n= 14,StSt) and E.elongata(2n=14,EE) total genomic DNA as probe respectively. It showed that two chromosomes with green-yellow hybridization signal were observed among the 44 chromosomes, with the signal covering the whole chromosome in the GISH pattern with the Ps.strigosa as probe; no signal was observed with the E.elongata as probe. It indicated that Shannong87074-519 was one alien disomic addition line with two added chromosomes belonging to genome St of Elytrigia elongata.The SSR technique was further applied to verify Shannong87074-519, with 170 primer pairs testified. The results indicated that Shannong87074-519 contained genetic material transferred from Elytrigia elongata; the primer pairs BARC165 on chromosome 5AL could show the diversity between Shannong87074-519 and its parents, and the specific segment of Elytrigia elongata could be stably amplified in its DNA PCR products, whose size was about 270bp, assigned as BARC165₂₇₀ temporarily. The molecular maker BARC165₂₇₀ could be used in the verification of Shannong87074-519. Subsequently, the specific repeat segment of Elytrigia elongata was retrieved and cloned. DNASTAR software analysis showed that the nucleotide sequence length of specific segment is 268bp, then the marker was designated as BARC165₂₆₈, and the repeat sequence was submitted to the genbank with the accession number EF397435. BARC165₂₆₈ was prepared as specific probe in the GISH identification, strong yellow dot hybridization signal was observed in chromosome of Shannong87074-519, but not in the common wheat. Considering the GISH pattern by the probe St, it was inferred that the specific repeat sequence likely concentrated on chromosome St, and it was the specific repeat sequence of genome St. The repeat sequence could be used as a valuable marker to identify the germplasm of Elytrigia elongata in common wheat background.The identification of Shannong87074-519 by SDS-PAGE showed that no specific locus coded the Elytrigia elongata HWM-GS located on the added chromosome St.3. 592 primer pairs were amplified in the identification of the nine alien substitutions Shannong87074-513,-526,-541,-551,-554,-555,-557,-565,-576 by SSR molecular technique. Nine primer pairs among them could amplify the specific band of Elytrigia elongata in PCR products of the nine substitutions, which indicated the specific difference between the substitutions and their parents, and further verified that the substitutions were the progeny from Elytrigia elongata.The nine primer pairs could be used as molecular markers in the identification of the nine alien substitutions, such as:BARC066₂₃₀ and BARC023₂₇₀ of Shannong87074-513, BARC148₂₄₀, BARC066₂₃₀, BARC168_150/180, BARC232₄₅₀, BARC195₂₀₀ and BARC023₂₇₀ of Shannong87074-526, BARC148₍2400, BARC099₁₅₀, BARC232₄₅₀ and BARC023₂₇₀ of Shannong87074-541, BARC148₂₄₀, BARC099₁₅₀, BARC040₁₈₀ and BARC023₂₇₀ of Shannong87074-551, BARC148₂₄₀, BARC040₁₈₀ and BARC023₂₇₀ of Shannong87074-554, BARC148₂₄₀ and BARC232₄₅₀ of Shannong87074-555, BARC066₂₃₀, BARC040₁₈₀ and BARC023₂₇₀ of Shannong87074-557, BARC100₁₅₀, BARC040₁₈₀ and BARC232₄₅₀ of Shannong87074-565, BARC148₂₄₀, BARC066₂₃₀ and BARC023₂₇₀ of Shann0ng87074-576.The identification of the nine alien substitutions by A-PAGE and SDS-PAGE showed that the substitutions contained genetic material of Elytrigia elongata, and the substituted chromosome did not belong to the first homologous group. One set of Chinese Spring nullisomic-tetrasomic lines were utilized to further verify the homologous substitution chromosome. The results showed that, the primer pair SWES240 on 2B chromosome could amplify one specific segment in the PCR products to distinguish the differences among the tested materials, with the segment size about 400bp;The specific band could not be amplified in the PCR products of Elytrigia elongata, E.elongata(2n=14), substitutions Shannong87074-513,-526,-541,-551,-554,-555,-557,-576, and the 2B nullisomics 2D tetrasomics(N2BT2D) of Chinese Spring, but appeared in Ps.strigosa, Chinese Spring, the other 22 Chinese Spring nullisomic-tetrasomic lines, common wheat Lumai5 and Jinanl3, substitution line Shannong87074-565. It concluded that the 2B chromosomes of the eight lines Shannong87074-513,-526,-541,-551,-554,-555,-557,-576 were substituted by 2E chromosome of Elytrigia elongata, the eight lines were alien disomic substitutions with 2B/2E.4. The chromosomal pairing behavior of PMC MI of the three octaploid Tritielytrigia was analyzed and the results showed that Shannong20 and Shannong122 had the same chromosomal genomes, and belonged to the same type as xiaoyan68(previously designated with the basic genomes ABDE2 ), however, Shannongl 9 was the other type different from Shannong20, Shannong122, and the former xiaoyan68,xiaoyan693 and xiaoyan7430. The GISH patterns with Elytrigia elongata(2n=70), E. elongata(2n=14,EE) and Ps.strigosa(2n=14,StSt) total genomic DNA as probe respectively indicated that Shannongl9 was added by one set St genome, Shannong122 probably was added by one set E genome. The basic chromosomal genome of Shannongl9 could be designated as ABDSt, and that of Shannong20 and Shannong 122 was assigned as ABDE temporarily.5. The inoculation assessment, cytological analysis, SSR molecular marker technique, and GISH identification were used in the identification and chromosomal location of resistant genes. Three resistant genes derived from decaploid Elytrigia elongata, YrSt andYr2E with the yellow rust resistance, and Pm2E with the powdery mildew resistance, were located on St chromosome and 2E chromosome respectively. It could be inferred that the three resistant genes likely all were novel genes.The F₂ segregating population of alien substitution line Shannong87074-557 and common wheat huixianhong was employed to further analyze the dominant resistant gene on the substitution chromosome by SSR technique. Two primer pairs that linked to the substitution chromosome were singled out from 592 SSR primer pairs of wheat, one was Xgwm344_120/150 on the 7B chromosome of wheat, it linked to the powdery mildew resistant gene Pm2E and yellow rust resistant gene Yr2E; the other was Barc074₁₅₀ on the 5BL chromosome, it linked to the leaf rust resistant gene.