Identification And Sequence Characterization Of Alien DNA Segments Introgression From Secale Cereale Into Wheat With Resistance Against Stripe Rust

Stripe (yellow) rust, caused by Puccinia striiformis f.sp. tritici West. (Pst), is the most important fungal disease that causes great yield losses in wheat production. Plant Breeding Programs have been using specific resistant genes to control this disease. However, this resistance is frequently lost due to the appearance of new races, in addition to the modification of pathogen-host relationship. The most damaging of wheat stripe rust to grain yields indicates the lack of resistance sources in Southwestern China. The primary focus of breeding for disease control is new durable genetic resistance, which often involves race-nonspecific, slow-rusting mechanisms and is best identified in the adult-plant stage.Rye (Secale cereale L.) is the close sib plant of wheat, and it involves rich gene pools that can be used to improve the resistance, production, quality, and environmental toleration of wheat in breeding program. The resistance sources widely used in China most introduced from ryes cultivated in Europe. The resistance gene Yr9 located at 1BL/1RS chromosome of wheat translocation lines, has lost the resistance to new prevailed races in China. Abundance of rye cultivars haven't been used efficiently in China. So to transfer new alien resistance genes from rye to wheat and cultivate new resistance materials is the urgent task in wheat breeding program at present.Wheat lines 96-1054 used in this research are distant hybridization progenies of wheat cultivar My11 and rye cultivar 'Weining' inbred-line, and lines 96-1054 show high resistance to current stripe rust races. This article reports the resistance investigation and genetic analysis of resistance genes involved, molecular identification of alien chromatin introgression, cloning of DNA segments related to resistance, and sequence homologous comparisons. The origin of resistance to stripe rust and productive values in wheat genetic improving also be discussed. The main results as followed:1. Investigation of resistance and genetic analysis reveals that wheat lines 96-1054 show immunity to mixed races of Tiaozhong 29, 30, 31, and Shuiyuan isolates in seedling stage, and high resistance (infection type (IT) is from 0; to 1) in adult stage. The resistance of the lines 96-1054-4, 96-1054-9, and 96-1054-10 is controlled by two recessive genes and upwards. And other lines' F₁ progenies of direct cross and backcross with their wheat parent My11 are all susceptible (IT is 4) to stripe rust, and F₂ progenies shows the 1: 3 (resistance : susceptible) segregation ratio. So their resistance to stripe rust is deemed to be controlled by recessive single gene. The highresistance phenotype of wheat lines 96-1054 are related to rye parent R3.2. Genome Amplification used rye-specific repetitive sequence primers pScll9.1, pSclOC and pSc20H reveals that three DNA segments 745bp, 1012bp, and 1494bp are amplified in lines 96-1054-9 and 96-1054-11, respectively. No amplification is present in other wheat lines. So it means that the two lines involve insertions of several different length repetitive sequences. The amplification intensity of the two lines isn't identical, and the 96-1054-9 is stronger than 96-1054-11.3. The SSR analysis used 27 pairs chromosome located microsatellite primers reveals that only SCM9 located in IRS chromosome displays the amplification band of 210bp in wheat lines 96-1054-9 and 96-1054-11. So simple repetitive sequences are present in their genome.4. To clone the rye-specific sequence pSM9 from line 96-1054-9, sequence and execute BLAST search in GenBank for homologous comparison, the results reveals that pSM9 has strong homology (Identity=91%) to the 5'LTR of long terminal repeats (LTRs) of a gypsy-like retrotransposon reported in einkom {Triticum monococcum L.), and also shows 94% homology with pLmIs51, a Sa&rma-family transposon in tritileymus {Leymus mollis (Trin.) Hara). All these above suggest that the resistance to stripe rust of wheat lines 96-1054 is aroused by the insertion of retrotransposon elements, coming from rye cultivar R3.5. Using 157 pairs of EST-SSRs primers from rye and 12 pairs of representative RGA primers to assay wheat lines 96-1054 and their parents, the results reveal that no specific sequences related to R genes and other constitutive genes present in lines 96-1054.6. In 65 pairs of EST-SSRs screened from 157 pairs of EST-SSRs, seven rye-specific EST-SSR primers located in seven different chromosomes of rye respectively was identified using a set of wheat-rye ('Chinese Spring'-'Imperial') diploid addition lines. It provides a new valuable analysis mothod for identification and fine location of rye EST-SSRs under wheat background.