Artificially Synthesized Wheat RSP And Its Preharvest Sprouting Tolerance Derived From Aegilops Tauschii Cosson

Aegilops tauschii Cosson is the wild donor species of D genome of hexaploid wheat (Triticum aestivum L.), which distributes in Asia Minor, Caspian Sea, Turkey, Iran, Iraq, Turkmenistan, Afghanistan, Tajikistan, Azerbaijan, Pakistan, etc. It also distributes in Yellow River Valley and Xinjiang in China. The genetic diversity of Ae. tauschii have been analyzed in morphology, isozyme, storing protein and molecular level, and it was found that there were larger variations in D genomes of Ae. tauschii than that of T. aestivum. It suggested that only a few Ae. tauschii accessions have participated in the origin of common wheat during forming of hexaploid wheat by crossing between tetraploid wheat and Ae. tauschii.There are a lot of biotic and abiotic resistance genes in Ae. tauschii, such as resistant to strip rust, brown leaf rust, black stem rust, powdery mildew, ocular spot, Hessian fly, aphid, mite, root-lesion nematode, freezing hardiness, salt stress, drought stress, preharvest sprouting, etc. These desirable variations in Ae. tauschii are very useful for the improvement of common wheat.Hybridization was carried out between female parent, tetraploid landrace Ailanmai (Triticum turgidum L., 2n=28, AABB) and Ae. tauschii accession (2n=14, DD) with long dormancy. The hybrid amphidiploid, named as RSP, was obtained via the seedlings treatment by 0.05% colchicine for 24 hours. RSP had 42 chromosomes by detecting the root tip cells, and has the meiosis configuration ofpollen mother cell with 2.04 1+19.91 II+0.04 III and a few cells appeared lag chromosomes at anaphase I or micronuclei at tetrad, indicating that a few cells were abnormal at meiosis.The morphological and agronomic characters of RSP were similar to parents, or situsted between the parents, however, plant height, spike length, spikelet length, glume length, seed length and 1000 grain weight were higher than those of its parents.Germination percentages at 5 filling periods of RSP were tested. The germination ability of embryos increased constantly following filling period. The high peak stages of germinations including intact spikes, seeds with glume and seed occurred on 35 days after anthesis. The germination percentages of embryos, seeds, seeds with glume and intact spikes were 53.2%, 7.8%, 4.8% and 2.9%, respectively.According to the calculations of inhibition percentages, seed had the highest inhibition percentage, up to 85.3%; followed by intact spike, up to 63.5%. It indicated that the preharvest sprouting tolerance of RSP was mostly resulted from the seed dormancy. At the high peak stage, the germination percentage of intact spike of RSP was only 6.1%. The cultivar Chuanyu 12, which was considered as a breeding resource of tolerance to sprouting in Sichuan, was 27.6%. Thus, the tolerance from RSP is expressed and much stronger than that of commercial cultivars.RSP was crossed with 6 wheat cultivars, which were diversely sensitive to preharvest sprouting. Genetic analysis indicated that the preharvest sprouting tolerance was inherited as a recessive trait controlled by one gene. The gene of tolerant sprouting was located on 2D chromosome via genetic analysis using 'Mianyang 11' monosomes (D-genome set).Simple sequence repeats (SSRs) were employed to detect molecular markers on 2D linked to the tolerance gene by analyzing F2 plants from RSP * 88-1643. Nine out of 15 microsatellite markers were polymorphic between the tolerant and susceptible parents. After evaluating these polymorphic markers in F2 population,all of the 9 microsatellite markers were positive correlation to the sprouting tolerance, and Xgwm261 mapped on chromosome 2DS was found to be linked to the tolerance gene, with an estimated genetic distance of 31.17 cM.Using PCR amplification of the genomic DNA, three novel low-molecular-weight glutenin genes (i.e. LMWRSP-1, LMWRSP-2 and LMWRSP-3) were isolated from RSP, in which, LMWRSP-2 (GenBank accession number: AY676682) is a pseudogene due to one stop code in its coding regions. The coding regions of LMWRSP-1 (GenBank accession number: AY676681) and LMWRSP-3 (GenBank accession number: AY676683) are 825 and 915 bp, encoding two LMW glutenin subunits with 274 and 304 amino acid residues, respectively. Sequence multiple alignment analysis indicated that LMWRSP-1 and LMWRSP-3 have the highest similarity scores to the known LMW-GS genes encoded by Glu-A3 and Glu-D3, respectively.Synthetic wheat RSP with sprouting tolerance derived from Ae. tauschii was crossed with red seed cultivar Mianyang 11, which has desirable agronomic characters and white seed lines YY2 and 88-1643, which was susceptible to sprouting (over 50% germinations in normal years). The preharvest sprouting tolerance of F2 plants and F3 lines from the three crossing combinations was evaluated. Twenty-one homozygous tolerance lines and 27 plants with 0% germination were screened from 'RSPXMianyangll'. Five white kernel lines with a germination lower than 7% were selected from 'RSP X YY2' and 'RSP X 88-1643'. Their agronomic characters have been improved in comparison with that of RSP. They are useful breeding materials for transferring preharvest sprouting tolerance of Ae. tauschii into commercial white kernel wheat cultivars.