Development And Molecular Cytogenetic Analysis On Derivatives Of Triticum Aestivum-Leymus Mollis

Leymus mollis(2n = 4x = 28, Ns Ns Xm Xm) is an important tetraploid species in Leymus(Poaceae: Triticeae) and a useful genetic resource for wheat breeding because of the desirable characters, such as a perennial growth habit, strong rhizomes, vigorous growth, a high tillering ability, good fecundity, a high number of seeds per spike, stress tolerance and good resistance to fungal diseases. By wide hybridization and chromosome engineering technology, it can transfer the excellent genes of L.mollis into common wheat, which will be useful for enriching germplasm resources and enhancing genetic diversity. In the present study, the different derivatives were identified and evaluated by the methods of morphology, cytology, molecular marker techniques and in situ hybridization technology(GISH/FISH). The main results were as follows:(1) Preliminary karyotype analysis of L.mollis was carried out and its karyotype formula was 2n = 4x = 28 = 22m(6sat) + 6sm, m means metacentric chromosome, sm means submetacentric chromosome, sat means satellite chromosome, belonging to "2A" of Stebbins. In total 825 primers were used to amplify the L.mollis, Psathyrostachys huashanica(Ns), Thinopyrum bessarabicum(J), Th.elongatum(E), Psathyrostachys huashanica(St). The polymorphic analysis results showed that the genetic relationship between L.mollis and Ps.huashanica was much closer than the other three species.(2) Three derivatives of common wheat 7182 and L. mollis, M47, M42 and M51, were subjected to chromosomal characterization via cytogenetic identification, the analysis of molecular markers and genomic in situ hybridization(GISH). These derivatives were all morphologically and cytogenetically stable. M47 was highly resistant to powdery mildew and nearly immune to stripe rust at the adult stage, and the chromosome constitution of this derivative can be expressed as 2n = 56 = 42 T.a + 14 L.m(where T.a=Triticum aestivum chromosomes; L.m=L. mollis chromosomes). Compared to M47, M42 was also resistant to stripe rust but was susceptible to powdery mildew; the chromosome constitution of M42 was 2n = 54 = 42 T.a + 12 L.m, in which a pair of homoeologous group 7 L.m chromosomes was eliminated. Finally, M51 was susceptible to powdery mildew and stripe rust and had a chromosome constitution of 2n = 48 = 42 T.a + 6L.m, in which four pairs of L.m chromosomes from homoeologous groups 2, 4, 5 and 7 were eliminated. The differing disease resistances of the three derivatives are discussed in this report in the context of their chromosomal variations; this information can thus contribute to breeding disease resistant wheat with the potential for applying these derivatives as useful bridge materials.(3) We developed a new disomic addition line M11028-1-1-5(2n = 44 = 21 " + 1") from a cross between wheat cv. 7182 and octoploid Tritileymus M47. Cytological observations demonstrated that M11028-1-1-5 contained 44 chromosomes and formed 22 bivalents during meiotic metaphase I. The genomic in situ hybridization(GISH) investigations showed this line contained 42 wheat chromosomes and a pair of L. mollis chromosomes. SSR, EST and PCR-based Landmark Unique Gene(PLUG) markers were screened to determine the homoeologous relationships of the introduced L.mollis chromosomes in wheat background. Eleven markers, i.e., Xwmc256, Xgpw312, Swes123, CD452568, CD491981, BG604865, BF483643, BQ169205, TNAC1748, TNAC1751 and TNAC1752, all of which were located on the homoeologous group 6 chromosomes of common wheat, amplified bands unique to L. mollis in M11028-1-1-5. Gliadin analysis also confirmed that the added chromosomes in M11028-1-1-5 were correlated with the sixth group chromosome. This indicated that M11028-1-1-5 contained a pair of introduced L. mollis chromosome belonging to homoeologous group 6, which we designated it as Lm#6Ns disomic addition line.(4) M11003-3-1-15-8 and M11003-3-1-12-10 both were developed and selected from the BC1F5 progeny between wheat cv. 7182 and octoploid Tritileymus M47(2n = 8x = 56, AABBDDNs Ns). Cytological observations suggested that the chromosome constitution of M11003-3-1-15-8 and M11003-3-1-12-10 was 2n = 6x = 42 = 21Ⅱ and 2n = 44 =22Ⅱ, respectively. The sequential FISG-GISH investigations and nulli-tetrasomic analysis showed that M11003-3-1-15-8 contained 40 wheat chromosomes and a pair of L. mollis chromosomes, while M11003-3-1-12-10 carried a pair of L. mollis chromosomes and a whole set of 42 wheat chromosomes. EST-STS multiple loci markers and PLUG(PCR-based Landmark Unique Gene) markers confirmed that the introduced L. mollis chromosomes in both lines belonged to homoeologous group 7, it was designated as Lm#7Ns. Therefore, it was deduced that M11003-3-1-15-8 was a wheat – L. mollis Lm#7Ns(7D) disomic substitution line and M11003-3-1-12-10 was a wheat – L. mollis Lm#7Ns disomic addition line. Field disease resistance demonstrated that the introduced L. mollis chromosomes Lm#7Ns were responsible for the stripe rust resistance at the adult stage, they could be exploited as important genetic materials in wheat resistance breeding programs and genetic resources.(5) A common wheat-L. mollis double monosomic addition line M11003-4-3-8/13/15(2n = 44 = 42 T.a + L.m2 +L.m3) with resistance to stripe rust was developed in 2014(where T.a=Triticum aestivum chromosome, L.m=L. mollis chromosome; L.m2/L.m3=L. mollis chromosome of homoeologous group 2/3). The progenies of this line were characterized by cytogenetics, molecular markers, florescence in situ hybridization(FISH) and genomic in situ hybridization(GISH). Among the progenies, there existed five different types(Ⅰ, Ⅱ,Ⅲ, ΙV and V) of chromosomes constitution, and that mean 2n = 44 = 42 T.a + 1L.m2 + 1L.m3, 2n = 43 = 42 T.a + 1L.m2, 2n = 43 = 42 T.a + 1L.m3, 2n = 42 = 42 T.a and 2n = 44 = 42 T.a +2L.m3, respectively. Field disease screening showed that type Ⅰ, Ⅲ and V were resistant to stripe rust, while type Ⅱ and ΙV were susceptible to that. And meanwhile, a wheat-Leymus mollis disomic addition line M11005-1-2-7-10 introduced one pair of L.m3 chromosomes, also performed resistant to stripe rust. From the above results, we can conclude that Lm3 chromosome was responsible for stripe rust resistance at the adult stage. These various materials could be fully exploited as important genetic resources in wheat disease breeding analysis.