Development Of Germplasms For Triticum-Secale-Thinopyrum Trigeneric Hybrids And Molecular Cytogenetic Research

Wheat breeding in China has entered a "bottleneck" stage,as indicated by the fact that most newly released cultivars have poor disease resistance and grain quality.Two causes are responsible for this situation:a relative narrow germplasm basis and a greater emphasis on grain yield than on grain quality.Germplasm development is a key to wheat genetic improvement and wild relatives of wheat harbor numerous useful genes,including genes for biotic and abiotic stresses.It is estimated that only 10 to 15%of the gene resources in the wild relatives of wheat has been utilized in wheat improvement.We believe that the development of new germplasm with high yield potential,pest resistance and superior disease(resistance to Fusarium Head Blight in particular),and superior abiotic stress tolerance will facilitate to overcome the current bottleneck in wheat breeding.Triticale(X Triticosecale Wittmack)is an artificial cereal species,which has combined many good characteristics from wheat and rye,including high grain yield,good grain quality,and superior winter hardness,rust and powdery mildew resistance,and abiotic stress tolerance.However,it also shares the same weakness as wheat in poor resistance to Fusarium Head Blight.On the other hand,Thinopyrum elongatum,an important wheat relative,has many desirable characteristics such as good growth vigor,high prolificacy,high photosynthetic efficiency,good resistance to diseases,and good tolerance to salinity and draught.It is especially resistant to Fusarium Head Blight.It is,therefore,potentially an excellent gene donor for wheat genetic improvement.In this study we have developed a Triticum-Secale-Thinopyrum trigeneric hybrid by crossing a hexaploid triticale genotype T182(AABBRR)and a hexaploid amphiploid Triticum trititrigia(AABBEE)aided by the embryo rescue technique.We have developed a large number of molecular makers specific to rye and Th.elongatum chromosomes,using the high throughput Specific-Locus Amplified Fragment Sequencing(SLAF-seq)technique.We identified the trigeneric hybrids using cytogenetic techniques,in situ hybridization,and chromosome specific molecular markers.We have screened the trigeneric hybrids for reactions to Fusarium Head Blight,leaf rust,and stem rust(Ug99).Main conclusions and more details follow,1.The study showed that crossing two inter-genic amphiploids followed by embryo rescue was an efficient approach to obtain trigeneric hybrid.An F1 plant was obtained between a hexaploid triticale genotype(AABBRR)and a hexaploid amphiploid T.trititrigi T.(AABBEE)by crossing the two followed by embryo rescue.Chromosome counting and genomic in situ hybridization confirmed that it carried full sets of A and B genomes,plus 7 R chromosomes from rye and 7 E chromosomes from Th.elongatum.Chromosome count and agronomic trait measurements of F2,F4,and F5 plants revealed chromosome segregation and recombination and highly variable phenotypes.The 14 F2 plants had variable number of chromosomes from 2n=41 to 2n=50,all being greater than 2n=40.The number of chromosomes in the F4 and F5 plants varied from 2n=28 to 2n=56,with the greatest frequency occurred between 2n=40 and 2n=44 and peaked at 2n=42.Among the F4 plants,some had a phenotype resembled Th.elongatum and some resembled triticale;this was related to the number of chromosomes of Th.elongatum and rye they carried.The jointed use of GISH,ND-FISH,and chromosome specific molecular markers allowed systematic,rapid,and accurate identification of chromosome constitutions in the offspring and disease resistant trigeneric hybrids.2.Using the SLAF-seq technique a large number of DNA segment sequences specific to rye and Th.elongatum were obtained,which were used to design molecular markers specific to rye and Th.elongatum chromosomes.A total of 350 primer pairs were designed for the rye genome,and 253(72.29%)pairs of rye-specific makers were obtained.Among these 44 were genome specific,33 were multi-chromosome specific,and 176 were individual chromosome specific.Genome and chromosome specific markers account for 62.86%of the total number of primer pairs.A total of 525 primer pairs were designed for the E genome,and 280(53.33%)specific markers were obtained.Among these 49 were genome specific,80 were multi-chromosome specific,and 151 were individual chromosome specific.The ratio of specific makers over the total primer pairs was 38.1%.These markers are stable and can be used to reliably detect individual chromosomes in the wheat-rye-Th.elongatum hybrid,and will be useful in wheat breeding utilizing these trigeneric materials.3.In wheat breeding utilizing wild wheat relatives,it is essential to detect the presence of alien chromosomes and any change in the chromosome constitution.We have examined the chromosome constitutions of all eight trigenericlines using both chromosome specific markers and GISH and found that the two methods were fully consistent.It was found that all eight lines carried full sets of A and B chromosomes.Four of the eight lines(RE24-1,RE26-1,RE26-2,and RE33-3)carried a full set of rye chromosomes and carried no Th.elongatum chromosomes.The line RE36-lcarried all rye chromosomes but 2R,and carried no Th.elongatum chromosome.RE33-2 carried all R chromosomes plus a pair of 1R/1E translocated chromosome.RE62-1 had 6 pairs of rye chromosomes plus a pair of 5R/5E translocated chromosome.Line RE24-2 was not stable yet.At F6 it carried 6 pairs of rye chromosomes(1R to 6R),plus one Th.elongatum chromosome(7E)and one 7R/7E translocated chromosome(2n =7ⅡA+7ⅡB+6ⅡR+1I7E+1I7R/7E=42).It was found that the wheat genomes were intact in the trigeneric lines and the alien genome was made up of chromosomes from different homeologous chromosome groups,and it was not possible to have both R and E chromosomes from the same homeologous chromosome group in an individual.In other words,chromosome translocation occurred only within homeologous chromosome groups.4.Two phenotypically stable trigeneric lines were obtained,named RE21 and RE62,respectively.GISH and ND-FISH analysis indicated that RE21 had 2n=42 chromosomes,consisted of intact sets of A and B genomes from wheat plus 1E,2E,3E,and 5E from Th.elongatum and 4R,6R,and 7R from rye.Line RE62 also carried 42 chromosomes,consisted of full sets of A and B genome,intact chromosomes 1R to 4R,6R,and 7R,plus a translocation chromosome 5R/5E.Disease screening in a greenhouse indicated that RE21 was resistant to leaf rust,stem rust(Ug99),and in particular,Fusarium Head Blight.RE62 was also resistant to leaf rust and stem rust but was susceptible to Fusarium Head Blight.This indicates that the alien chromosomes in the trigeneric lines carries disease resistance genes and can be used in breeding disease resistant wheat cultivars,and the chromosome specific molecular makers developed in this study can be useful for this purpose.