| As a secondary genetic resource of common wheat (Triticum aestivum L.,2n=6x=42,AABBDD), wild emmer (Triticum dicoccoides L.,2n=4x=28, AABB) possesses abundantgenetic diversity and many excellent beneficial genes. The genetic background of commonwheat have been narrowed and declined the adaption ability to the environment. Whatmore,wheat varieties is the low processing quality. Therefore, new genetic resources are extremelyneeded to be explored and exploited, in order to enrich the wheat genetic background andenhance its quality. In this work, the genetic diversity of110wild emmer materials from theMiddle East as well as genetic differentiation of A, B genome in evolution process ofcommon wheat are analyzed through SSRs from the nuclear genome and chloroplast genome,respectively. The relationship between HMW-GS and flour quality of wild emmer wasexplored through the analysis of its flour quality and the differences of HMW-GS composition.The molecular basis between HMW-GS and quality effect was investigated through cloningsix HMW-GS genes of wild emmer Glu-1B locus, and the evolution of common wheat Bgenome was explored based on the multiple alignments of HMW-GS gene homologoussequence. The main results are as followed:1. Forty-three of58pairs of the nuclear genome SSR markers amplified stable andpolymorphic types. Totally,545allelic variations are detected in these primers. The averageNei’s genetic diversity index (He) and polymorphic information content (PIC) based on SSRprimers are0.842and0.825, respectively. Among them, the average Nei’s genetic diversityindex (He) of hexaploid and tetraploid wheat are0.431and0.600, respectively, while that ofthe wild emmer is0.836. The phylogenetic tree is built according to Nei’s genetic distancesobtained from the nuclear genome SSR loci, in which110wild emmers are divided into three categories with the genetic distance from0.3534to0.3807and an average of0.3664. In Agenome, the genetic distance of tetraploid wheat and wild emmer is0.4209, while that oftetraploid wheat and common wheat is0.7056. Comparatively, the tetraploid wheat and wildemmer exhibits a genetic distance of0.4497, and tetraploid wheat and common wheat showsa value of0.5441in B genome. This implies that A, B genomes of wild emmer appear acertain extent of differentiation in the evolution of common wheat with different evolutionaryrate.Twenty-one of24pairs of the chloroplasts genome SSR markers amplified stable andpolymorphic types. Totally,56allelic variations are detected in these primers. The averageNei’s genetic diversity index (He) and polymorphic information content (PIC) based on SSRprimers are0.279and0.248, respectively. The phylogenetic tree is built according to Nei’sgenetic distances obtained from the chloroplasts genome SSR loci, in which110wild emmersare divided into three categories with the similarity coefficient is0.904. The genetic distancesof wild emmer and tetraploid wheat, wild emmer and common wheat are0.27and0.3504,respectively. This indicates that their chloroplast genome has a high similarity.2. The HMW-GS composition of110wild emmers is identified by SDS-PAGEelectrophoresis. Altogether9subunit types are detected on the Glu-A1locus, in which the1subunit appears the highest frequency. Moreover,0.8,0.9,1.1and1.2subunits, which arerarely appeared in the Glu-A1locus of common wheat, are also detected. Additionally,30kinds of subunits and subunit combinations are detected on the Glu-B1locus, among whichthe subunit combination of13+16appears the highest frequency of12.8%. Relatively highfrequencies occur in13+18,7+8,7+16,7+18,6+15and6+18subunit combinations. Inaddition, many individual subunits appear on the Glu-B1locus. The results show thatHMW-GS of wild emmer from Israel, Syria and Lebanon present abundant allelic variationtypes. 3. Contrasted to Yumai34grown under the same conditions, flour quality parameters ofwile emmer (dough development time, dough stability time, degree of softening) areextensively and systematically compared and analyzed. The results show that doughdevelopment time and stability time of most the materials are rather short. The averagedevelopment and stability time are1.73and1.68minutes, respectively. Whereas, the degreeof softening is high with an average value of116.6FU. This indicates that the flour quality ofthese materials is poor. However, some materials with good flour quality parameters are found,such as J129, J135, and TD-20. Especially, the dough stability time of J129reaches5.3minutes, even better than that of Yumai34. This shows that wild emmer can provide somevaluable germplasms for wheat quality breeding.4. Six HMW-GS genes on the Glu-1B locus are obtained through the method of"subcloning of middle segments", namely TD-1Bx6, TD-1Bx13, TD-1Bx14, TD-1By15,TD-1By16and TD-1By18. Six recombinant expression vectors are constructed and theirprokaryotic expression is induced, demonstrating that the cloned genes are encoded by1Bx6+1By18,1Bx13+1By16and1Bx14+1By15subunits. The β-turn sequences in the middlerepeat region of these HMW-GS from the wild emmer and common wheat (1Bx7,1Bx13,1Bx14,1Bx17,1By8,1By15,1By16,1By18subunit) are compared. It is observed that thetotal number of four repeat sequences in1Bx subunit is more than those of1By subunit,indicating that the x-type subunits of HMW-GS contain more β-turn structure and havegreater influence on dough quality in comparison with y-type subunits. The quantity of β-turnstructure is the most in TD-1Bx13and1Bx13subunits, and those of TD-1Bx6, TD-1Bx14and1Bx14subunit are nearly the same. However, the flour quality of TD-1Bx14subunit ispoor since it only contains two cysteine residues. In1By subunit, the repeat sequence inTD-1By18, TD-1By16and1By15subunit are comparatively abundant, indicating that thethree subunits have a relatively large impact on flour processing quality. While the repeat sequence in TD-1By15,1Bx8,1Bx18subunits are relatively few. The β-turn sequences of theGlu-1B loci in wild emmer J127, J129, TD256and common wheat were compared. Theresults show that TD-1Bx13+TD-1By16subunit combination contains the most repetitivesequences, and the quantities of β-turn sequences of TD-1Bx6+TD-1By18and1Bx14+1By15subunit combinations are close. This indicates that the TD-1Bx13+TD-1By16andTD-1Bx6+TD-1By18subunit combinations posses superior quality, which can enhance thedough flexibility. The β-turn sequences of TD-1Bx14+TD-1By15subunit combination arerelatively few. Moreover, TD-1Bx14subunit only contains two cysteine residues. Thus, it isnot difficult to understand the poor flour quality of TD-1Bx14+TD-1By15subunitcombination. The above analysis partly explains the flour processing quality differences ofwild emmer decided by HMW-GS.The genetic relationship of wild emmer and other Aegilops species is compared throughx-and y-type HMW-GS gene sequences, respectively. The graph analysis shows that the wildemmer and common wheat have a high similarity coefficient, indicating that the B genome ofwild emmer and common wheat has a closer relationship. The genetic similarity between wildemmer and common wheat improves significantly in the evolution process of common wheat.In addition, the phylogenetic tree is built based on the alignment of gene sequence of x-typeHMW-GS subunit, from which it is obvious that the Aegilops.speltoides, Aegilops.longissimaof Aegilops and wild emmer gather together, indicating that it is most possibly at least theabove mentioned species involve in the origin of common wheat B genome. Similarly, thephylogenetic tree is established according to the alignment of gene sequence of y-typeHMW-GS subunit, wherein the Aegilops species and wild emmer have low sequencesimilarity. This means the sequences of common wheat B donor genome undergoessignificant change in the genome evolution process of common wheat. Compared with thex-type HMW-GS gene sequence, that of the y-type from wild emmer experienced greater sequence variation.Currently, great focus has been put on the wheat quality breeding with strong glutenwheat varieties. Besides A.tauschiii contained superior subunits can provide germplasmresources for improvement of common wheat D genome, this work provides excellentexogenous germplasm resources for improvement of common wheat A, B genome, which setsa theoretical basis for wheat quality breeding. |
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