The Germplasm Diversity, Genetic Analysis And Molecular Markers Of The Important Characters Of Zoysiagrass

Zoysiagrass is a well-known warm season turfgrass all over the world and belongs to the subfamily Chloridoideae in Gramineae. Because of the late beginning on the research of zoysiagrass, the development of variety could not meet the need of users and many questions were still existed and waited for further studying and resolving. In this study, based on the previous research, molecular markers were used to analyze the genetic diversity of96accessions of Zoysia Willd including5species and1variety covering all distributing and applying regions of China which provide a scientific basis in the molecular level for the development, utilization and genetic improvement of zoysiagrass germplasms in China. On this basis, the heredity of vegetative characters, reproductive characters, cold tolerance and green period were analyzed by the major gene and polygene mixed genetic model, and the molecular markers related to the cold tolerance and green period were studied by the method of association analysis. The results were reported as follows:1The germplasms relationship and genetic diversity of zoysiagrass1.1Optimization of the SSR-PCR system and its application in zoysiagrassThe concentration of Mg2+, dNTP, primer, Taq DNA polymerase and template DNA in the SSR-PCR system was optimized for Zoysia Willd. using orthogonal design. The results showed that the optimal concentration was10×Buffer, Mg2+2.5mmol/L,dNTP200μmol/L, Primer0.6μmol/L, Taq DNA polymerase0.5U, template DNA30～90ng with a total volume of10μl reaction solution. The molecular identification of zoysiagrass germplasms including10cultivars (lines) of4zoysia species was conducted using30SSR primers with the optimal SSR marker system. It showed that the SSR primers of Xgwm and its optimized system would play an important role in zoysia germplasm identification and genetic diversity analysis.1.2Germplasms relationship and genetic diversity of zoysiagrass revealed by SSR markersThe genetic diversity and relationship of96germplasms of Zoysia Willd. belonging to 5species and1variety were analyzed with SSR markers. The results showed that:①Total282bands were detected with29pairs of SSR primers, and272of282were polymorphic with PPB as96.45%, the Nei’s gene diversity index (H) was0.2203and the Shannon diversity index (I) was0.3504, which indicated that a high level of zoysiagrass diversity was revealed by SSR markers.②The genetic similarity (GS) among96zoysia germplasms ranged from0.5922to0.9362with the average of0.7811, which showed that more genetic polymorphism existed among germplasms of zoysiagrass. GS between Z. japonica and Z. sinica, Z. matrella and Z. tenuifolia were relatively high, both Z. machrostachya (Z010) and Z. sinica.var nipponica (Z122) had a relatively lower GS to other species. The range of GS within species of Z. japonica, Z. sinica and Z. matrella were0.5922～0.9362,0.6879～0.9078and0.6738～0.8582respectively.③Based on the presence of bands,96zoysia germplasms were classified into6major groups by cluster analysis. Group Ⅰ included Z. japonica, Z. sinica, and some accessions of Z. matrella, account for90.6%of all germplasms. Some accessions of Z. matrella and Z. tenuifolia form one group. Z. machrostachya (Z010), Z. sinica.var nipponica (Z122), Z. matrella (Z095) and Z. sinica (Z115) form one group by itself respectively. The results of cluster for some secondary groups showed that most accessions of Z. japonica and Z. Sinica were clustered into one group, whereas, Z. matrella and Z. tenuifolia were clustered into one group, and some accessions of Z. japonica, Z. Sinica and Z. matrella were clustered into one group. Genetic distance and relationship among96zoysia germplasms were showed clearly in the cluster dendrogram. By this research, the diversity and relationship among96germplasms were displayed, meanwhile, a preliminary discussion was made on the ownership of part accessions. Then, the findings will provide a scientific basis on the molecular level for future study and application of zoysiagrass germplasms.1.3Germplasms relationship and genetic diversity of zoysiagrass revealed by SRAP markersThe genetic diversity and relationship of98germplasms of zoysia Willd. belong to5species and1vareity were analyzed using SRAP markers. The results showed that:①A total of362bands were detected with54pairs of SRAP primers and337of them were polymorphic(PPB=93.09%), the Nei’s gene diversity index(H) was0.2409and the Shannon diversity index (I) was0.3746, which means a high level of genetic diversity of zoysiagrass revealed by SRAP markers.②The genetic similarity(GS) among96zoysia germplasms ranged from0.5939to0.9834with the average of0.7588, which showed that a greater genetic polymorphism existed among germplasms of zoysiagrass. GS between Z. japonica and Z. sinica, Z. matrella and Z. tenuifolia were relatively high compared with the GS of other species. The variance range of GS within species of Z. japonica, Z. sinica and Z. matrella were0.5994～0.9834,0.6243～0.9807and0.6630～0.9475, respectively.③Based on the presence of bands,96zoysia germplasm were classified into7major groups by cluster analysis, in which group I and group II included3species of Z. Japonica, Z. sinica and a few accessions of Z. matrella. Group III included4accessions introduced from America. Group IV mainly comprised Z. matrella and Z. tenuifolia. Z. machrostachya (Z010), z.sinica.var nipponica (Z122) and z.sinica (Z115) form a group by itself respectively. The results of cluster showed that some accessions of Z. japonica, Z. Sinica and Z. matrella clustered into one group, one species were not always clustered into one group, moreover, a few groups contained only one accession which difference from other accessions.1.4Comparison on the results of the diversity of zoysiagrass revealed by SSR and SRAP markersCompared with the diversity of zoysiagrass revealed by SSR and SRAP markers, the results revealed by two markers were consistent that the accessions belong to one species clustered into one group basically, and some accessions of Z. japonica and Z. Sinica clustered into one group, some accessions of Z. matrella and Z. tenuifolia clustered into one group, Z. machrostachya, z.sinica.var nipponica form one group by itself respectively. Some accessions, such as Z115(z.sinica) form one group by itself for its specific genetic basis. Correlation analysis was conducted between the GS based on two markers, and the results showed that the GS based on two markers had a significant correlation (r=0.699>r0.01, n=4560) which futher verified the similarity of clustered results by two markers. SSR and SRAP markers are both effient methods in revealing the relationship and genetic diversity of zoysiagrass.2Genetic analysis for some important characters of zoysiagrass2.1Hybrids identification of zoysiagrassCross breeding were conducted by two zoysia accessions Z136(Z. japonica) and Z039(Z.sinica) differing from vegetative characters, reproductive characters, cold tolerance and green period, in order to further analyze of the genetic mechanisms of these characters in zoysiagrass. The authenticity of184progenies of Z136×Z039and103progenies of Z039×Z136were identified by SRAP markers.26primer combinations with paternal characteristic bands which present in Z039and absent in Z136and35primer combinations with paternal characteristic bands which present in Z136and absent in Z039were screened from200SRAP primer combinations, and11primer combinations with the paternal characteristic bands of Z039and Z136respectively were further utilized to identify the true hybrids. The results showed that168progenies of Z136×Z039and99progenies of Z039×Z136have paternal characteristic bands which showed they were true hybrids. The rest progenies were deduced to be self-hybrids because of the absent of paternal characteristic bands. The results of hybrids identification provided a solid evidence for further studying and applying these true hybrids.2.2Genetic analysis of vegetative characters of zoysiagrassThe heredity of vegetative characters, i.e. density, turf height, leaf length, leaf width, leaf length/width, internode length, internode diameter, and internode length/diameter in two F1populations of Z136×Z039and Z039×Z136were analyzed by the major gene and polygene mixed genetic model, in order to reveal the genetic mechanisms of theses characters of zoysiagrass. The results showed:①Variance range of every character of reciprocal progenies was far beyond that of their two parents, coefficient of variance (CV) is different among8characters, the variation of density is widest, followed by internode length/diameter and internode length, the coefficient variation of leaf width is the least, and other characters is in the middle.②The significant difference were existed between two reciprocal crosses for turf height, leaf length, leaf width, leaf length/width, internode diameter and internode length/diameter, which further explain that there may be maternal genetic phenomenon for these characters in zoysiagrass, and no significant differences were found between two reciprocal crosses for density and internode length.③The density both reciprocal cross of Z136×Z039were controlled by two additive-dominance-epistasis major genes model (B-1), and the heritability of major genes of positive cross and negative cross were93.67%and63.22%, respectively. No major gene model (A-0) was the most suitable model for the turf height, leaf length, leaf width, leaf length/width, internode length of reciprocal crosses, for the internode diameter of negative cross and for the internode length/diameter of positive cross. Internode diameter of positive cross and internode length/diameter of negative cross of Z136×Z039was controlled by one major gene model.2.3Genetic analysis of reproductive characters of zoysiagrassThe heredity of reproductive characters, i.e. inflorescence density, reproductive branch height, inflorescence length, specule No. of each spike, specule length, specule width, specule length/width in two F1populations of Z136×Z039and Z039×Z136were analyzed by the major gene and polygene mixed genetic model, in order to reveal the genetic mechanism of theses characters of zoysiagrass. The results showed:①Variance range of each character of reciprocal progenies was far beyond that of their parents, coefficient of variance (CV) was different among7characters, the variation of inflorescence density was widest, followed by reproductive branch height and specule No. of each spike, the coefficient variation of specule length and specule width was the least, and inflorescence length was in the middle.②The significant difference were existed between two reciprocal crosses for inflorescence density, reproductive branch height, specule length, specule length/width, which further explain that there may be maternal genetic phenomenon for these characters in zoysiagrass, and no significant differences were found between two reciprocal crosses for inflorescence length, specule No. of each spike and specule width.③The inflorescence density both reciprocal cross of Z136×Z039were controlled by two major genes model. No major gene model (A-0) was the most suitable model for the reproductive branch height, inflorescence length, and specule width of reciprocal crosses, the specule No. of each spike of negative cross and the specule length of positive cross. The specule No. of each spike of positive cross and the specule length of negative cross of Z136×Z039were controlled by one major gene model. The most suitable model for the specule length/width of positive cross was two additive-dominance-epistasis major genes model (B-1), and the heritability of major genes is42.72%, and two additive-dominance major genes model (B-2) was the most suitable model for the specule length/width of negative cross.2.4Genetic analysis of cold tolerance of zoysiagrassThe cold tolerance of F1populations were identified by the means of leaf electrolyte leakage rate, and the heredity of cold tolerance in two F1populations of Z136×Z039and Z039×Z136were analyzed by the major gene and polygene mixed genetic model. The results showed that both two crosses had higher variation range of cold tolerance which were-0.3～-11.6℃and-1.7～-10.9℃respectively, and far beyond that of their two parents-9.1℃and-3.3℃. The average LT50of reciprocal crosses were-6.0℃and-6.3℃, both of them were intermediate between two parents, no obvious maternal genetic phenomenon for cold tolerance were observed in zoysiagrass. The frequency distribution of cold tolerance in F1population of two crosses showed characteristics of a mixed normal distribution, which indicated that the inheritance of cold tolerance followed a major gene plus poly-genes model. The results of genetic analysis showed that the genetic model B-4was the most suitable model for the trait, i.e. the cold tolerance of zoysia was controlled by two equal additive major genes, the heritability of major genes were77.27%and79.60%in two crosses。2.5Genetic analysis of green period of zoysiagrassThe heredity of green period in two F, populations of Z136×Z039and Z039×Z136were analyzed by the major gene and polygene mixed genetic model, in order to reveal the genetic mechanism of green period of zoysiagrass. The results showed:①variation range of green period of both two crosses were178～261d and188～2533d, and far beyond that of their two parents248and231d. The average of green period of reciprocal crosses were223.6d and216.8d, respectively, and significant differences were existed between two reciprocal crosses, which further explain that there have maternal genetic phenomenon for green period in zoysiagrass.②There is a great difference of genetic models between two reciprocal crosses, the effect of maternal genetic and environment were significant to green period. No major gene model (A-0) was the most suitable model for the green period of Z136×Z039, but the green period of reciprocal cross Z039×Z136were controlled by two equal additive major genes, the most suitable model is B-4, and the heritability of major genes of green period of reciprocal cross was94.35%.3. Association analysis of cold tolerance and green period with molecular markers in zoysiagrassThe genome DNA of96zoysia germplasms were amplified by29pairs of SSR markers and54pairs of SRAP markers, and254SSR polymorphic loci and338SRAP polymorphic loci were detected. Then all these polymorphic loci were classified according to8types of data, i.e. original data, species, collection site and molecular dendrogram by NTSYS software (96zoysia germplasms were classified into two, three, four, five and six groups respectively), association analysis were conducted between green period and molecular markers according to8results of classified. The results showed that3SSR loci and1SRAP loci were identified to be associated with the cold tolerance of zoysiagrass, in which, Xgwml31-3B-187and Xgwm469-6D-194were associated with the cold tolerance of zoysiagrass in8classified conditions, Xgwm234-5B-244and Me19+Em5-359were associated with the cold tolerance in7conditions except collection site classification.3SSR loci and2SRAP loci were identified to be associated with the green period of zoysiagrass, in which, Xgwml32-6B-225was associated with the green period of zoysiagrass in8classified conditions, Xgwmlll-7D-34and Me19+Em5-359were associated with the green period in7conditions except the condition of classifying the accessions into six group classification by NTSYS software, Xgwm102-2D-97and Mel6+Em8-483were associated with the green period in6and5conditions respectively.