结缕草属植物种质资源多样性及重要性状的遗传分析与分子标记
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摘要
结缕草属(Zoysia Willd.)植物是禾本科画眉草亚科的多年生草本植物,是世界公认的优良暖季型草坪草,由于结缕草属植物的研究工作起步较晚,目前结缕草品种的开发还远远不能满足市场的需求,还有许多问题有待于进一步研究和解决。本研究充分利用已拥有的结缕草属植物种质资源,在前期工作的基础上,首先通过分子标记的手段对涵盖了中国各个分布和应用区域的5种1变种共96份种源材料的遗传多样性进行了研究,为中国丰富的结缕草属植物种质资源的开发利用及遗传改良提供科学依据。在此基础上,针对目前关于结缕草属植物抗寒性、青绿期及外部性状遗传基础和遗传机制的研究缺乏这一现状,利用“主基因+多基因混合遗传模型”分析方法,对结缕草属植物重要的外部性状(包括营养性状、生殖性状、抗寒性、青绿期)进行遗传分析,并通过关联分析法对与结缕属植物抗寒性和青绿期相关联的分子标记进行了研究,有关研究结果如下：
1结缕草属植物种质资源多样性研究
1.1结缕草属植物SSR反应体系的优化及其应用
以结缕草属植物DNA为模板,应用正交设计法对简单重复序列(simple sequence repeats, SSR)反应体系中的各个主要影响因子进行了优化筛选,并通过比较不同浓度的模板DNA对聚合酶链式反应(polymerase chain reaction, PCR)的影响,确立了适合结缕草属植物SSR-PCR反应的最佳体系。结果表明,10uL的SSR反应体系中各组分的最适浓度分别为：10×PCR缓冲液,Mg2+2.5mmol/L, dNTP200μmol/L,正反向引物分别为0.6μmol/L, Taq DNA聚合酶0.5U,模板DNA的用量在30-9Ong均可。并利用该优化体系,通过30对SSR引物对包含结缕草属植物4个种的10份材料进行了种质鉴定,结果发现Xgwm系列SSR引物,可以有效地用于结缕草属植物不同种源间的鉴定及遗传多样性研究。
1.2结缕草属植物种质资源多样性的SSR标记分析
通过SSR分子标记技术对结缕草属植物5种1变种共96份种源进行种质资源多样性分析,结果表明,①结缕草属种质间存在丰富的遗传多样性,29对SSR引物共扩增出282条清晰的用于多样性分析的谱带,其中多态性条带272条,多态性比率(PPB)为96.45%,Nei’S(1973)基因多样性指数(H)为0.2203,Shannon信息指数(Ⅰ)为0.3504。②应用Nei—Li相似系数法估算了96份材料间的遗传相似系数(GS),其GS在0.5922-0.9362,平均为0.7811。不同种之间,结缕草与中华结缕草、沟叶结缕草与细叶结缕草中的部分材料遗传相似系数较高,遗传距离较近。大穗结缕草Z010和长花结缕草Z122与其它结缕草的遗传相似系数都相对较低。同一个种内,结缕草、中华结缕草和沟叶结缕草种内遗传变异均较大,GS分别为0.5922-0.9362,0.6879-0.9078和0.6738-0.8582。③通过分子系统聚类法,将96份种源分为6个大的类群,其中第一大类为包含结缕草、中华结缕草和少量沟叶结缕草三个种的87份种质材料,占所有材料的90.6%。沟叶结缕草和细叶结缕草聚为一小类。大穗结缕草Z010、长花结缕草Z122、沟叶结缕草Z095以及结缕草Z115分别单独聚为一类。从亚群来看,除个别材料自行一类,遗传基础与其它材料差异较大外,其余材料也并不是同一个种的材料完全相聚,基本上是结缕草与中华结缕草交叉相聚,沟叶结缕草与细叶结缕草交叉相聚,沟叶结缕草与结缕草、中华结缕草也有交叉聚类现象。
1.3结缕草属植物种质资源多样性的SRAP标记分析
通过相关序列扩增多态性(Sequence-related Amplified Polymorphism, SRAP)分子标记技术对结缕草属植物5种1变种共96份种源进行多样性分析,结果表明,①结缕草属种质间存在丰富的遗传多样性,54对SRAP引物共扩增出362条清晰的用于多样性分析的谱带,其中多态性条带337条,多态性比率(PPB)为93.09%,Nei′S(1973)基因多样性指数(H)为0.2409,Shannon信息指数(Ⅰ)为0.3746。②应用Nei-Li相似系数法估算了96份材料间的遗传相似系数(GS),其GS在0.5939-0.9834之间,平均为0.7588。不同种之间,结缕草与中华结缕草、沟叶结缕草与细叶结缕草中的部分材料遗传相似系数较高,遗传距离较近。大穗结缕草Z010和长花结缕草Z122与其它结缕草的遗传相似系数都相对较低。同一个种内,结缕草、中华结缕草和沟叶结缕草种内遗传变异均较大,GS分别为0.5994-0.9834,0.6243-0.9807和0.6630-0.9475。③通过分子系统聚类法,将96份种源分为七个大的类群,其中第一大类和第二大类均包含结缕草、中华结缕草和少量的沟叶结缕草种源；第三大类群主要包括4份美国引进的材料；第四大类主要包括沟叶结缕草和细叶结缕草；大穗结缕草Z010、长花结缕草Z122和结缕草Z115都单独聚为一类。从聚类结果可以看出,结缕草、中华结缕草和沟叶结缕草均有交叉聚类现象,并不是同一个种的材料完全相聚,个别种源自行一类,遗传基础与其它材料差异较大,从遗传聚类图可以很明确地看出96份种源间的遗传距离及亲缘关系。
1.4SSR标记与SRAP标记对结缕草属植物种质资源多样性研究结果的比较
比较两种标记的多样性分析结果,发现两种标记的研究结果非常一致,从聚类结果来看,两种标记均表现为同一个种的材料基本上聚在一起,其中结缕草与中华结缕草交叉聚类,沟叶结缕草与细叶结缕草交叉聚类,而大穗结缕草和长花结缕草单独相聚,个别材料如结缕草Z115,因具有遗传特异性而单独聚为一类。对基于这两种标记的样品遗传相似系数矩阵进行相关性分析,结果表明,两种标记检测的遗传相似性呈极显著的正相关(r=0.699>r0.01,n=4560),进一步验证了两种标记聚类结果的相似性。两种标记都可以有效地用于结缕草属植物种质资源多样性的研究中。
2结缕草属植物重要性状的遗传分析
2.1结缕草属植物杂交后代杂种真实性鉴定
通过在抗寒性、青绿期、营养性状和生殖性状各个方面均存在差异的两份结缕草属种源材料,即结缕草Z136和中华结缕草Z039相互杂交,获得正反交F,群体,拟通过所得F,群体对结缕草属植物营养性状、生殖性状、抗寒性和青绿期的遗传特性作进一步的研究。本文首先通过SRAP分子标记技术对结缕草属植物Z136×Z039的184个正交后代和103个反交后代的杂种真实性进行了鉴定,在200对SRAP引物组合中筛选出26对父本(Z039)有特异带的引物组合,35对母本(Z136)有特异带的引物组合,从正反交父本有特异带的引物组合中,分别选取了11对引物组合对后代真实性进行鉴定,结果有168个正交后代和99个反交后代因具有父本的特异带被鉴定为真杂种,其余的16个正交后代和4个反交后代因不具有父本的特异带被认为是自交种。杂种真实性的鉴定结果为杂交后代的进一步研究及应用奠定了良好的基础。
2.2结缕草属植物营养性状的遗传分析
应用植物数量性状主基因+多基因混合遗传模型分析方法对F,群体的密度、草层高度、叶长、叶宽、叶长／叶宽、节间长度、节间直径和节间长度／节间直径进行遗传分析,以初步明确这些性状的遗传特性。结果表明：①在调查的8个性状中,正反交杂交后代中每一个性状的变异范围均超出了双亲的变异范围,变异系数不同性状差异较大,密度的变异系数最大,其次为节间长度／直径和节间长度,叶宽的变异系数最小,其它性状的变异系数居中。②草层高度、叶长、叶宽、叶长／叶宽、节间直径和节间长度／直径的正反交后代的表型值存在显著差异,可能有母体遗传效应,密度和节间长度正反交后代间无显著差异。③密度的最佳遗传模型正反交均为B-1模型,即两对主基因的加性-显性-上位性遗传模型,正交的主基因遗传率为93.67%,反交的主基因遗传率为63.22%。草层高度、叶长、叶宽、叶长／叶宽和节间长度正反交后代群体的最佳遗传模型均为A-0模型,即无主基因模型。节间直径的正交为一对主基因的遗传模型,反交为无主基因模型,节间长度的正交为无主基因模型,反交为一对主基因模型。
2.3结缕草属植物生殖性状的遗传分析
应用植物数量性状主基因+多基因混合遗传模型分析方法对F1群体的花序密度、生殖枝高度、花序长度、每穗小穗数、小穗长度、小穗宽度、小穗长度／宽度进行遗传分析,以初步明确这些性状的遗传特性。结果表明：①在调查的7个性状中,正反交杂交后代中每一个性状的变异范围均超出了双亲的变异范围,变异系数不同性状差异较大,花序密度的变异系数最大,其次为生殖枝高度和每穗粒数,小穗长度和宽度的变异最小,花序长度的变异居中。②花序密度、生殖枝高度、小穗长度和小穗长／宽正反交后代的观测值存在显著差异,可能有母体遗传效应,花序长度、每穗粒数和小穗宽度正反交后代间的观测值无显著差异。③花序密度正反交后代群体的最佳遗传模型为存在两对主基因控制的遗传模型,生殖枝高度、花序长度和小穗宽度正反交后代群体的最佳遗传模型均为A-0模型,即无主基因模型。每穗粒数正交为一对主基因的遗传模型,反交为无主基因模型,小穗长度的正交为无主基因模型,反交为一对主基因模型。小穗长／宽正交的最适遗传模型为B-1模型,即两对主基因的加性-显性-上位性遗传模型,主基因遗传率为42.72%,反交群体的最适遗传模型为B-2模型,即两对主基因的加性-显性遗传模型,主基因遗传率为98.81%。
2.4结缕草属植物抗寒性的遗传分析
通过改良电导率外渗法对F,群体的抗寒性进行鉴定,并应用植物数量性状主基因+多基因混合遗传模型分析方法对F,群体进行遗传分析。结果表明：①正反交后代的抗寒性变异范围均较大,正交的变异范围为-0.3～-11.6℃,反交的变异范围为-1.7～-10.9℃,均远远超出了双亲的抗寒性(-9.1℃和-3.3℃),正反交后代的平均值相差不大,正交后代LT50的平均值为-6.0℃,反交后代LT50的平均值为-6.3℃,均间于双亲之间,没有发现明显的母性遗传现象。②次数分布分析结果表明,正反交F,群体的抗寒性均呈混合正态分布,表现出明显的主基因+多基因的遗传特征。③遗传分析结果表明,结缕草属植物的最适遗传模型为B-4,即结缕草属植物的抗寒性受两对等加性的主效基因控制,正反交主基因遗传率分别为77.27%和79.60%。
2.5结缕草属植物青绿期的遗传分析
应用植物数量性状主基因+多基因混合遗传模型分析方法对F1群体青绿期进行遗传分析,以初步明确结缕草属植物青绿期的遗传特性。结果表明：①正反交后代的青绿期变异范围均较大,正交的变异范围为178-261天,反交的变异范围为188-253天,均远远超出了双亲的青绿期(248天和231天),正反交后代的平均值分别为223.6天和216.8天,两者存在显著差异,结缕草属植物的青绿期有母体遗传效应。②正反交的遗传模型差异较大,母体遗传效应和环境效应对青绿期的影响明显,正交F,群体青绿期的最适遗传模型为A-0模型,即无主基因模型,而反交F,群体青绿期的最适遗传模型为B-4模型,即两对主基因的等加性遗传模型,且反交的主基因遗传率较高,为94.35%。
3与结缕草属植物抗寒性和青绿期相关联的分子标记的研究
利用29对SSR引物和54对SRAP引物对96份结缕草属植物种质资源的基因组变异进行扩增扫描,共检测到254个SSR多态性标记位点和338个SRAP多态性标记位点,对这些多态性位点按原始数据、种分类、采集地分类、NTSYS软件分类(分别分为2、3、4、5、6类),共8种情况下与抗寒性和青绿期作多标记联合分析,结果检测到与抗寒性相关联SSR标记位点3个、SRAP标记位点1个,分别为：Xgwm131-3B-187、Xgwm469-6D-194、Xgwm234-5B-244和Mell+Em7-406,其中前两个标记在8种分类情况下均与抗寒性存在关联性,后两个标记除了按采集地分类外,在其余7种分类情况下均与抗寒性相关联；检测到与青绿期相关联的SSR标记位点3个、SRAP标记位点2个,分别为：Xgwmlll-7D-34、Xgwm102-2D-97、Xgwm132-6B-225和Mel9+Em5-359,Mel6+Em8-483.其中标记位点Xgwm132-6B-225在8种分类情况下均与青绿期有关联性,标记位点Xgwm111-7D-34和Me19+Em5-359,除了按分子聚类图分为6类的情况下外,在其余7种情况下都检测到了与青绿期的相关性,标记位点Xgwm102-2D-97和Me16+Em8-483分别在6种和5种分类情况下检测到与青绿期的关联性。
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 zoysiagrass
1.1Optimization of the SSR-PCR system and its application in zoysiagrass
The 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 markers
The 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 markers
The 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 markers
Compared 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 zoysiagrass
2.1Hybrids identification of zoysiagrass
Cross 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 zoysiagrass
The 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 zoysiagrass
The 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 zoysiagrass
The 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 zoysiagrass
The 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 zoysiagrass
The 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.
1结缕草属植物种质资源多样性研究
1.1结缕草属植物SSR反应体系的优化及其应用
以结缕草属植物DNA为模板,应用正交设计法对简单重复序列(simple sequence repeats, SSR)反应体系中的各个主要影响因子进行了优化筛选,并通过比较不同浓度的模板DNA对聚合酶链式反应(polymerase chain reaction, PCR)的影响,确立了适合结缕草属植物SSR-PCR反应的最佳体系。结果表明,10uL的SSR反应体系中各组分的最适浓度分别为：10×PCR缓冲液,Mg2+2.5mmol/L, dNTP200μmol/L,正反向引物分别为0.6μmol/L, Taq DNA聚合酶0.5U,模板DNA的用量在30-9Ong均可。并利用该优化体系,通过30对SSR引物对包含结缕草属植物4个种的10份材料进行了种质鉴定,结果发现Xgwm系列SSR引物,可以有效地用于结缕草属植物不同种源间的鉴定及遗传多样性研究。
1.2结缕草属植物种质资源多样性的SSR标记分析
通过SSR分子标记技术对结缕草属植物5种1变种共96份种源进行种质资源多样性分析,结果表明,①结缕草属种质间存在丰富的遗传多样性,29对SSR引物共扩增出282条清晰的用于多样性分析的谱带,其中多态性条带272条,多态性比率(PPB)为96.45%,Nei’S(1973)基因多样性指数(H)为0.2203,Shannon信息指数(Ⅰ)为0.3504。②应用Nei—Li相似系数法估算了96份材料间的遗传相似系数(GS),其GS在0.5922-0.9362,平均为0.7811。不同种之间,结缕草与中华结缕草、沟叶结缕草与细叶结缕草中的部分材料遗传相似系数较高,遗传距离较近。大穗结缕草Z010和长花结缕草Z122与其它结缕草的遗传相似系数都相对较低。同一个种内,结缕草、中华结缕草和沟叶结缕草种内遗传变异均较大,GS分别为0.5922-0.9362,0.6879-0.9078和0.6738-0.8582。③通过分子系统聚类法,将96份种源分为6个大的类群,其中第一大类为包含结缕草、中华结缕草和少量沟叶结缕草三个种的87份种质材料,占所有材料的90.6%。沟叶结缕草和细叶结缕草聚为一小类。大穗结缕草Z010、长花结缕草Z122、沟叶结缕草Z095以及结缕草Z115分别单独聚为一类。从亚群来看,除个别材料自行一类,遗传基础与其它材料差异较大外,其余材料也并不是同一个种的材料完全相聚,基本上是结缕草与中华结缕草交叉相聚,沟叶结缕草与细叶结缕草交叉相聚,沟叶结缕草与结缕草、中华结缕草也有交叉聚类现象。
1.3结缕草属植物种质资源多样性的SRAP标记分析
通过相关序列扩增多态性(Sequence-related Amplified Polymorphism, SRAP)分子标记技术对结缕草属植物5种1变种共96份种源进行多样性分析,结果表明,①结缕草属种质间存在丰富的遗传多样性,54对SRAP引物共扩增出362条清晰的用于多样性分析的谱带,其中多态性条带337条,多态性比率(PPB)为93.09%,Nei′S(1973)基因多样性指数(H)为0.2409,Shannon信息指数(Ⅰ)为0.3746。②应用Nei-Li相似系数法估算了96份材料间的遗传相似系数(GS),其GS在0.5939-0.9834之间,平均为0.7588。不同种之间,结缕草与中华结缕草、沟叶结缕草与细叶结缕草中的部分材料遗传相似系数较高,遗传距离较近。大穗结缕草Z010和长花结缕草Z122与其它结缕草的遗传相似系数都相对较低。同一个种内,结缕草、中华结缕草和沟叶结缕草种内遗传变异均较大,GS分别为0.5994-0.9834,0.6243-0.9807和0.6630-0.9475。③通过分子系统聚类法,将96份种源分为七个大的类群,其中第一大类和第二大类均包含结缕草、中华结缕草和少量的沟叶结缕草种源；第三大类群主要包括4份美国引进的材料；第四大类主要包括沟叶结缕草和细叶结缕草；大穗结缕草Z010、长花结缕草Z122和结缕草Z115都单独聚为一类。从聚类结果可以看出,结缕草、中华结缕草和沟叶结缕草均有交叉聚类现象,并不是同一个种的材料完全相聚,个别种源自行一类,遗传基础与其它材料差异较大,从遗传聚类图可以很明确地看出96份种源间的遗传距离及亲缘关系。
1.4SSR标记与SRAP标记对结缕草属植物种质资源多样性研究结果的比较
比较两种标记的多样性分析结果,发现两种标记的研究结果非常一致,从聚类结果来看,两种标记均表现为同一个种的材料基本上聚在一起,其中结缕草与中华结缕草交叉聚类,沟叶结缕草与细叶结缕草交叉聚类,而大穗结缕草和长花结缕草单独相聚,个别材料如结缕草Z115,因具有遗传特异性而单独聚为一类。对基于这两种标记的样品遗传相似系数矩阵进行相关性分析,结果表明,两种标记检测的遗传相似性呈极显著的正相关(r=0.699>r0.01,n=4560),进一步验证了两种标记聚类结果的相似性。两种标记都可以有效地用于结缕草属植物种质资源多样性的研究中。
2结缕草属植物重要性状的遗传分析
2.1结缕草属植物杂交后代杂种真实性鉴定
通过在抗寒性、青绿期、营养性状和生殖性状各个方面均存在差异的两份结缕草属种源材料,即结缕草Z136和中华结缕草Z039相互杂交,获得正反交F,群体,拟通过所得F,群体对结缕草属植物营养性状、生殖性状、抗寒性和青绿期的遗传特性作进一步的研究。本文首先通过SRAP分子标记技术对结缕草属植物Z136×Z039的184个正交后代和103个反交后代的杂种真实性进行了鉴定,在200对SRAP引物组合中筛选出26对父本(Z039)有特异带的引物组合,35对母本(Z136)有特异带的引物组合,从正反交父本有特异带的引物组合中,分别选取了11对引物组合对后代真实性进行鉴定,结果有168个正交后代和99个反交后代因具有父本的特异带被鉴定为真杂种,其余的16个正交后代和4个反交后代因不具有父本的特异带被认为是自交种。杂种真实性的鉴定结果为杂交后代的进一步研究及应用奠定了良好的基础。
2.2结缕草属植物营养性状的遗传分析
应用植物数量性状主基因+多基因混合遗传模型分析方法对F,群体的密度、草层高度、叶长、叶宽、叶长／叶宽、节间长度、节间直径和节间长度／节间直径进行遗传分析,以初步明确这些性状的遗传特性。结果表明：①在调查的8个性状中,正反交杂交后代中每一个性状的变异范围均超出了双亲的变异范围,变异系数不同性状差异较大,密度的变异系数最大,其次为节间长度／直径和节间长度,叶宽的变异系数最小,其它性状的变异系数居中。②草层高度、叶长、叶宽、叶长／叶宽、节间直径和节间长度／直径的正反交后代的表型值存在显著差异,可能有母体遗传效应,密度和节间长度正反交后代间无显著差异。③密度的最佳遗传模型正反交均为B-1模型,即两对主基因的加性-显性-上位性遗传模型,正交的主基因遗传率为93.67%,反交的主基因遗传率为63.22%。草层高度、叶长、叶宽、叶长／叶宽和节间长度正反交后代群体的最佳遗传模型均为A-0模型,即无主基因模型。节间直径的正交为一对主基因的遗传模型,反交为无主基因模型,节间长度的正交为无主基因模型,反交为一对主基因模型。
2.3结缕草属植物生殖性状的遗传分析
应用植物数量性状主基因+多基因混合遗传模型分析方法对F1群体的花序密度、生殖枝高度、花序长度、每穗小穗数、小穗长度、小穗宽度、小穗长度／宽度进行遗传分析,以初步明确这些性状的遗传特性。结果表明：①在调查的7个性状中,正反交杂交后代中每一个性状的变异范围均超出了双亲的变异范围,变异系数不同性状差异较大,花序密度的变异系数最大,其次为生殖枝高度和每穗粒数,小穗长度和宽度的变异最小,花序长度的变异居中。②花序密度、生殖枝高度、小穗长度和小穗长／宽正反交后代的观测值存在显著差异,可能有母体遗传效应,花序长度、每穗粒数和小穗宽度正反交后代间的观测值无显著差异。③花序密度正反交后代群体的最佳遗传模型为存在两对主基因控制的遗传模型,生殖枝高度、花序长度和小穗宽度正反交后代群体的最佳遗传模型均为A-0模型,即无主基因模型。每穗粒数正交为一对主基因的遗传模型,反交为无主基因模型,小穗长度的正交为无主基因模型,反交为一对主基因模型。小穗长／宽正交的最适遗传模型为B-1模型,即两对主基因的加性-显性-上位性遗传模型,主基因遗传率为42.72%,反交群体的最适遗传模型为B-2模型,即两对主基因的加性-显性遗传模型,主基因遗传率为98.81%。
2.4结缕草属植物抗寒性的遗传分析
通过改良电导率外渗法对F,群体的抗寒性进行鉴定,并应用植物数量性状主基因+多基因混合遗传模型分析方法对F,群体进行遗传分析。结果表明：①正反交后代的抗寒性变异范围均较大,正交的变异范围为-0.3～-11.6℃,反交的变异范围为-1.7～-10.9℃,均远远超出了双亲的抗寒性(-9.1℃和-3.3℃),正反交后代的平均值相差不大,正交后代LT50的平均值为-6.0℃,反交后代LT50的平均值为-6.3℃,均间于双亲之间,没有发现明显的母性遗传现象。②次数分布分析结果表明,正反交F,群体的抗寒性均呈混合正态分布,表现出明显的主基因+多基因的遗传特征。③遗传分析结果表明,结缕草属植物的最适遗传模型为B-4,即结缕草属植物的抗寒性受两对等加性的主效基因控制,正反交主基因遗传率分别为77.27%和79.60%。
2.5结缕草属植物青绿期的遗传分析
应用植物数量性状主基因+多基因混合遗传模型分析方法对F1群体青绿期进行遗传分析,以初步明确结缕草属植物青绿期的遗传特性。结果表明：①正反交后代的青绿期变异范围均较大,正交的变异范围为178-261天,反交的变异范围为188-253天,均远远超出了双亲的青绿期(248天和231天),正反交后代的平均值分别为223.6天和216.8天,两者存在显著差异,结缕草属植物的青绿期有母体遗传效应。②正反交的遗传模型差异较大,母体遗传效应和环境效应对青绿期的影响明显,正交F,群体青绿期的最适遗传模型为A-0模型,即无主基因模型,而反交F,群体青绿期的最适遗传模型为B-4模型,即两对主基因的等加性遗传模型,且反交的主基因遗传率较高,为94.35%。
3与结缕草属植物抗寒性和青绿期相关联的分子标记的研究
利用29对SSR引物和54对SRAP引物对96份结缕草属植物种质资源的基因组变异进行扩增扫描,共检测到254个SSR多态性标记位点和338个SRAP多态性标记位点,对这些多态性位点按原始数据、种分类、采集地分类、NTSYS软件分类(分别分为2、3、4、5、6类),共8种情况下与抗寒性和青绿期作多标记联合分析,结果检测到与抗寒性相关联SSR标记位点3个、SRAP标记位点1个,分别为：Xgwm131-3B-187、Xgwm469-6D-194、Xgwm234-5B-244和Mell+Em7-406,其中前两个标记在8种分类情况下均与抗寒性存在关联性,后两个标记除了按采集地分类外,在其余7种分类情况下均与抗寒性相关联；检测到与青绿期相关联的SSR标记位点3个、SRAP标记位点2个,分别为：Xgwmlll-7D-34、Xgwm102-2D-97、Xgwm132-6B-225和Mel9+Em5-359,Mel6+Em8-483.其中标记位点Xgwm132-6B-225在8种分类情况下均与青绿期有关联性,标记位点Xgwm111-7D-34和Me19+Em5-359,除了按分子聚类图分为6类的情况下外,在其余7种情况下都检测到了与青绿期的相关性,标记位点Xgwm102-2D-97和Me16+Em8-483分别在6种和5种分类情况下检测到与青绿期的关联性。
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 zoysiagrass
1.1Optimization of the SSR-PCR system and its application in zoysiagrass
The 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 markers
The 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 markers
The 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 markers
Compared 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 zoysiagrass
2.1Hybrids identification of zoysiagrass
Cross 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 zoysiagrass
The 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 zoysiagrass
The 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 zoysiagrass
The 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 zoysiagrass
The 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 zoysiagrass
The 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.
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