陆地棉耐盐性状与SSR分子标记的关联分析

本研究以134份陆地棉栽培种为试验材料,测定其在0.3%盐浓度(质量分数)下的出苗率,并使用74对SSR引物对这些材料进行基因组变异扫描。利用Structure2.3.4软件分析该自然群体的遗传结构,在此基础上采用Tassel2.1软件对耐盐性状与SSR分子标记进行关联分析,寻找与棉花耐盐性状相关的分子标记。研究结果表明:(1)134份陆地棉栽培种的出苗率呈极显著差异,并筛选出27个盐敏感材料和10个耐盐材料。(2)74个SSR分子标记共检测出148个多态性位点,涉及246个等位变异,变异范围为2~7,平均每个标记3.32个;基因多样性指数变异范围为0.0295~0.4959,平均值为0.2897;SSR分子标记多态性信息含量(PIC)变幅为0.0290~0.3729,平均值为0.2381。(3)通过群体结构分析,将该自然群体划分2个亚群体,分别包含89份和45份材料。(4)关联分析共发现8个与棉花耐盐性状相关的SSR分子标记位点,表型变异解释率变幅为2.91%~7.82%,平均值为4.32%。此研究结果可以为棉花耐盐性状分子标记辅助选择育种提供参考。     

新疆早熟陆地棉种质资源遗传多样性及纤维品质性状SSR关联分析

【目的】探究新疆早熟陆地棉种质资源遗传多样性,发掘特异种质资源和与纤维品质相关的优异等位变异。【方法】以219份新疆早熟陆地棉种质资源为材料,在3个环境下对此群体的15个农艺性状进行鉴定,用128对简单序列重复(Simple sequence repeat,SSR)引物对该群体进行基因型鉴定,使用NTsys-pc2.1进行遗传多样性分析,用Structure 2.3.1和Tassel 5.0对此群体纤维品质性状进行关联分析,依据表型效应值挖掘携带优异等位变异的典型材料。【结果】128对标记在219份资源材料群体中共扩增出244个位点,平均每个标记扩增出1.91个位点;多态信息含量范围为0.13～0.86,平均为0.63;此群体材料间遗传相似性系数分布范围为0.42～0.99,平均为0.61,遗传相似系数在0.5～0.7的占90.19%。通过关联分析检测到11个与纤维品质性状显著关联的标记(P0.01),发掘出7个携带特异等位变异的典型材料。【结论】219份新疆早熟陆地棉种质资源遗传多样性低,基于SSR关联分析发掘了一些与纤维品质性状相关的优异等位变异及典型材料。     

新疆陆地棉经济性状优异等位基因位点的遗传解析

【目的】基于SSR(Simple sequence repeat,简单序列)标记展开对陆地棉经济性状的关联分析,挖掘优异等位变异位点,解析新疆陆地棉经济性状的遗传基础,为新疆陆地棉的遗传机理研究和高效的陆地棉分子设计育种提供理论依据。【方法】利用筛选出覆盖棉花全基因组的73对SSR标记对新疆156份陆地棉品种进行多态性扫描;采用R语言绘制boxplot图,利用TASSEL软件进行关联分析,挖掘与产量、纤维品质性状相关联的优异等位变异位点。【结果】通过对6个环境下新疆陆地棉品种的产量、品质相关性状进行关联分析,获得与产量性状相关的等位变异位点10个,表型变异解释率为6.69%～9.88%,平均值为8.43%;与纤维品质性状相关的等位变异位点23个,表型变异解释率为3.73%～13.22%,平均值为7.52%。其中22个QTL(Quantitative trait locus)已被报道,有10个QTL的关联性状与前人研究一致。【结论】新疆陆地棉品种的群体遗传结构简单,连锁不平衡水平低,6个环境条件下表型性状变化趋势较稳定。基于SSR的关联分析,发掘了与产量和纤维品质相关的优异等位变异基因及聚合优异等位基因位点的典型材料。     

陆地棉产量及农艺性状的SSR标记关联分析

【目的】挖掘与陆地棉产量和重要农艺性状密切关联的分子标记,为棉花分子标记辅助育种提供依据。【方法】本研究以147份陆地棉材料为供试群体,分别调查了7个环境下该群体的铃重和衣分数据,3个环境下的单株果枝数、单株铃数和株高数据。利用237对SSR标记对上述材料进行基因型检测,采用Tassel 2.1中的一般线性模型程序对上述5个性状进行关联分析。【结果】铃重、衣分、单株果枝数、单株铃数和株高的平均变异系数的变幅为7.28%～21.06%,平均值为13.47%。这表明供试群体具有较为丰富的表型多样性。237个标记在147份陆地棉中共检测到281个多态性位点,各位点上等位基因数的范围为2～6个,涉及690个等位基因,平均每个位点2.455 0个;多态性信息含量平均值为0.216 5;Structure2.2群体结构分析将147份供试材料划分为7个亚群;在3个及以上环境检测到的与铃重和衣分显著关联的标记位点分别有45个和1个,解释表型变异率在不同环境中最高分别达19.30%和11.58%(P0.01);在2个及以上环境检测到与单株果枝数、单株铃数和株高显著关联的标记位点分别有1、4和4个,表型变异解释率在不同环境中分别最高达9.96%、7.00%和5.75%,在BNL2448附近同时检测到与铃重、单株果枝数和株高显著关联的位点。【结论】通过关联分析挖掘了多个可重复检测到的与陆地棉产量及重要农艺性状关联的分子标记位点,此研究结果可为棉花高产育种分子辅助选择提供有益参考。     

陆地棉SSR标记遗传多样性及其与农艺性状的关联分析

分析陆地棉栽培种遗传多样性,通过关联分析寻找与棉花农艺性状相关联的分子标记,为分子标记辅助选择育种和提高棉花育种效率奠定基础。本文采用74个Simple sequence repeat(SSR)标记对172份陆地棉栽培种的基因组变异进行扫描,使用NTSYS-pc 2.20进行聚类,分析该群体遗传多样性;利用Structure 2.3.4软件分析群体结构,在此基础上结合田间表型数据,采用Tassel 2.1的一般线性模型(General linear model,GLM)进行关联分析,定位与农艺性状相关的QTLs。74个标记共检测到148个多态性位点,涉及246个等位变异,变异范围2~7个,平均等位变异数为3.32;引物的多态性信息含量(PIC)为0.0281~0.3733,平均值为0.2370;遗传相似系数变异在0.2816~1,平均值为0.5369,平均遗传相似系数为0.5369,表明我国陆地棉遗传基础狭窄,尽管国外及西北内陆棉区部分材料具有较丰富的遗传变异。聚类分析将该群体划分为12个亚群,不同棉区的材料交叉分布,且聚类结果基本与系谱吻合。群体结构分析却将172份供试材料划分为3个亚群;通过关联分析,发现30个位点与铃重、衣分、黄萎病抗性显著相关(P0.05),各位点对表型变异贡献率为2.24%~5.27%。     

引进陆地棉种质材料纤维品质及农艺性状关联分析

引进陆地棉(Gossypium hirsutum)种质材料的性状-标记关联分析是发掘其优异基因并在分子标记辅助育种中使用的科学依据。本研究利用199个SSR标记和42个In Del标记对94份国外陆地棉种质材料和27份新疆本地品种的纤维品质和农艺学性状进行全基因组关联分析。遗传结构和Neighbor-Join聚类分析将陆地棉种质群体划分为2个亚群,连锁不平衡分析发现有9.42%的位点组合存在LD(p0.01,r2≥0.05);基于混合线性模型的关联分析结果显示,能够同时在2个播期检测到的与6个纤维品质性状、4个产量性状和4个农艺性状显著关联的标记位点分别有18个、10个和5个,平均表型变异解释率分别为6.06%(1.17%~13.86%)、4.62%(1.18%~15.28%)和6.75%(1.87%~9.98%)。上述关联位点中共检测到188个等位变异,其中表型效应值大于0.6的优异等位变异有12个。研究结果可进一步用于分子辅助育种,并为基因精细定位提供参考资料。     

粳稻品种资源耐盐性状的SSR标记关联分析

为了发掘影响粳稻芽期和幼苗期耐盐相关性状的分子标记关联位点。以新疆历年选育及主栽的46份粳稻品种组成的自然群体为研究材料,1.2%氯化钠溶液对芽期和幼苗期进行胁迫处理,并对其相对发芽势(RGV)、相对盐害率(RSI)、相对苗高(RSH)及幼苗胁迫的盐害级别(STL)进行调查。同时鉴定46份品种的基因型和标记与性状的关联分析,挖掘其优异变异。结果表明,相对盐害率的变异幅度最大,相对苗高变化呈现较均匀的正态分布,相对盐害率与相对发芽势和相对苗高呈显著正相关;SSR标记关联分析结果表明,分别检测到6、12、8和8个与粳稻相对发芽势、相对苗高、相对盐害率和盐害等级相关联的位点;其中有13个与前人报道的位点重合。其中,位点RM211同时与3个耐盐性状显著关联,可能是一个重要的粳稻芽期耐盐性相关联的位点。     

SSR标记与陆地棉田间黄萎病抗性的关联分析

通过关联分析挖掘与陆地棉黄萎病抗性关联的分子标记,为陆地棉抗黄萎病性状的分子检测及标记辅助选择育种提供有益参考。选用在棉花基因组上均匀分布多态性较好的237个SSR标记对214份陆地棉材料的基因组变异进行了扫描,并使用Power Marker v3. 25分析群体的遗传多样性,Structure 2. 2分析群体结构,在此基础上结合3年黄萎病抗性鉴定数据,采用Tassel 2. 1中的GLM(Q)方法挖掘与黄萎病抗性相关的QTLs。结果表明,不同陆地棉材料黄萎病发病情况差异显著; 237个标记共检测到280个多态性位点,共计695个等位变异,变异范围2～6个,平均等位变异数2. 479 0;按照基因型数据可将该群体划分为2个亚群;通过关联分析,在不同年份中发掘与黄萎病抗性显著关联(P 0. 01)的SSR位点27个,其中有2个位点(BNL3442和BNL1064)在3年均被重复检测到,表型变异解释率最高分别为12. 10%和8. 02%。这些在不同年份中稳定存在的SSR标记位点有可能与抗病基因紧密连锁,有望用于棉花黄萎病抗性材料筛选和抗病基因挖掘。     

陆地棉农艺性状与SSR标记的关联分析

利用关联分析方法以160对SSR引物对68份陆地棉品种2个播期组成的自然群体进行基因型检测,在利用STRUCTURE 2.3.4软件分析群体结构的基础上,采用TASSEL 4.0软件的GLM程序和MLM程序对17个农艺性状进行关联分析。结果显示,STRUCTURE群体结构分析将68份供试材料划分为3个亚群,基于Nei's遗传距离的聚类分析也将68份供试材料划分为3个亚群,与群体结构划分结果基本一致。6个SSR位点均能通过GLM和MLM程序检测到,且在2个播期下稳定出现,其中,CER0098-400与全生育期显著关联,DPL0375-250和HAU2414-147与果枝始节显著关联,DPL0504-135、HAU0883-120和NAU1298-524与衣分显著关联。本研究结果为棉花分子标记辅助选择提供了依据。     

毛棉苗期抗旱性状的QTL定位

【目的】通过分析毛棉苗期抗旱相关性状的数量性状位点(Quantitative trait locus,QTL),以期检测稳定的主效QTL,促进栽培品种抗旱性状遗传改良及提高抗旱育种效率。【方法】以四倍体野生种毛棉(Gossypium tomentosum)和陆地棉品种中棉所12(CCRI 12)的种间杂种F2及其F2:3家系为研究材料,用于基因型分型的F2有188个系,用于表型分型的F2:3家系有149个株系。分别在干旱胁迫和正常灌水2个环境下调查表型数据。采用复合区间作图法对F2:3家系苗期相关性状抗旱系数进行QTL定位。【结果】对苗期相关性状抗旱系数的QTL定位分析,共得到16个QTL,其中与株高、叶片数、叶绿素含量、脯氨酸含量、丙二醛含量抗旱系数相关的QTL分别有5个、1个、3个、3个、4个,分布在13条染色体上。来自毛棉的5个加性QTL分别为qSHDC-19-1、qSHDC-19-2、qSLNDC-5-1、qMDADC-24-1、qMDADC-24-2,其加性效应值为0.10~0.22,解释变异9.4%~25.8%。【结论】这些与抗旱相关的QTL有助于棉花抗旱分子标记辅助选择。     

中棉所70分离群体农艺性状相关QTL定位

【目的】定位棉花产量相关性状的数量性状基因座(Quantitative trait locus,QTL)。【方法】以中棉所70的F_2分离群体为遗传作图群体,利用从14 820对简单序列重复(Simple sequence repeat,SSR)引物中筛选出的267对两亲本间的多态性引物检测F_2群体250个单株的标记基因型,利用Joinmap 4.0进行连锁分析,并通过WinQTLCart 2.5复合区间作图法对F_(2:3)群体的株高、单株结铃数和单株果枝数性状进行QTL定位。【结果】在F_2群体中共获得342个SSR标记位点,并构建了包括312个标记、35个连锁群,总长1 929.9 cM的遗传连锁图谱(标记间平均距离为9.2 cM,覆盖棉花基因组的43.4%)。经QTL定位,共检测到19个QTL,其中涉及株高的7个、单株果枝数4个、单株结铃数8个,这些QTL分布在8条染色体上,解释0.25%~11.28%的表型变异。【结论】这些与农艺性状相关的QTL有助于棉花产量分子标记辅助选择。     

Association Analysis of Agronomic and Yield-Related Traits in an Upland Cotton (Gossypium hirsutum L.) Germplasm Population Based on SSR Markers

[Objective] Molecular markers associated with agronomic and yield-related traits could provide useful references for improving the efficiency of Gossypium hirsutum breeding. [Method] In 147 upland cotton materials, 237 Simple Sequence Repeat(SSR) markers were assayed for polymorphisms. The population structure was analyzed using Structure 2.2. An association analysis between SSR markers and the agronomic and yield-related traits was performed using the Tassel 2.1 General Linear Model program. [Result] The average coefficients of variation for the five indicators varied from 7.28% to 21.06%, with an average of 13.47%, which indicated that the test population had a rich phenotypic diversity. The 237 SSR markers included 690 alleles belonging to 281 loci, with an average of 2.455 0 alleles per SSR marker (ranging from 2 to 6), and the average polymorphism information content value was 0.216 5. The test group could be divided into seven subgroups using Structure software. A genome-wide association analysis was conducted on five agronomic and yield-related traits from 2013 to 2015. There were 45 SSR loci and 1 locus significantly (P＜0.01) associated with boll weight and lint percentage, which were detected in 3 or more environments, and they explained 19.30% and 11.58%, respectively, of the phenotypic variation in the different environments (P＜0.01). Nine loci were significantly (P＜0.01) associated with agronomic traits at two or more bowing times, and one, four and four markers were associated with fruit branch number, boll number and plant height, explaining 9.96%, 7.00% and 5.57%, respectively, of the phenotypic variation in different environments. Sites significantly associated with boll weight, fruit branch number and plant height were simultaneously detected near BNL2448. [Conclusion] Many loci associated with the yield and other important agronomic traits of upland cotton were repeatedly detected in different environments using an association analysis. The results provide useful reference information for cotton molecular marker-assisted selection-based breeding.     

Quantitative trait locus mapping of drought and salt tolerance in an introgressed recombinant inbred line population of Upland cotton under the greenhouse and field conditions

Drought and salt tolerances are complex traits and controlled by multiple genes, environmental factors and their interactions. Drought and salt stresses can result in more than 50% yield loss in Upland cotton (Gossypium hirsutum L.). G. barbadense L. (the source of Pima cotton) carries desirable traits such as tolerance to abiotic and biotic stress along with high fiber quality. However, few studies have been reported on mapping quantitative trait loci (QTL) for abiotic stress tolerance using a permanent bi-parental population in multiple tests. The transfer of drought and salt tolerance from Pima to Upland cotton has been a challenge due to interspecific hybrid breakdown. This issue may be overcome by using introgression lines with genes transferred from Pima to Upland cotton. In this study, four replicated tests were conducted in the greenhouse each for drought and salt tolerance along with another test conducted in a field for drought tolerance using an Upland recombinant inbred line population of TM-1/NM24016 that has a stable introgression from Pima cotton. The objectives of the study were to investigate the genetic basis of drought and salt tolerance and to identify genetic markers associated with the abiotic stress tolerance. A total of 1004 polymorphic DNA marker loci including RGA-AFLP, SSR and GBS-SNP markers were used to construct a genetic map spanning 2221.28 cM. This population together with its two parents was evaluated for morphological, physiological, yield and fiber quality traits. The results showed that drought under greenhouse and field conditions and salt stress in the greenhouse reduced cotton plant growth at the seedling stage, and decreased lint yield and fiber quality traits in the field. A total of 165 QTL for salt and drought tolerance were detected on most of the cotton chromosomes, each explaining 5.98–21.43% of the phenotypic variation. Among these, common QTL for salt and drought tolerance were detected under both the greenhouse and field conditions. This study represents the first study to report consistent abiotic stress tolerance QTL from multiple tests in the greenhouse and the field that will be useful to understand the genetic basis of drought and salt tolerance and to breeding for abiotic stress tolerance using molecular marker-assisted selection in cotton.     

Mapping of QTL associated with waterlogging tolerance and drought resistance during the seedling stage in oilseed rape (Brassica napus)

Soil waterlogging and drought are major environmental stresses that suppress rapeseed (Brassica napus) growth and yield. To identify quantitative trait loci (QTL) associated with waterlogging tolerance and drought resistance at the rapeseed seedling stage, we generated a doubled haploid (DH) population consisting of 150 DH lines from a cross between two B. napus lines, namely, line No2127-17 × 275B F₄ (waterlogging-tolerant and drought-resistant) and line Huyou15 × 5900 F₄ (waterlogging-sensitive and drought-sensitive). A genetic linkage map was constructed using 183 simple sequence repeat and 157 amplified fragment length polymorphism markers for the DH population. Phenotypic data were collected under waterlogging, drought and control conditions, respectively, in two experiments. Five traits (plant height, root length, shoot dry weight, root dry weight and total dry weight) were investigated. QTL associated with the five traits, waterlogging tolerance coefficient (WTC) and drought resistance coefficient (DRC) of all the traits were identified via composite interval mapping, respectively. A total of 28 QTL were resolved for the five traits under control conditions, 26 QTL for the traits under waterlogging stresses and 31 QTL for the traits under drought conditions. Eleven QTL were detected by the WTC, and 19 QTL related to DRC were identified. The results suggest that the genetic bases of both waterlogging tolerance and drought resistance are complex. Some of the QTL for waterlogging tolerance-related traits overlapped with QTL for drought resistance-related traits, indicating that the genetic bases of waterlogging tolerance and drought resistance in the DH population were related in some degree.     

Genetic Diversity and Association Analysis of Fiber Quality Traits with SSR Markers in Germplasm Resources of Early Maturity Upland Cotton in Xinjiang

[Objective] We explored the genetic diversity of early maturity upland cotton germplasm resources in Xinjiang, China. We discovered specific germplasm resources and elite allelic variation related to fiber quality. [Method] We used 219 early maturity upland cotton accessions in our experiments and investigated 15 agronomic traits in three environments. We used a total of 128 pairs of simple sequence repeat (SSR) primers to scan for polymorphism. We then used NTsys-pc2.1 software to analyze genetic diversity, and Structure2.3.1 and Tassle5.0 software to perform association analysis of fiber quality traits based on phenotypic effect values to identify elite allele variation and conventional materials. [Result] A total of 244 loci were amplified by 128 marker pairs in 219 samples with an average of 1.91 loci per marker. The polymorphic information content ranged from 0.13 to 0.86 with an average of 0.63. The distribution range of the genetic similarity coefficient between materials in this population was 0.42-0.99 with an average of 0.61. The genetic similarity coefficient was between 0.5 and 0.7 and accounted for 90.19%. Through association analysis, we detected 11 markers that were significantly (P<0.01) associated with fiber quality traits, and discovered seven typical materials with specific allele. [Conclusion] In total, 219 Xinjiang early-maturing upland cotton germplasm resources have low genetic diversity. Based on SSR association analysis, we discovered some specific allele variations and conventional materials related to fiber quality traits.     

陆地棉种质资源抗旱性状的关联分析

干旱是导致全世界棉花严重减产、纤维品质下降的重要因素,因此获得高产、优质、耐旱的棉花新品种一直是棉花的育种目标。本研究选取217份陆地棉栽培种组成的自然群体为研究对象,采用全生育期处理组灌水量为对照组50%的干旱胁迫处理,并在处理后期对217份材料的株高、衣分、单铃重等18个性状进行2年2点的表型鉴定,干旱胁迫后,群体间响应差异明显,多个表型性状在对照和处理间表现显著差异。通过BLUP分析表型数据并计算各性状的抗旱系数;全基因组范围选取的214对多态性SSR分子标记扫描群体,共检测到393个多态性位点,基因多样性系数平均值为0.402,范围为0.072~0.631,PIC值平均为0.329,范围为0.070~0.560;群体结构分析表明,该群体可分为2个亚群。用上述SSR标记分别对18个性状的抗旱系数进行关联分析,共关联到76个极显著位点(P<0.01),表型变异解释率为2.930%~7.218%,其中共有14个标记位点能同时被2种或以上性状检测到。研究结果可为后期棉花杂交育种亲本选择及抗旱分子标记辅助育种提供理论基础及参考依据。     

Genetic Dissection of Allelic Loci Associated with Economic Traits of Upland Cottons in Xinjiang

[Objective] We completed anassociation analysis of economic traits for upland cotton using simple sequence repeat (SSR) markers. We then explored the allelic variation sites to analyze the genetic basis of economically important traits, studied the genetic mechanism of Xinjiang upland cotton, and aimed to accelerate efficient breeding of upland cotton. [Method] We carried out polymorphic scanning on 156 upland cotton varieties in Xinjiang by screening 73 pairs of SSR markers encompassing the whole cotton genome. We constructed boxplot maps using R statistical computing software and graphics language and used TASSEL software to correlate yield or fiber quality traits with significant allelic variation loci. [Results] We obtained 10 allelic variation loci related to yield traits using the correlation analysis of Xinjiang upland cotton varieties from six different environments. The interpretation rate of phenotypic variation ranged from 6.69% to 9.88% with an average of 8.43%. Twenty-three allelic variation loci associated with fiber quality traits and phenotypic variation interpretation rates ranged from 3.73% to 13.22% with an average of 7.52%. The 22 detected quantitative trait loci were reported in previous studies and 10 showed the same associated traits as previously reported. [Conclusion] The population genetic structure of Xinjiang upland cotton varieties is simple, the linkage disequilibrium level is low, and the phenotypic traits show a stable trend under six environments. Using association analysis, we discovered unique allelic variation genes related to yield and fiber quality and diverse allele loci.     

棉花种质资源收集鉴定与创新利用

十二五期间通过对我国西南地区、南海诸岛、及国外野生棉起源中心实地考察和广泛征集,新收集国内外棉花种质资源1220份,其中陆地棉986份、海岛棉176份、亚洲棉58份,所收集种质均已入国家中期库保存。收集的棉花种质资源分别在河南安阳、海南三亚、新疆阿拉尔进行主要农艺经济性状鉴定和特性鉴定。通过SSR(Simple sequence repeat)分子标记、重测序等技术对收集到的种质进行了分子鉴定,筛选到大量与纤维品质、抗黄萎病、抗旱、抗草甘膦显著关联的基因。在收集保存的种质资源基础上,通过辐射诱变、远缘杂交、复合杂交创造出优质、高产、抗逆等优异种质资源。通过展示向全国科研院所及公司发放棉花种质19 191份次。     

三个栽培种中天然彩色棉的遗传多样性及关联分析

本研究利用主要农艺性状和SSR标记分析了137份陆地棉、13份海岛棉以及27份亚洲棉等不同纤维色泽的种质资源。170对SSR引物共检测到1036个等位基因变异。其中有多态性的等位基因923个,平均每个SSR位点有5.43个等位变异,变化范围为1~10。位点多态性信息量(PIC)的变化范围为0.56~0.93,PIC在0.85以上的多态性SSR位点为122个。结果表明,实验材料存在着丰富的遗传多样性。在群体结构分析基础上,使用TASSEL软件中的混合线性模型(MLM),在陆地棉中得到与11个性状相关联的SSR标记23个;在海岛棉群体得到与5个性状关联的17个标记;在亚洲棉中检测到与8个性状关联的15个标记位点。在三个栽培种中与纤维色泽度相关的标记位点一共有6个,分别是CIR51-4、BNL1421-2、BNL2656-2、DPL570-2、GH268-6和TMB131-1,表型变异解释率从3.12%到18.97%。三个栽培种中天然彩色棉种质具有丰富的遗传多样性,为棉花的种间、种内杂交育种以及拓宽棉花新品种的遗传背景提供了物质基础和理论依据。