【作者】 王艳玲；

【导师】 胡建斌；

【作者基本信息】 河南农业大学 ， 园艺学， 2019， 硕士

【摘要】 甜瓜（Cucumis melo L.）为葫芦科（Cucurbitaceae）的一种重要水果,因其香甜的口感和丰富的营养受到大众的普遍欢迎,人们对甜瓜周年供应的要求日益迫切。尽管园艺设施已经在甜瓜生产中推广普及,但我国北方地区早春栽培仍然时常受到低温的侵害,长江流域的“倒春寒”已是春季甜瓜生产的重要环境障碍,低温已经成为限制我国甜瓜春季栽培和周年供应的重要环境因素之一。培育和推广强耐冷的甜瓜品种,是解决这一问题的有效途径,但其前提是明确耐冷性的遗传规律,发掘控制耐冷性的主效基因QTL。为此,本研究从甜瓜核心种质中筛选到一个强耐冷的种质ZTG00581,通过分析该种质耐冷性的遗传规律,并定位耐冷性QTL,主要结果如下:本试验以耐低温材料ZTG00581（581）和低温敏感材料ZTG00906（906）为研究对象,构建六世代（P1、P2、F1、Bi、B2、F2）群体,进行多世代联合分析;对140个F2单株和亲本进行RAD-seq技术的简化测序,构建遗传连锁图谱。实验结果如下:1.耐冷、冷敏亲本的差异分析。耐冷亲本ZTG00581（581）和冷敏亲本ZTG00906（906）的冷害指数分别为0.39和0.86;低温胁迫下,581的叶绿素叶绿素荧光参数普遍比906高,Fo、Fm、QY_max分别比906高出1.48、1.82、1.94倍;581的电导率显著低于906,其相对叶绿素含量SPAD和可溶性蛋白含量均高于906;超微结构观察表明,低温胁迫下906叶绿体等细胞器结构受损伤程度较581严重。2.耐冷性遗传分析。对581×906组合（区分正交（O）、反交（R））幼苗六世代（P1、P2、F1、Bi、B2、F2）的耐冷性进行多世代联合遗传分析。在CⅡ 1和CⅡ₂两个冷害指标下发现,CIⅡ₁o（第一次统计的冷害指数）和CⅡ 1R的最适遗传模型均为两对加性-显性-上位性主基因+加性-显性多基因;CⅡ 2o（第二次统计的冷害指数）的最适遗传模型为加性-显性-上位性模型;CⅡ 2R的最适遗传模型为两对加性-显性-上位性主基因+加性-显性-上位性多基因模型。正、反交试验结果均表明,甜瓜幼苗耐冷性主要由主基因控制又有多基因调控的复杂数量性状。3.遗传图谱构建。构建了包含140个单株F2群体,对群体及亲本进行RAD-seq简化基因测序。亲本581测得5,788,680条高质量的reads,占总reads的93.57%;906测得5,824,098条高质量的reads,占总reads的94.28%;以及F2群体平均测得7,136,845条高质量的reads;占总reads的93.92%。与甜瓜参考基因组（DHL92）的比对后,经检测共得到379,585个SNP;将筛选的SNP进一步过滤,得到6443个多态性标记,最终筛选出566个Bin标记;通过添加额外的SSR标记进行连锁分析,构建一组包括12个连锁群的遗传图谱,覆盖基因组全长3843.64 cM,标记间平均距离为5.9cM。不同连锁群上的标记数目在31-75之间,长度在225.82cM-439.72cM之间。4.耐冷性QTL定位。以140个单株为基础构建了 F2:3家系群体,对F2:3幼苗进行了 3次独立的耐冷性鉴定。以CⅡ 1和CⅡ 2两个指标进行QTL定位,分别检测到10、8个位点,位于第2、3、4、6、9和11号染色体,其中两个指标均检测到的区段位于2号染色体的chr02 2920027和chr02 2647384标记之间,以及11号染色体的CmSSR26454和CmSSR26330标记之间,其中qCⅡ₁ 11-3可解释12.48%表型变异,qCⅡ₁11-1的表型变异解释率为16.37%。更多还原

【Abstract】 Melon（Cucumis melo L.）is an important fruit of Cucurbitaceae.Because of its sweet taste and abundant nutrition,it is widely welcomed by the public.The demand for annual supply of melon is increasingly urgent.Although horticultural facilities have been popularized in Muskmelon production,early spring cultivation in northern China is still often encroached by low temperature.The "late spring cold" in the Yangtze River Basin has become an important environmental obstacle to muskmelon production in spring,and low temperature has become one of the important environmental factors restricting muskmelon cultivation in spring and annual supply in China.Breeding and popularizing melon varieties with strong cold tolerance is an effective way to solve this problem,but the premise is to clarify the genetic law of cold tolerance and explore the major gene QTL controlling cold tolerance.In this study,a strong cold-tolerant germplasm ZTG00581 was screened from the core germplasm of melon.The inheritance of cold-tolerance of the germplasm was analyzed,and the cold-tolerant QTL was mapped.The main results were as follows:In this experiment,low-temperature resistant material ZTG00581（581）and low-temperature sensitive material ZTG00906（906）were used as research objects to construct six generations（P1,P2,F1,B1,B2,F2）population and conduct multi-generational joint analysis;140 F2 plants and parents were sequenced by RAD-seq technology to construct genetic linkage map.The experimental results are as follows:1.Differential analysis of cold-tolerant and cold-sensitive parents.The chilling injury index of cold-tolerant parent ZTG00581（581）and cold-sensitive parent ZTG00906（906）were 0.39 and 0.86,respectively.Under low temperature stress,chlorophyll chlorophyll fluorescence parameters of 581 were generally higher than 906,Fo,Fm and QY_max were 1.48,1.82 and 1.94 times higher than 906,respectively;conductivity of 581 was significantly lower than 906,and its relative chlorophyll content SPAD and soluble protein content were higher than 906;ultrastructure observation The results showed that the damage degree of organelles such as chloroplast 906 was more serious than 581 under low temperature stress.2.Genetic analysis of cold tolerance.The cold tolerance of 5 81 x 906 combinations(distinguishing orthogonal（O）and backcross（R）seedlings in six generations（Pi,P2,Fi,B i,B2,F2）was analyzed by multi-generation combined genetic analysis.Under the two chilling injury indices of CⅡ₁ and CⅡ₂,it was found that the optimal genetic models of CⅡ₁O（Chilling injury index of the first statistics）and CⅡ₁R were two pairs of additive-dominant-epistatic major genes+additive-dominant polygenes,the optimum genetic model of CⅡ₂O（Chilling injury index of second statistics）is additive-dominant-epistatic model,and the optimum genetic model of CⅡ₂R is two additive-dominant-epistatic major genes+additive-dominant-epistatic polygene models.The results of both positive and negative crosses showed that cold tolerance of melon seedlings was a complex quantitative trait controlled by multiple genes.3.Construction of genetic map.A F2 population consisting of 140 individual plants was constructed and RAD-seq simplified gene sequencing was carried out for the population and parents.The parents 581 measured 5,788,680 high-quality reads,accounting for 93.57%of the total reads;906 measured 5,824,098 high-quality reads,accounting for 94.28%of the total reads;and the F2 population averaged 7,136,845 high-quality reads,accounting for 93.92%of the total reads.Compared with the reference genome of melon（DHL92）,379,585 SNPs were detected.;the screened SNP was further filtered and 6443 polymorphic markers were obtained;finally 566 Bin markers were screened out.By adding additional SSR markers for linkage analysis,a set of genetic maps including 12 linkage groups was constructed,covering the genome length of 3843.64 cM and the average distance between markers was 5.9 cM.The number of markers on different linkage groups ranged from 31 to 70,and the length ranged from 225.82 cM to 439.72 cM.4.Location of cold tolerance QTL.F2:3 family population was constructed based on 140 individual plants.Three independent cold tolerance tests were carried out for F3 seedlings.Mapping of QTL with CⅡ₁ and CⅡ₂,10 and 8 loci were detected,respectively,on chromosomes 2,3,4,6,9 and 11.The segments detected by both markers were between chr02 2920027 and chr02 2647384 markers on chromosome 2,and between CMSSR26454 and CMSSR26330 markers on chromosome 11.Among them,qCⅡ₁11-3 could explain 12.48%of phenotypic variation,and qCⅡ₁11-1 could explain 12.48%of phenotypic variation.The explanatory rate of phenotypic variation was 16.37%.更多还原