罗汉果遗传图谱构建及农艺性状QTL定位
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摘要
罗汉果(Siraitia grosvenorii (Swingle) C. Jeffrey)是葫芦科多年生雌雄异株植物,是广西特有的经济植物。罗汉果是高度杂合的二倍体植物(2n=28),其果实称为“罗汉果”,具有重要的医疗和保健价值。罗汉果具有清热润肺等功效,常用于治疗咳嗽,咽喉痛,肠燥便秘等症。其果实中含有热量极低而甜度很高的天然甜味物质—罗汉果苷,它是一种葫芦烷三萜甙类化合物。其中,罗汉果甜苷V甜度是蔗糖的300倍,被用作肥胖症患者和糖尿病患者的食糖代用品,其药理作用已明确并已开始工业化生产
本研究以野红一号为母本,长滩果为父本构建的150个F1子代为作图群体,利用ISSR和SRAP技术,构建了罗汉果的第一张遗传连锁图谱,并首次对10个农艺性状进行了QTL定位。主要结果如下：
1、建立了罗汉果第一份遗传图谱构建的F1群体材料。该群体以野生红毛一号和长滩果杂交获得了150株F1子代,种植于广西桂林兴安县实验基地,用于罗汉果遗传图谱构建及农艺性状QTL定位。
2、从100条ISSR引物中筛选出17条扩增清晰且具有多态性的引物,扩增出51条ISSR多态性条带,平均每条引物扩增出3条,片段大小200-2000 bp；从196对SRAP引物组合中筛选出74对引物对子代进行扩增,共获得222个多态性位点,平均每个引物获得3.07个多态性位点,122对引物未扩增出多态性位点。此外,我们还调查了5个质量性状用于遗传图谱构建。
3、本研究总共获得278个多态性位点,其中包括51个ISSR位点,222个SRAP位点和5个质量性状位点,有203个位点定位到27个连锁群上,包括29个ISSR位点,173个SRAP位点和1个性别标记。连锁群总长度为1474.1cM,平均图距是7.3 cM,最大图距是52.6 cM。大部分标记在连锁群上均匀分布,只有LG2一端的标记稍有聚集。27个连锁群上的标记为2-36个,连锁群长度为19.5-152.6 cM,遗传图谱覆盖率为65.2%。
4、利用该图谱,运用软件Windows QTL Cartographer V2.5的复合区间作图法对所考察罗汉果的茎粗,叶柄长,叶宽,叶长,叶绿素含量,叶片全氮含量,果实鲜重,果实横径,果实纵径,果形指数10个性状进行QTL定位研究。取LOD临界值为2.5,共获得QTLs位点三十三个,分别位于LG1, LG2, LG3, LG4, LG5, LG7, LG9, LG10, LG11, LG13, LG14, LG17, LG22, LG27十三个不同的连锁群上单位点遗传贡献率8.40%-32.95%。三十三个QTLs的LOD值介于2.60-7.40之间,其中控制茎粗的QTL有1个,控制叶柄长的QTL有4个,控制叶宽的QTL有5个,控制叶长的QTL有4个,控制叶绿素含量的QTL有3个,控制果实鲜重的QTL有3个,控制果实横径的QTL有3个,控制果实纵径的QTL有5个,控制果形指数的QTL有2个,控制叶片全氮的QTL有3个。
Siraitia grosvenorii (Swingle) C. Jeffrey (Cucurbitaceae family), a dioecious, perennial and liana plant with root tubers, is an economically important species endemic to southern China, with the most planting in Guilin. It is a highly heterozygous diploid species with a chromosome number of 2n=28. The fruits of Siraitia grosvenorii, called "Luohanguo" have long been used as a food and beverage material as well as a traditional Chinese medicine. The pharmacological characteristics of Luohanguo make it a pulmonary demulcent and emollient for the treatment of dry cough, sore throat, dire thirst and constipation. The fruit contains zero-calorie high intensive natural sweeteners known as mogrosides, a group of terpene glycosides. Among these, the mogrosides v is about 300 times sweeter than sucrose and pharmacological effects have been known, and have been produced on an industrial scale. As such, Luohanguo has been used as a sugar substitute for patients with adiposity and diabetes.
The first genetic linkage map of Luohanguo (Siraitia grosvenorii [Swingle] C. Jeffrey) was constructed with 150 F1 individuals derived from a cross between the cultivars Yehong 1 and Changtanguo using inter-simple sequence repeat (ISSR) and sequence related amplified polymorphism (SRAP) markers, and the QTL of ten agronomic traits was analyzed for the first time. The main conclusions are as follows:
1. The first F1 mapping population of Luohanguo was constructed. The population consisted of 150 F1 progenies derived from a cross between the wild species Hongmaoyihao and cultivar Changtanguo performed at Xing'an County, Guilin City, Guangxi Province, for genetic map construction and QTLs analysis of ten agronomic traits.
2. Out of 100 ISSR primers examined, only 17 (17%) of the ISSR primers were selected for efficient amplification and detection of polymorphism between the two parents and 150 F1 progenies. They produced 51 ISSR markers. The average number of markers produced per primer was 3. Fragment sizes ranged from 200 to 2,000bp. A total of 196 SRAP primer combinations were used to screen for polymorphisms between parents and 74 primer combinations showed polymorphisms.The 74 primer pairs yielded 222 polymorphic loci with an average of 3.07 polymorphic loci, and 122 primer pairs produced no polymorphic loci. In addition, we investigated five qualitative traits for genetic map construction.
3. The genetic map of Luohanguo (Siraitia grosvenorii [Swingle] C. Jeffrey) was constructed using 278 polymorphic loci(51 ISSR and 222 SRAP polymorphic loci and 5 qualitative traits). Among the 278 markers obtained,203 markers (29 ISSRs,173 SRAPs and 1 sex marker) were mapped to 27 linkage groups. The map covered 1474.1 cM with an average map distance of 7.3 cM between adjacent markers and a maximum map distance of 52.6 cM between two markers. The markers distributed randomly in 27 groups except for minor clusters in the distal region of LG2. All 27 linkage groups consisted of 2-36 loci ranging in length from 19.5 to 152.6 cM and accounted for 65.2% of the total map distance.
4. Composite interval mapping method of Windows QTL cartographer V2.5 was employed in QTL analysis of ten agronomical traits including stem diameter, petiole length, leaf width, leaf length, chlorophyll content, fruit fresh weight, fruit width, fruit length, fruit index and leaf total nitrogen content in Siraitia grosvenorii. A total of thirty-three QTLs were mapped on thirteen linkage groups for the ten triats with LOD≧ 2.5. Phenotypic variation explained by the single putative QTL varied from 8.40% to 32.95%, and the LOD score ranged from 2.60 to 7.40. These QTL included one for stem diameter,four for petiole length,five for leaf width, four for leaf length, three for chlorophyll content, three for fruit fresh weight, three for fruit width, five for fruit length,two for fruit index and three for leaf total nitrogen content.
本研究以野红一号为母本,长滩果为父本构建的150个F1子代为作图群体,利用ISSR和SRAP技术,构建了罗汉果的第一张遗传连锁图谱,并首次对10个农艺性状进行了QTL定位。主要结果如下：
1、建立了罗汉果第一份遗传图谱构建的F1群体材料。该群体以野生红毛一号和长滩果杂交获得了150株F1子代,种植于广西桂林兴安县实验基地,用于罗汉果遗传图谱构建及农艺性状QTL定位。
2、从100条ISSR引物中筛选出17条扩增清晰且具有多态性的引物,扩增出51条ISSR多态性条带,平均每条引物扩增出3条,片段大小200-2000 bp；从196对SRAP引物组合中筛选出74对引物对子代进行扩增,共获得222个多态性位点,平均每个引物获得3.07个多态性位点,122对引物未扩增出多态性位点。此外,我们还调查了5个质量性状用于遗传图谱构建。
3、本研究总共获得278个多态性位点,其中包括51个ISSR位点,222个SRAP位点和5个质量性状位点,有203个位点定位到27个连锁群上,包括29个ISSR位点,173个SRAP位点和1个性别标记。连锁群总长度为1474.1cM,平均图距是7.3 cM,最大图距是52.6 cM。大部分标记在连锁群上均匀分布,只有LG2一端的标记稍有聚集。27个连锁群上的标记为2-36个,连锁群长度为19.5-152.6 cM,遗传图谱覆盖率为65.2%。
4、利用该图谱,运用软件Windows QTL Cartographer V2.5的复合区间作图法对所考察罗汉果的茎粗,叶柄长,叶宽,叶长,叶绿素含量,叶片全氮含量,果实鲜重,果实横径,果实纵径,果形指数10个性状进行QTL定位研究。取LOD临界值为2.5,共获得QTLs位点三十三个,分别位于LG1, LG2, LG3, LG4, LG5, LG7, LG9, LG10, LG11, LG13, LG14, LG17, LG22, LG27十三个不同的连锁群上单位点遗传贡献率8.40%-32.95%。三十三个QTLs的LOD值介于2.60-7.40之间,其中控制茎粗的QTL有1个,控制叶柄长的QTL有4个,控制叶宽的QTL有5个,控制叶长的QTL有4个,控制叶绿素含量的QTL有3个,控制果实鲜重的QTL有3个,控制果实横径的QTL有3个,控制果实纵径的QTL有5个,控制果形指数的QTL有2个,控制叶片全氮的QTL有3个。
Siraitia grosvenorii (Swingle) C. Jeffrey (Cucurbitaceae family), a dioecious, perennial and liana plant with root tubers, is an economically important species endemic to southern China, with the most planting in Guilin. It is a highly heterozygous diploid species with a chromosome number of 2n=28. The fruits of Siraitia grosvenorii, called "Luohanguo" have long been used as a food and beverage material as well as a traditional Chinese medicine. The pharmacological characteristics of Luohanguo make it a pulmonary demulcent and emollient for the treatment of dry cough, sore throat, dire thirst and constipation. The fruit contains zero-calorie high intensive natural sweeteners known as mogrosides, a group of terpene glycosides. Among these, the mogrosides v is about 300 times sweeter than sucrose and pharmacological effects have been known, and have been produced on an industrial scale. As such, Luohanguo has been used as a sugar substitute for patients with adiposity and diabetes.
The first genetic linkage map of Luohanguo (Siraitia grosvenorii [Swingle] C. Jeffrey) was constructed with 150 F1 individuals derived from a cross between the cultivars Yehong 1 and Changtanguo using inter-simple sequence repeat (ISSR) and sequence related amplified polymorphism (SRAP) markers, and the QTL of ten agronomic traits was analyzed for the first time. The main conclusions are as follows:
1. The first F1 mapping population of Luohanguo was constructed. The population consisted of 150 F1 progenies derived from a cross between the wild species Hongmaoyihao and cultivar Changtanguo performed at Xing'an County, Guilin City, Guangxi Province, for genetic map construction and QTLs analysis of ten agronomic traits.
2. Out of 100 ISSR primers examined, only 17 (17%) of the ISSR primers were selected for efficient amplification and detection of polymorphism between the two parents and 150 F1 progenies. They produced 51 ISSR markers. The average number of markers produced per primer was 3. Fragment sizes ranged from 200 to 2,000bp. A total of 196 SRAP primer combinations were used to screen for polymorphisms between parents and 74 primer combinations showed polymorphisms.The 74 primer pairs yielded 222 polymorphic loci with an average of 3.07 polymorphic loci, and 122 primer pairs produced no polymorphic loci. In addition, we investigated five qualitative traits for genetic map construction.
3. The genetic map of Luohanguo (Siraitia grosvenorii [Swingle] C. Jeffrey) was constructed using 278 polymorphic loci(51 ISSR and 222 SRAP polymorphic loci and 5 qualitative traits). Among the 278 markers obtained,203 markers (29 ISSRs,173 SRAPs and 1 sex marker) were mapped to 27 linkage groups. The map covered 1474.1 cM with an average map distance of 7.3 cM between adjacent markers and a maximum map distance of 52.6 cM between two markers. The markers distributed randomly in 27 groups except for minor clusters in the distal region of LG2. All 27 linkage groups consisted of 2-36 loci ranging in length from 19.5 to 152.6 cM and accounted for 65.2% of the total map distance.
4. Composite interval mapping method of Windows QTL cartographer V2.5 was employed in QTL analysis of ten agronomical traits including stem diameter, petiole length, leaf width, leaf length, chlorophyll content, fruit fresh weight, fruit width, fruit length, fruit index and leaf total nitrogen content in Siraitia grosvenorii. A total of thirty-three QTLs were mapped on thirteen linkage groups for the ten triats with LOD≧ 2.5. Phenotypic variation explained by the single putative QTL varied from 8.40% to 32.95%, and the LOD score ranged from 2.60 to 7.40. These QTL included one for stem diameter,four for petiole length,five for leaf width, four for leaf length, three for chlorophyll content, three for fruit fresh weight, three for fruit width, five for fruit length,two for fruit index and three for leaf total nitrogen content.
引文
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