基于胚抢救技术的枳雀×枳杂交群体创建及鉴定
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摘要
【目的】新资源获得是开展育种的前提和基础,通过两种砧木杂交,创造出聚合两种砧木优良性状的新型砧木资源材料。【方法】以枳雀为母本、枳为父本进行杂交实验,结合胚抢救技术获得子一代植株,利用形态学观察及SSR分子标记技术对子一代进行鉴定。【结果】三年胚抢救实验共接种3 146粒种子,获得胚抢救幼苗1250株,成苗率为39.73%。其中通过形态学鉴定出41株杂种苗,共筛选出8对SSR引物来对父本母本进行区分,其中的CS014RCS014F、CS065R-CS065F引物组合可以准确地鉴定出杂种后代,通过SSR分子标记鉴定出20株有性杂种植株。所获得的61株杂种资源为将来深入开展柑橘砧木育种研究工作奠定了材料基础。【结论】以多胚性柑橘为母本的杂交组合获得的成熟种子,难以通过直接播种的方式获得杂种苗。将胚拯救与分子鉴定相结合,通过SSR分析,找到可靠的SSR引物对鉴定杂交后代,为柑橘砧木育种的进一步研究奠定了物质基础。
【Objective】The research and utilization of resistant rootstock resources of Citrus in China started relatively late, and the breeding rootstock is still relatively delayed in comparison with that in the developed countries. In order to obtain hybrid offspring population of Zhique( Citrus wilsonii Tanaka) Raf.) and Trifoliate orange[Poncirus trifoliata(L.) Raf], we conducted this study. Trifoliate orange is used as a kind of citrus rootstock widely in China. It has good affinity and general resistance. But the major defect is the insufficiency of iron absorption. Zhique is a kind of local citrus rootstock resource reported in Chenggu county, Shanxi province recently. It has good ability in using iron. In order to fully combine the beneficial properties, we carried out hybridization combined with embryo rescue technique to obtain hybrid offspring.【Methods】The species Zhique was planted in Chenggu county, Shanxi province. In this experiment, the Zhique's plants, leaves, flowers, fruits and other materials used for morphological description were all collected from a tree which was estimated more than 40 years old. Zhique was used as female parent and Trifoliate orange was used as male parent. The pollen of Trifoliate orange was collected in HZAU Germplasm Resources Nursery during morning. After the collection, the pollen was dried in dryer with no light at 27-29 ℃. We stored the pollen in 1.5 mL centrifuge tubes at4 ℃ when they were fully dried. We used acetic acid magenta to test the activity before the pollination and bagged the flowers after pollination. The hybrid fruits were picked 80-85 days after the pollination.The fruits were stored in 4 ℃ refrigerator before the embryo rescue was done. The embryos were inoculated on the shooting medium(MT+KT 0.5 mg · L~(-1)+BA 0.5 mg · L~(-1)+NAA 0.5 mg · L~(-1)) under illumination culture until the bud grew up. The shoots were moved on rooting medium(l/2 MT+IBA 0.1 mg · L~(-1)+NAA 0.5 mg · L~(-1)+Activated Carbon 0.5 mg · L~(-1)) until the root grew up. After subculture the plants were transfered into water for 1 month adaptation. Finally we put the well adapted seedlings into small pots with nutritive soil, and moved the pots in greenhouse. There were 1 133 progeny seedlings survival in the end, some of them were identified with morphology of the male parent, but the rest had to be identified by molecular markers. 8 of the primer pairs of the 10 possible SSR primer pairs were able to be used for the identification of the true hybrid seedlings.【Results】61 hybrid offspring were obtained.Among them 41 of the hybrid plants were identified with the trifoliate morphologic characteristics. Another 20 hybrid plants were identified by SSR molecular marker primer pair CS014 R-CS014 F and CS065 R-CS065 F.he hybrid rate was 4.1%.【Conclusion】Hybrid offsprings derived from the cross between Zhique and Trifoliate orange were successfully identified by means of morphology and SSR marker selection from the seedlings obtained with embryo rescue which created the materials for the further study of citrus rootstock breeding.
【Objective】The research and utilization of resistant rootstock resources of Citrus in China started relatively late, and the breeding rootstock is still relatively delayed in comparison with that in the developed countries. In order to obtain hybrid offspring population of Zhique( Citrus wilsonii Tanaka) Raf.) and Trifoliate orange[Poncirus trifoliata(L.) Raf], we conducted this study. Trifoliate orange is used as a kind of citrus rootstock widely in China. It has good affinity and general resistance. But the major defect is the insufficiency of iron absorption. Zhique is a kind of local citrus rootstock resource reported in Chenggu county, Shanxi province recently. It has good ability in using iron. In order to fully combine the beneficial properties, we carried out hybridization combined with embryo rescue technique to obtain hybrid offspring.【Methods】The species Zhique was planted in Chenggu county, Shanxi province. In this experiment, the Zhique's plants, leaves, flowers, fruits and other materials used for morphological description were all collected from a tree which was estimated more than 40 years old. Zhique was used as female parent and Trifoliate orange was used as male parent. The pollen of Trifoliate orange was collected in HZAU Germplasm Resources Nursery during morning. After the collection, the pollen was dried in dryer with no light at 27-29 ℃. We stored the pollen in 1.5 mL centrifuge tubes at4 ℃ when they were fully dried. We used acetic acid magenta to test the activity before the pollination and bagged the flowers after pollination. The hybrid fruits were picked 80-85 days after the pollination.The fruits were stored in 4 ℃ refrigerator before the embryo rescue was done. The embryos were inoculated on the shooting medium(MT+KT 0.5 mg · L~(-1)+BA 0.5 mg · L~(-1)+NAA 0.5 mg · L~(-1)) under illumination culture until the bud grew up. The shoots were moved on rooting medium(l/2 MT+IBA 0.1 mg · L~(-1)+NAA 0.5 mg · L~(-1)+Activated Carbon 0.5 mg · L~(-1)) until the root grew up. After subculture the plants were transfered into water for 1 month adaptation. Finally we put the well adapted seedlings into small pots with nutritive soil, and moved the pots in greenhouse. There were 1 133 progeny seedlings survival in the end, some of them were identified with morphology of the male parent, but the rest had to be identified by molecular markers. 8 of the primer pairs of the 10 possible SSR primer pairs were able to be used for the identification of the true hybrid seedlings.【Results】61 hybrid offspring were obtained.Among them 41 of the hybrid plants were identified with the trifoliate morphologic characteristics. Another 20 hybrid plants were identified by SSR molecular marker primer pair CS014 R-CS014 F and CS065 R-CS065 F.he hybrid rate was 4.1%.【Conclusion】Hybrid offsprings derived from the cross between Zhique and Trifoliate orange were successfully identified by means of morphology and SSR marker selection from the seedlings obtained with embryo rescue which created the materials for the further study of citrus rootstock breeding.
引文
[1]朱世平,陈娇,马岩岩,闫树堂,钟广炎.柑橘砧木评价及应用研究进展[J].园艺学报,2013,40(9):1669-1678.ZHU Shiping,CHEN Jiao,MA Yanyan,YAN Shutang,ZHONG Guangyan. Advances in evaluation and application of Citrus Rootstocks[J]. Acta Horticulturae Sinica,2013,40(9):1669-1678.
[2] FRIEDERIKE B,MARíA J A,JOSEP A J,ALBERTO U. Replacement of CTV-susceptible sour orange rootstock by CTV-tolerant ones may have triggered outbreaks of Tetranychus urticae in Spanish citrus[J]. Agriculture,Ecosystems and Environment,2010,137(1):93-98.
[3] GMITTER F G,XIAO S Y,HUANG S,HU X L,GARNSEY S M,DENG Z. A localized linkage map of the citrus tristeza virus resistance gene region[J]. Theoretical&Applied Genetics,1996,92(6):688-695.
[4] O'BANNON J H,FORD H W. Resistance in citrus rootstocks to Radopholus similis and Tylenchulus semipenetrans(Nematoda)[C]. Proceedings of the International Society of Citriculture,1978:544-549.
[5] JI Q H,LI Y T,WANG Y Q. Research Progress on Citrus iron deficiency chlorosis in calcareous soil[J]. Journal of Sichuan Agricultural University,1998,16(3):365-369.
[6] FU L N,CHAI L J,DING D K,PAN Z Y,PENG S A. A Novel Citrus rootstock tolerant to iron deficiency in calcareous soil[J].Journal of the American Society for Horticultural Science,2016,141(2):112-118.
[7]朱世平,陈娇,刘小丰,曹立,陆智明,赵晓春,15种柑橘砧木出苗期耐盐碱性评价[J].西南大学学报(自然科学版),2014,36(6):47-52.ZHU Shiping,CHEN Jiao,LIU Xiaofeng,CAO Li,LU Zhiming,ZHAO Xiaochun,Evaluation of salt tolerance and alkalinity of 15 Citrus Rootstocks at Seedling Stage[J]. Journal of Southwestern University(Natural Science Edition),2014,36(6):47-52.
[8]李航,杨晓明,丁德宽,王小丽,谢宗周,叶俊丽,彭抒昂,邓秀新,柴利军.基于cpSSR和nSSR标记的地方柑橘资源枳雀’亲本分析[J].果树学报,2018,35(10):265-274.LI Hang,YANG Xiaoming,DING Dekuan,WANG Xiaoli,XIE Zongzhou,YE Junli,PENG Shu’ang,DENG Xiuxin,CHAI Lijun. Parentage analysis of indigenous citrus species‘Zhi Que’based on nuclear and chloroplast SSR markers[J]. Journal of Fruit Science,2018,35(10):265-274.
[9]谭美莲.柑橘有性杂种群体的获得及分子遗传连锁框架图的构建[D].武汉:华中农业大学,2007.TAN Meilian. Creation of sexual hybrid populations and construction of molecular genetic linkage framework map in citrus[D]. Wuhan:Huazhong Agricultural University,2007.
[10]李航.宜昌橙及两个地方柑橘种的资源调查与评价[D].武汉:华中农业大学,2015.LI Hang. Resources investigation and evaluation of Yichang oranges and two local citrus species[D]. Wuhan:Huazhong Agricultural University,2015.
[11]孙世航,林苗苗,齐秀娟,孙雷明,钟云鹏,方金豹.应用InDel标记进行软枣猕猴桃杂交子代真实性鉴定[J].果树学报,2018,35(1):32-37.SUN Shihang,LIN Miaomiao,QI Xiujuan,SUN Leiming,ZHONG Yunpeng,FANG Jinbao. Authenticity identification of hybrid progeny of Actinidia kiwifruit by InDel markers[J]. Journal of Fruit Science,2018,35(1):32-37.
[12]庄恩及,姚强.早熟和特早熟桃胚珠培养研究[J].园艺学报,1991,18(4):303-308.ZHUANG Enji,YAO Qiang. Ovule culture of early maturing and very early maturing peach[J]. Acta Horticulturae Sinica,1991,18(4):303-308.
[13]赵艳华,李春敏,吴雅琴.植物的胚抢救[J].河北农业科学,2003,7(b9):55-58.ZHAO Yanhua,LI Chunmin,WU Yaqin. Embryo rescue of plants[J]. Hebei Agricultural Sciences,2003,7(b9):55-58.
[14]王家福,陈振光.柑桔杂交的胚胎发育[J].福建农林大学学报(自然版),1992,21(3):302-308.WANG Jiafu,CHEN Zhenguang. Embryonic development of Citrus hybrids[J]. Journal of Fujian Agriculture And Forestry University(Natural Version),1992,21(3):302-308.
[15]王家福,陈振光.影响柑桔球形合子胚离体培养的若干因素[J].热带作物学报,1999,20(2):27-30.WANG Jiafu,CHEN Zhenguang. Factors affecting the in vitro culture of spherical zygotic embryos of Citrus[J]. Chinese Journal of Tropical Crops,1999,20(2):27-30.
[2] FRIEDERIKE B,MARíA J A,JOSEP A J,ALBERTO U. Replacement of CTV-susceptible sour orange rootstock by CTV-tolerant ones may have triggered outbreaks of Tetranychus urticae in Spanish citrus[J]. Agriculture,Ecosystems and Environment,2010,137(1):93-98.
[3] GMITTER F G,XIAO S Y,HUANG S,HU X L,GARNSEY S M,DENG Z. A localized linkage map of the citrus tristeza virus resistance gene region[J]. Theoretical&Applied Genetics,1996,92(6):688-695.
[4] O'BANNON J H,FORD H W. Resistance in citrus rootstocks to Radopholus similis and Tylenchulus semipenetrans(Nematoda)[C]. Proceedings of the International Society of Citriculture,1978:544-549.
[5] JI Q H,LI Y T,WANG Y Q. Research Progress on Citrus iron deficiency chlorosis in calcareous soil[J]. Journal of Sichuan Agricultural University,1998,16(3):365-369.
[6] FU L N,CHAI L J,DING D K,PAN Z Y,PENG S A. A Novel Citrus rootstock tolerant to iron deficiency in calcareous soil[J].Journal of the American Society for Horticultural Science,2016,141(2):112-118.
[7]朱世平,陈娇,刘小丰,曹立,陆智明,赵晓春,15种柑橘砧木出苗期耐盐碱性评价[J].西南大学学报(自然科学版),2014,36(6):47-52.ZHU Shiping,CHEN Jiao,LIU Xiaofeng,CAO Li,LU Zhiming,ZHAO Xiaochun,Evaluation of salt tolerance and alkalinity of 15 Citrus Rootstocks at Seedling Stage[J]. Journal of Southwestern University(Natural Science Edition),2014,36(6):47-52.
[8]李航,杨晓明,丁德宽,王小丽,谢宗周,叶俊丽,彭抒昂,邓秀新,柴利军.基于cpSSR和nSSR标记的地方柑橘资源枳雀’亲本分析[J].果树学报,2018,35(10):265-274.LI Hang,YANG Xiaoming,DING Dekuan,WANG Xiaoli,XIE Zongzhou,YE Junli,PENG Shu’ang,DENG Xiuxin,CHAI Lijun. Parentage analysis of indigenous citrus species‘Zhi Que’based on nuclear and chloroplast SSR markers[J]. Journal of Fruit Science,2018,35(10):265-274.
[9]谭美莲.柑橘有性杂种群体的获得及分子遗传连锁框架图的构建[D].武汉:华中农业大学,2007.TAN Meilian. Creation of sexual hybrid populations and construction of molecular genetic linkage framework map in citrus[D]. Wuhan:Huazhong Agricultural University,2007.
[10]李航.宜昌橙及两个地方柑橘种的资源调查与评价[D].武汉:华中农业大学,2015.LI Hang. Resources investigation and evaluation of Yichang oranges and two local citrus species[D]. Wuhan:Huazhong Agricultural University,2015.
[11]孙世航,林苗苗,齐秀娟,孙雷明,钟云鹏,方金豹.应用InDel标记进行软枣猕猴桃杂交子代真实性鉴定[J].果树学报,2018,35(1):32-37.SUN Shihang,LIN Miaomiao,QI Xiujuan,SUN Leiming,ZHONG Yunpeng,FANG Jinbao. Authenticity identification of hybrid progeny of Actinidia kiwifruit by InDel markers[J]. Journal of Fruit Science,2018,35(1):32-37.
[12]庄恩及,姚强.早熟和特早熟桃胚珠培养研究[J].园艺学报,1991,18(4):303-308.ZHUANG Enji,YAO Qiang. Ovule culture of early maturing and very early maturing peach[J]. Acta Horticulturae Sinica,1991,18(4):303-308.
[13]赵艳华,李春敏,吴雅琴.植物的胚抢救[J].河北农业科学,2003,7(b9):55-58.ZHAO Yanhua,LI Chunmin,WU Yaqin. Embryo rescue of plants[J]. Hebei Agricultural Sciences,2003,7(b9):55-58.
[14]王家福,陈振光.柑桔杂交的胚胎发育[J].福建农林大学学报(自然版),1992,21(3):302-308.WANG Jiafu,CHEN Zhenguang. Embryonic development of Citrus hybrids[J]. Journal of Fujian Agriculture And Forestry University(Natural Version),1992,21(3):302-308.
[15]王家福,陈振光.影响柑桔球形合子胚离体培养的若干因素[J].热带作物学报,1999,20(2):27-30.WANG Jiafu,CHEN Zhenguang. Factors affecting the in vitro culture of spherical zygotic embryos of Citrus[J]. Chinese Journal of Tropical Crops,1999,20(2):27-30.