乌塌菜和白菜臺核复等位基因雄性不育系的创制与利用
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摘要
乌塌菜和白菜薹是十字花科芸薹属A基因组的两种特色蔬菜,异花传粉,杂种优势显著。为了解决其杂种优势利用中的杂交制种手段问题,以复等位基因遗传的大白菜和奶白菜核基因雄性不育系为不育源,以乌塌菜和白菜薹高代自交系为转育目标品系,设计定向转育方案,采用杂交、回交和自交方法,在转育核不育复等位基因的同时转育园艺学性状,创制新的雄性不育系；探索将分子标记辅助选择技术应用于白菜薹核不育系转育,利用SSR标记鉴定目标植株的基因型；针对育成的乌塌菜雄性不育系花蕾败育问题,采用cDNA-AFLP技术分析了花蕾败育相关基因的差异表达特性。主要研究成果如下：
1.利用奶白菜核复等位基因遗传的雄性不育系‘GMS-1’,与乌塌菜自交系‘WT01’杂交,通过对F1植株雄蕊育性的鉴定,确定乌塌菜转育目标品系在核不育位点上的基因型为MsfMsf。以此为基础设计了乌塌菜核基因雄性不育系定向转育方案,在连续4代回交转育园艺学性状的基础上,育成了具有100%不育株率和100%不育度、园艺学性状与转育目标品系‘WT01’相近的乌塌菜核基因雄性不育系‘GMS-3’。
2.以育成的乌塌菜核基因雄性不育系为母本,与4个乌塌菜优良自交系配制杂交组合,以乌塌菜商品种'Vitamin'为对照进行品种比较试验,对杂交组合的整齐度、品质和产量进行分析。各杂交组合的植物学性状的整齐度高,可溶性糖、蛋白、Vc、有机酸、粗纤维以及微量元素含量均处于较高水平,大部分品质指标极显著高于对照。各杂交组合产量表现出明显的杂种优势,均显著高于对照品种。综合考察产量和品质性状,筛选出一个优良杂交组合C1。
3.以乌塌菜核基因雄性不育系为试材,取5株植株一级分枝的正常开花期花蕾和败蕾期花蕾分别建立混合池,利用cDNA-AFLP技术和生物信息学技术分析败蕾过程中的表达差异基因。共筛选到64个与花蕾败育相关的差异表达片段(TDF),片段大小在80-400bp之间。按照正常花蕾→败育花蕾的顺序,差异表达片段呈现出4类表达模式：a：有→无；b：无→有；c：强→弱；d：弱→强。其中,a类条带占43.0%,b类条带占36.5%,c类条带占10.8%,d类条带占9.7%。选取42个典型的TDF进行测序,获得的序列信息在BRAD和NCBI上进行同源性比对,其中同源性大于90%的TDF有16条,基因功能可分为8类：①能量与代谢相关的TDF1条；②逆境响应相关的TDF5条；③转录和翻译相关的TDF1条；④信号转导相关的TDF4条；⑤抗病与衰老相关的TDF2条；⑥氨基酸的合成与加工相关的TDF2条；⑦跨膜运输相关的TDF3条；⑧功能未知的TDF2条。
4.利用大白菜核复等位基因遗传的雄性不育系‘GMS-2’,与白菜薹自交系‘Br02’杂交,根据F1植株育性调查结果确定白菜薹转育目标品系的基因型为MsfMsf。以此为依据设计了白菜薹核基因雄性不育系定向转育方案,并利用分子标记辅助选择技术筛选目标基因型植株。通过对本实验室前期开发的26对SSR引物的筛选鉴定,证明了GSSR1在双亲间具有多态性,且与显性雄性不育基因Ms共分离,同样可以标记同一位点恢复基因Msf和可育基因ms。在连续3代回交转育园艺学性状的基础上,育成了具有100%不育株率和100%不育度、园艺学性状与转育目标品系‘Br02’相近的白菜薹核基因雄性不育系‘GMS-4’。
5.利用白菜薹核不育系GMS-4与6个白菜薹优良自交系配制杂交组合,对各个杂交组合的整齐度和小区产量进行测定。各杂交组合的整齐度均较高,产量优势明显。综合考察产量和其他园艺学性状,筛选出一个优良杂交组合C3。
Wutacai(Brassica campestris ssp. chinensis var rosularis Tsen et Lee) and Baicaitai (Brassica campestris ssp. chinenesis L.) are two spetical types of Brassica crops (AA,2n=2x=20) which own significant hybrid vigor. In order to resolve the problem of breeding in utilizing hybrid vigor, a "directional transfer program" was designed to breed the multiple-allele male sterile lines of Wutacai and Baicaitai. The multiple-allele male sterile lines of Naibaicai(Brassica campestris ssp. chinensis L., GMS-1) and Chinese cabbage (GMS-2) were used as the male sterile resourse, and the inbred lines of Wutacai (WT01) and Baitaicai (BrO2) were used as the target lines. Crossing, recurrent backcrossing and selfing were employed to transfer the male sterility and other botanical traits simultaneously. Several SSR markers were adopted for molecular marker-assisted selection (MAS) to solve the problem in identifying genotypes during the transferring of the male sterile line in Baicaitai. Aming at studying the molecular mechanism of aborted bud which found in male sterile line of Wutacai, cDNA-AFLP technology was used to analyze gene expression difference between normal bud and aborted bud. The main results were as follows.
1. The genotype of the inbred line'TW01'was cheked by using a male sterile line (Msms) of Naibaicai'GMS-1'. It indicated that genotype of'TW01'was MsfMsf. According to the genotype, a directional transfer program was designed to transfer male sterile line. After backcrossing for four generations, a new male sterile line,'GMS-3', with similar botanical traits to'WT01',100%sterility degree and100%sterility rate, was bred.
2. Four hybrid combinations were generated with'GMS-3'as female parent and four excellent inbred lines as male parents. The'Vitamin' was as a control. Evenness degree, quality and yield of hybrid combinations were analyzed through Variety comparison test. Four hybrid combinations showed high evenness drgree in botanical traits. The component analysis results show that the components contents contained soluble sugar, protein, vitamin C, organic acids, crude fiber and trace elements were also at a high level. Most quality index is significantly higher than that of control. Yield of hybrid combinations showed remarkable hybrid vigor and signify cant differences was found between combinations and control. An excellent one,'Cl'was developed from the new male sterile line.
3. Normal bud and aborted bud of the male sterile line of Wutacai were collected from primary branches of five plants, respectively. RNA was extracted from mixed buds to set up gene pools. cDNA-AFLP technology and bioinformatics analysis were used to analyze gene expression difference between normal bud and aborted bud. A total of64differential expressed transcript-derived fragments (TDFs) which were between80bp and400bp were obtained. They were divided into four types according to expression of "normal bud→aborted bud":a:"present→absent" type, approximately accounting for43.0%; b:"absent→present" type, approximately accounting for36.5%; c:"strong→weak" type, approximately accounting for10.8%; d:"weak→strong" type, approximately accounting for9.7%.42typical TDFs were chosen to sequence. The sequencing results were compared similarity by BRAD and NCBI.16TDFs similarity were more than90%. Gene functions involved eight types:①1TDF was related with energy and metabolism;②5TDFs was related with stress response;③1TDF was related with signal transduction;④4TDFs was related with transcription and translation;⑤2TDFs was related with disease resistance and senescence;⑥2TDFs was related with amino acid synthesizing and processing;⑦3TDFs was related with transmembrane transport;⑧2TDFs were function unknown.
4. The genotype of the inbred line'Br02'was cheked by using a male sterile line (Msms) of Chinese cabbage'GMS-2'. F1plants genotype identification results indicated that genotype of'Br02'was MsfMsf. According to the genotype, a directional transfer program was designed to transfer male sterile line. Recurrent backcrossing method was employed to transfer male sterility and botanical traits while the genotype was identified by MAS. The GSSR1, which is closely linked to the male sterility gene "Ms", was chosen from26pairs of SSR primers to apply in the selection. GSSR1also could select the fertile gene "ms" and the fertile restoration gene "Msf". After three times recurrent backcrossing, a new genetic male sterile line of Baicaitai with100%of male sterile plants and100%of male sterility,'GMS-4', was bred. The new male sterile line was similar to'Br02'in most botanical traits.
5. Six hybrid combinations were generated with'GMS-4' as female parent and excellent inbred lines as male parents. The evenness degree and yield of hybrid combinations were analyzed through Variety comparison test. High evenness drgree of botanical traits were showed between blocks and that showed much difference between hybrid combinations. Hybrid vigor was in hybrid combinations and signi-ficant differences were found between combinations and control. C3was developed as the excellent hybrid combination.
1.利用奶白菜核复等位基因遗传的雄性不育系‘GMS-1’,与乌塌菜自交系‘WT01’杂交,通过对F1植株雄蕊育性的鉴定,确定乌塌菜转育目标品系在核不育位点上的基因型为MsfMsf。以此为基础设计了乌塌菜核基因雄性不育系定向转育方案,在连续4代回交转育园艺学性状的基础上,育成了具有100%不育株率和100%不育度、园艺学性状与转育目标品系‘WT01’相近的乌塌菜核基因雄性不育系‘GMS-3’。
2.以育成的乌塌菜核基因雄性不育系为母本,与4个乌塌菜优良自交系配制杂交组合,以乌塌菜商品种'Vitamin'为对照进行品种比较试验,对杂交组合的整齐度、品质和产量进行分析。各杂交组合的植物学性状的整齐度高,可溶性糖、蛋白、Vc、有机酸、粗纤维以及微量元素含量均处于较高水平,大部分品质指标极显著高于对照。各杂交组合产量表现出明显的杂种优势,均显著高于对照品种。综合考察产量和品质性状,筛选出一个优良杂交组合C1。
3.以乌塌菜核基因雄性不育系为试材,取5株植株一级分枝的正常开花期花蕾和败蕾期花蕾分别建立混合池,利用cDNA-AFLP技术和生物信息学技术分析败蕾过程中的表达差异基因。共筛选到64个与花蕾败育相关的差异表达片段(TDF),片段大小在80-400bp之间。按照正常花蕾→败育花蕾的顺序,差异表达片段呈现出4类表达模式：a：有→无；b：无→有；c：强→弱；d：弱→强。其中,a类条带占43.0%,b类条带占36.5%,c类条带占10.8%,d类条带占9.7%。选取42个典型的TDF进行测序,获得的序列信息在BRAD和NCBI上进行同源性比对,其中同源性大于90%的TDF有16条,基因功能可分为8类：①能量与代谢相关的TDF1条；②逆境响应相关的TDF5条；③转录和翻译相关的TDF1条；④信号转导相关的TDF4条；⑤抗病与衰老相关的TDF2条；⑥氨基酸的合成与加工相关的TDF2条；⑦跨膜运输相关的TDF3条；⑧功能未知的TDF2条。
4.利用大白菜核复等位基因遗传的雄性不育系‘GMS-2’,与白菜薹自交系‘Br02’杂交,根据F1植株育性调查结果确定白菜薹转育目标品系的基因型为MsfMsf。以此为依据设计了白菜薹核基因雄性不育系定向转育方案,并利用分子标记辅助选择技术筛选目标基因型植株。通过对本实验室前期开发的26对SSR引物的筛选鉴定,证明了GSSR1在双亲间具有多态性,且与显性雄性不育基因Ms共分离,同样可以标记同一位点恢复基因Msf和可育基因ms。在连续3代回交转育园艺学性状的基础上,育成了具有100%不育株率和100%不育度、园艺学性状与转育目标品系‘Br02’相近的白菜薹核基因雄性不育系‘GMS-4’。
5.利用白菜薹核不育系GMS-4与6个白菜薹优良自交系配制杂交组合,对各个杂交组合的整齐度和小区产量进行测定。各杂交组合的整齐度均较高,产量优势明显。综合考察产量和其他园艺学性状,筛选出一个优良杂交组合C3。
Wutacai(Brassica campestris ssp. chinensis var rosularis Tsen et Lee) and Baicaitai (Brassica campestris ssp. chinenesis L.) are two spetical types of Brassica crops (AA,2n=2x=20) which own significant hybrid vigor. In order to resolve the problem of breeding in utilizing hybrid vigor, a "directional transfer program" was designed to breed the multiple-allele male sterile lines of Wutacai and Baicaitai. The multiple-allele male sterile lines of Naibaicai(Brassica campestris ssp. chinensis L., GMS-1) and Chinese cabbage (GMS-2) were used as the male sterile resourse, and the inbred lines of Wutacai (WT01) and Baitaicai (BrO2) were used as the target lines. Crossing, recurrent backcrossing and selfing were employed to transfer the male sterility and other botanical traits simultaneously. Several SSR markers were adopted for molecular marker-assisted selection (MAS) to solve the problem in identifying genotypes during the transferring of the male sterile line in Baicaitai. Aming at studying the molecular mechanism of aborted bud which found in male sterile line of Wutacai, cDNA-AFLP technology was used to analyze gene expression difference between normal bud and aborted bud. The main results were as follows.
1. The genotype of the inbred line'TW01'was cheked by using a male sterile line (Msms) of Naibaicai'GMS-1'. It indicated that genotype of'TW01'was MsfMsf. According to the genotype, a directional transfer program was designed to transfer male sterile line. After backcrossing for four generations, a new male sterile line,'GMS-3', with similar botanical traits to'WT01',100%sterility degree and100%sterility rate, was bred.
2. Four hybrid combinations were generated with'GMS-3'as female parent and four excellent inbred lines as male parents. The'Vitamin' was as a control. Evenness degree, quality and yield of hybrid combinations were analyzed through Variety comparison test. Four hybrid combinations showed high evenness drgree in botanical traits. The component analysis results show that the components contents contained soluble sugar, protein, vitamin C, organic acids, crude fiber and trace elements were also at a high level. Most quality index is significantly higher than that of control. Yield of hybrid combinations showed remarkable hybrid vigor and signify cant differences was found between combinations and control. An excellent one,'Cl'was developed from the new male sterile line.
3. Normal bud and aborted bud of the male sterile line of Wutacai were collected from primary branches of five plants, respectively. RNA was extracted from mixed buds to set up gene pools. cDNA-AFLP technology and bioinformatics analysis were used to analyze gene expression difference between normal bud and aborted bud. A total of64differential expressed transcript-derived fragments (TDFs) which were between80bp and400bp were obtained. They were divided into four types according to expression of "normal bud→aborted bud":a:"present→absent" type, approximately accounting for43.0%; b:"absent→present" type, approximately accounting for36.5%; c:"strong→weak" type, approximately accounting for10.8%; d:"weak→strong" type, approximately accounting for9.7%.42typical TDFs were chosen to sequence. The sequencing results were compared similarity by BRAD and NCBI.16TDFs similarity were more than90%. Gene functions involved eight types:①1TDF was related with energy and metabolism;②5TDFs was related with stress response;③1TDF was related with signal transduction;④4TDFs was related with transcription and translation;⑤2TDFs was related with disease resistance and senescence;⑥2TDFs was related with amino acid synthesizing and processing;⑦3TDFs was related with transmembrane transport;⑧2TDFs were function unknown.
4. The genotype of the inbred line'Br02'was cheked by using a male sterile line (Msms) of Chinese cabbage'GMS-2'. F1plants genotype identification results indicated that genotype of'Br02'was MsfMsf. According to the genotype, a directional transfer program was designed to transfer male sterile line. Recurrent backcrossing method was employed to transfer male sterility and botanical traits while the genotype was identified by MAS. The GSSR1, which is closely linked to the male sterility gene "Ms", was chosen from26pairs of SSR primers to apply in the selection. GSSR1also could select the fertile gene "ms" and the fertile restoration gene "Msf". After three times recurrent backcrossing, a new genetic male sterile line of Baicaitai with100%of male sterile plants and100%of male sterility,'GMS-4', was bred. The new male sterile line was similar to'Br02'in most botanical traits.
5. Six hybrid combinations were generated with'GMS-4' as female parent and excellent inbred lines as male parents. The evenness degree and yield of hybrid combinations were analyzed through Variety comparison test. High evenness drgree of botanical traits were showed between blocks and that showed much difference between hybrid combinations. Hybrid vigor was in hybrid combinations and signi-ficant differences were found between combinations and control. C3was developed as the excellent hybrid combination.
引文
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