CN101253268A - 提高植物育种的方法和组合物 - Google Patents
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Abstract
本发明提供提高植物种质的育种方法和组合物。该方法描述在农作物植物繁殖种群的种质中,转基因和有利的单倍型基因组区的鉴定和累积。
Description
发明背景
本申请要求2005年5月27日申请的美国临时申请No.60/685,584的权益,该申请的整个原文在此明确地引入作为参考。
1、发明领域
本发明涉及植物育种和植物生物技术领域,特别地涉及插入到与植物的基因组区连锁的遗传连锁内的转基因,以及涉及使用转基因/基因组区以提高种质以及在繁殖种群中累积其它有利的基因组区的用途。
2、相关技术的描述
从根据个体和其亲缘植物的表型记录选择植物和动物中经济上重要的性状,到使用分子遗传学以鉴别含有有价值的遗传性状的基因组区,育种方式在向前发展。DNA水平的信息已导致有价值的性状比仅根据表型数据所获得的性状更快地遗传累积到种质内。转基因作物的发展进一步使育种和农业作物生产发生彻底变革。基因工程作物的显著成功是显而易见的,专门用于转基因作物的农田面积从十年前微不足道的几英亩已经发展到在农业上很重要的国家如美国、加拿大、巴西和阿根廷中占主要作物耕种面积的一半以上。除了输入性状的发展,植物生物技术也很有希望未来发展将直接有益于消费者的输出性状,象营养优良的食物,如富含维生素A的稻米、不饱和的油以及对一些人有医学价值的农产品。编码新的或者改良的输入或输出性状的转基因的商业成功的潜能对于发展新的转基因及通过育种将其这些基因部署到原种种质内来说,具有极大的诱惑力。
在发展转基因农作物植物期间,这种努力集中在转基因的插入和表达的最优化,然后通过经典育种方法将转基因渐渗到整个繁殖种群。由于至少两个理由使得转基因插入到宿主基因组内的位点一直受到关注;(i)其插入的区域可能调节转基因的表达水平,和(ii)转基因的插入可能破坏邻近或者其插入处基因的正常功能或表达。对基因整合有益的基因组位置的选择为所引入的基因提供适当的稳定表达水平,并且通常不反向影响农作物植物的其它农艺学特征。
已插入转基因的基因组区也向农作物植物提供农艺学表型。这些表型在育种程序中具有其自己的价值并且当在多个转基因插入事件中进行选择时,应当考虑这些区域。与相对于农艺学性状或多个性状指数,具有改良的行为表现有关的转基因插入到基因组区内的事件,导致农作物植物中具有改良的表型,并且从该农作物植物衍生的后代含有转基因和相关的改良表型。对转基因事件的选择必然导致选择环绕其的宿主基因组片段,以及改良的表型效果。更进一步的改进涉及用于跟踪和维持具有相关转基因的基因组片段的分子标记的鉴定。这是在当前用转基因插入事件进行植物育种中一直没有被充分提到的领域。
在植物育种领域中需要鉴定与转基因插入事件相连,与相对于农艺学性状或多性状指数,具有改良的行为表现有关的基因组区,然后选择这些转基因染色体区用于分散到作物的繁殖种群内。本发明提供估计基因组区和转基因事件的价值的考虑因素。该值然后可用作在多个转基因事件中进行选择的标准。另一个好处是将转基因周围被牵涉的连锁最小化并且选择有价值的基因组区,其含有用于育种到作物种质内的转基因。
发明概述
本发明提供用转基因植物育种的方法。在一个方面,该方法包括提供一种鉴定一组种质基因组的至少两个不同单倍型的农艺学性状值的数据库。该方法进一步包括用重组DNA转化亲本植物以产生至少两个转基因事件,其中将重组DNA插入到具有亲本植物基因组的至少两个不同单倍型的连锁内。然后可参考该数据库以估计与不同单倍型相连事件的农艺学性状值(value of said agronomic trait),然后可选择具有更高参考育种价值的转基因事件用于育种到种质内。
本发明提供通过在种质中累积一个或多个单倍型改善植物种质的方法。该方法包括将转基因插入到第一个植物的基因组内,然后测定基因组中转基因的图谱位置。可将图谱位置与连锁的单倍型联系起来,其中转基因和单倍型包括T型基因组区。然后可将第一个植物与第二个植物杂交。第二个植物可含有至少一个T型基因组区或单倍型,其与第一个植物T型基因组区不同。然后可选择至少一个后代植物,该后代植物具有转基因的可检测到的表达或者其表型,并且在其基因组中包括第一个植物的T型基因组区和至少一个第二个植物的T型基因组或单倍型。然后可在与种质改良相关的活动中使用该后代植物,其中活动可选自使用植物用于制备育种杂交、植物的进一步检验、植物通过自我授粉的进化,使用植物或其部分用于转化、使用植物或其部分用于诱变,以及使用植物或其部分用于耕作,或者这些的任意组合。
本发明包括用于农作物植物育种的方法,特别是包括优选的T型基因组区的具有提高的农艺学和转基因性状的大豆或玉米植物。将T型基因组区的转基因进一步定义为赋予优选性状的转基因,象除草剂耐性、抗病性、昆虫或害虫抗性、改变的脂肪酸、蛋白质或碳水化合物代谢、增加谷粒产量、增加油量、增加营养含量、提高生长率、提高胁迫耐性,或者改变的形态特征,或者这些的任何组合。
本发明提供用于对至少一个插入转基因的基因组区进行作图的新方法。该方法包括间接作图并且不需要对转基因从头进行种群分离。该方法包括首先在作图种群的亲本品种之间,在转化的植物或品系中与转基因插入事件相邻的相应基因组区中鉴定至少一个第一多态性,然后分析作图种群的后代植物的多态性。可进行连锁分析以确定多态性的图谱位置并且由此确定转基因插入事件的图谱位置。然后可将作图种群中的图谱位置与转化的植物及其后代的单倍型联系起来。
说明性实施方案的描述
所提供的定义和方法详细说明本发明并且在本发明的实施中指导本领域的普通技术人员。除非另有说明,术语应当由相关领域中的普通技术人员根据常规用法进行理解。也可在Rieger等(1991);和Lewin(1994)中找到分子生物学中普通术语的定义。使用如在37CFR§1.822中所述的DNA碱基命名法。
如在这里所使用的,术语“玉米”意思是玉米(Zea mays或maize)并且包括可与玉米育种的所有植物变种,包括野生型玉米种。
如在这里所使用的,术语“大豆”意思是大豆(Glycine max)并且包括可与大豆育种的所有植物变种,包括野生型大豆种。
如在这里所用的,术语“包括”意思是“包括但不限于”。
通过用异源DNA,即,包括目的转基因的核酸构建体转化植物细胞,再生由转基因插入到植物基因组内所得的植物种群,以及选择以插入到特定基因组位置内为特征的特定植物,产生转基因“事件”。术语“事件”指包括异源DNA的原始转化体和该转化体的后代。术语“事件”也指通过转化体和包括异源DNA的另一变体之间的有性异型杂交所产生的后代。
本发明克服当前转基因育种方法的不足,通过描述定义为转基因和连锁的单倍型基因组区的T型基因组区,通过其选择遗传连锁的转基因和单倍型,然后通过育种渐渗到种质内。T型基因组区的选择是基于评价T型基因组区向农作物植物的种质所提供的T值。该评价的基础区别并选择用于在育种方法中使用的改良的T型基因组区,并且选择和发展包括改良的T型基因组区的植物。基于为所引入的基因提供适当的稳定表达水平,基因组位置对于基因整合是有利,并且对于鉴定与也向种质提供有益的农艺学特征的有利的单倍型区联合的转基因也是有利的。通过考虑转基因和与其遗传连锁的基因组区两者的有益方面,可将另外的值作为转基因事件的组成部分并且该值用于发展优良种质。在用转基因植物育种的大量经验的意想不到的结果中,本发明人意识到应当考虑与转基因插入连锁的基因组区的其它因素。由于通过育种方法转基因被扩散到植物种质内,因此与该转基因连锁的一部分遗传区也被扩散了。通过考虑与转基因连锁的遗传区,可实施生物技术和育种策略以提高转基因和与其连锁的遗传区的全部价值,提高种质改良并将发展不良遗传区的风险减少到最小,通常称作连锁累赘(drag)。
例如,在本发明的一个方面,已经鉴定了新的草甘膦耐受的大豆事件的T型基因组区,其包括与单倍型连锁的具有适当表达水平的草甘膦耐性转基因。将最易受影响的T型鉴定为事件19788(也称作MON89788),并且提供用同一基因组区中具有如在并行比较中所测定的改良的产量的单倍型替换事件40-3-2的T型基因组区。该发现对提高草甘膦耐受大豆的种质将具有显著的影响。最近大豆育种的一个重要部分已经利用含有在事件40-3-2中所发现的Roundup Ready性状的品种(Padgette等,1995),可能在提供用于在美国销售的差不多80-95%的大豆种质当前都含有该转基因事件。为了继续以提高大豆种质,期望能鉴定也具有有利的单倍型基因组区的草甘膦耐受事件并且替换种质中的40-3-2T型基因组区,因此向后代提供亲本品种的优良农艺学性状。
在本发明的另一个方面,鉴定昆虫耐受的大豆事件的T型基因组区,其包括与单倍型连锁的具有适当表达水平的昆虫抗性转基因。从全体转基因大豆事件中选择事件GM 19459。这些事件含有插入到大豆基因组内的转基因,其表达对大豆的鳞翅目昆虫害虫有毒的蛋白质。已经对多种单倍体基因组区进行了作图以有助于选择具有最有利的T型基因组区的事件。
在本发明的另一个方面,鉴定昆虫耐性玉米事件的T型基因组区,其包括与单倍型连锁的具有适当表达水平的昆虫抗性转基因。从全体转基因玉米事件中选择昆虫耐性玉米事件。这些事件含有插入到玉米基因组内的转基因,其表达对玉米的鳞翅目昆虫害虫有毒的蛋白质。对多种单倍型基因组区进行作图以便有助于选择具有最有利的T型基因组区的事件。
然后可将任何插入到可对基因组位置进行作图的农作物植物基因组内的转基因与在该位置中所发展的单倍型标记进行比较,以确定该位置是否包括具有提高的育种价值的单倍型。
在一个实施方案中,本发明提供用于大豆中T型基因组区的鉴定和育种的遗传标记和方法。本发明因此允许第一次产生结合转基因的价值和农艺学上优良的,或者有利的单倍型的大豆植物。至少将有利的单倍型鉴定为比在植物种群中所预计的更频繁地被遗传的那些。使用本发明的方法,可将包括T型基因组区的基因座引入到任何潜在期望的大豆植物内。提供分子标记,当用于辅助育种程序的标记时,其提供一种鉴定并维持有利的单倍型和转基因的联系,以提供有价值的T型基因组区的方法。本发明提供转基因的实例,其中转基因向大豆植物提供除草剂和昆虫抗性表型,也期望提供胁迫耐性、疾病耐性、提高蛋白质、油、氨基酸或其它饲料质量、营养或加工性状的其它转基因作为本发明的方面,并且将包括这些T型的种质杂交以提供具有优选的T型基因组区的堆积的(stacked)性状的产物。
在另一个实施方案中,本发明提供用于玉米中T型基因组区的鉴定和育种的遗传标记和方法。本发明因此允许第一次产生结合转基因的价值和农艺学优良的,或者有利的单倍型的玉米植物。使用本发明的方法,可将包括T型基因组区的基因座引入到任何潜在期望的玉米植物内。提供分子标记,当用于辅助育种程序的标记时,其提供一种鉴定和维持有利的单倍型和转基因的联系以提供有价值的T型基因组区的方法。本发明提供转基因的实例,其中转基因向玉米植物提供昆虫抗性表型,也期望提供胁迫耐性、除草剂耐性、提高蛋白质、油、氨基酸或其它饲料品质、营养或者加工性状的其它转基因作为本发明的方面,并且将包括这些T型的种质杂交以提供具有优选的T型基因组区的堆积的性状的产物。
T型基因组区和T型值的概念
T型基因组区是一种新的遗传组成,包括至少一种与单倍型遗传连锁的,具有适当的表达水平的转基因。在优选的实施方案中,由于转基因的局部插入,转基因与单倍型的连锁应当对单倍型的功能完整性没有可观察到的有害效应。另外,由于整合到转基因的遗传连锁内,可在功能上提高T型基因组区的单倍型。T型基因组区组成具有转基因和与其连锁的单倍型的益处。该T型基因组区是一种遗传组成,通过其转基因经育种扩散到种质内。
在本发明的优选实施方案中,T型基因组区的单倍型包括至少两个间隔大约10cM的双等位基因标记,或者在转基因的5cM内至少一个多等位基因的基因座并且具有高多态性信息含量。单倍型中的改变,例如通过重组引起的,可导致单倍型的修饰使得其仅包括与转基因在物理上连锁的原始(亲本)单倍型的一部分。在我们所定义的什么构成T型基因组区的定义中将包括单倍型中的任何这种改变,只要T型基因组区的功能完整性未被改变或改良。提供期望表型的转基因与单倍型或其功能部分的连锁优选地在单倍型区的约5cM内,或者在约2cM内,或者在约1cM内。如果单倍型的值相对于产量不产生负影响,或者相对于成熟不产生正影响,或者相对于成熟无影响,或者当与一组种质(育种种质、繁殖种群、优良自交系集合、全体随机交配个体、双亲本杂交)中同一染色体节段的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围,或者当与一组种质中的全部基因组中的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围,或者在繁殖种群或一组种质中存在具有可被认为是其高价值证据的50%或更高频率的单倍型时,或者这些的任何组合,那么认为单倍型的功能完整性未改变。
通过T值评估在其基因组中包括T型基因组区的植物的益处或价值,这取决于转基因性状的价值和与该转基因连锁的单倍型的价值。可从转基因编码的性状的价值中评估T型基因组区的转基因的价值。该值取决于转基因性状(例如,包括但不限于:除草剂耐性、昆虫抗性、抗病性、改善营养、提高产量、改良的加工性状或者胁迫耐性)并且可从增加的农作物植物产量,或者降低农作物培育所需的输入,或者这些的任何组合中进行评估。转基因性状也具有作为选择或评分标记的价值。对本领域技术人员来说,这在育种应用中有价值,因为对于转基因性状的选择或评分的能力导致同时选择连锁的单倍型。例如在用包括T型的植物进行杂交的情况中,其中转基因编码除草剂耐性,给该杂交的后代喷除草剂将具有选择转基因和紧密连锁的亲本或重组单倍型的高度可能性。可使用开发的用于定义单倍型的DNA标记物用于确定杂交后代中T型的完整性。
包括重组构建体的转基因可进一步包括选择性标记或评分标记。该核酸序列用作选择性或评分标记以便在细胞中产生表型,这便于它们相对于不含有该标记的细胞的鉴别。
选择标记的实例包括,但不限于,neo或nptII基因(Potrykus等,1991),其编码卡那霉素抗性并且可使用卡那霉素,G418等进行选择;编码双丙磷(bialaphos)抗性的bar基因;草甘膦抗性EPSP合酶、草甘膦抗性突变体EPSP合酶(Hinchee等,1988),其编码草甘膦抗性,灭活草甘膦的酶;赋予溴苯腈抗性的腈水解酶基因(Stalker等,1988);赋予咪唑啉酮或磺酰脲抗性的突变体乙酰乳酸合酶基因(ALS)(欧洲专利申请No.0154204);和氨基蝶呤抗性DHFR基因(Thillet等,1988)。
其它示范性的评分标记包括:β-葡糖醛酸酶或uidA基因(GUS),其编码已知有多种显色底物的酶(Jefferson,1987;Jefferson等,1987);R基因座基因,其编码调节植物组织中花色素苷色素(红色)产量的产物(Dellaporta等,1988);β-内酰胺酶基因(Sutcliffe等,1978),其编码已知有多种显色底物的酶(例如,PADAC,一种产色头孢菌素);荧光素酶基因(Ow等,1986);xylE基因(Zukowsky等,1983),其编码可转化产色儿茶酚的儿茶酚双加氧酶;酪氨酸酶基因(Katz等,1983),其编码能将酪氨酸氧化成DOPA和多巴醌(其依次浓缩成黑色素)的酶;以及β-半乳糖苷酶,其将转变显色的β-半乳糖底物。
编码分泌性标记的基因也包括在术语“选择或评分标记”内,可检测其分泌作为鉴定或选择转化细胞的方法。实例包括编码可通过抗体相互作用鉴定的分泌性抗原,或者甚至是可通过催化检测的分泌性酶的标记。选择性分泌的标记蛋白分为许多种,包括可检测的小的,可扩散的蛋白质,(例如,通过ELISA),可在细胞外溶液中检测的小的活性酶(例如,β-淀粉酶、β-内酰胺酶、膦丝菌素转移酶),或者是嵌入或被扣留(trapped)在细胞壁内的蛋白质(如包括前导序列的蛋白质,其中前导序列如在伸展蛋白的表达单元或者烟草PR-S中所发现的)。其它可能的选择标记基因对于本领域技术人员来说将是显而易见的。
标记优选地是GUS、绿色荧光蛋白(GFP)、新霉素磷酸转移酶II(nptII)、荧光素酶(LUX)、抗生素抗性基因编码序列,或者除草剂抗性基因编码序列。选择剂可以是抗生素,例如包括但不限于,卡那霉素、潮霉素,或者除草剂,例如包括但不限于,草甘膦、草铵膦(glufosinate)、2,4-D和麦草畏(dicamba)。
T型基因组区在标记辅助的选择和标记辅助的育种应用中有价值。在它们包括T型基因组区的情况中,选择转基因和有利的单倍型仅需要一个标记,而如果转基因和有利的单倍型未连锁,那么将需要至少两个标记。这种潜在的价值将随着种质中累积或者堆积起来更多的T型基因组区而增加。
可通过改变转基因的表达改变或修饰T值,其中改变发生在转基因的表达水平,或者在转基因表达的时机,或者在转基因表达的定位,或者这些的任何组合。本发明预期可通过顺式作用(局部的)或反式作用(可在间隔一定距离起作用,不仅仅对它们发生作用的DNA分子)因子,或者这些的组合,影响通过转基因表达的任意组分的改变所导致的T值的改变。
另外,可通过改变与转基因紧密连锁的单倍型,改变或修饰T值。本发明优选的实施方案是通过选择或指导存在的T型基因组的转基因与不同受体单倍型形成紧密连锁提高T值,其中不同的单倍型与额外的值有关并且如在并行的或者头对头(head-to-head)的比较中所测定的,相对于农艺学特性或多性状指数,比存在的T型单倍型有改进。通过产生或者选择至少一种重组体T型单倍型,其如在重复进行的并行的或者头对头(head-to-head)的比较中所测定的,相对于农艺学特性或多性状指数,比存在的T型单倍型有改进,也可导致单倍型中的改变。
本发明的另一个优选的实施方案是通过选择或者指导转基因与具有育种价值的受体单倍型形成连锁,使另外的价值成为新转基因事件的组成部分,其中育种价值对于产量不产生负影响的、对于成熟期不产生正影响的、对于成熟期无影响的、当与同一染色体节段的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围的,当与全部基因组中的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围的,或者是赋予农作物植物农艺学适度的等位基因或者在繁殖种群或一组种质中存在具有可被认为是其高价值证据的50%或更高频率的单倍型,或者这些的任何组合。
本发明的另一个实施方案是用至少一个第一转基因转化的植物或品种的选择,其中植物或品种的选择基于其基因组中是否包括高比例的具有育种价值的受体单倍型,其中育种价值相对于产量不是负数,或者对于产量不产生负影响的、对于成熟期不产生正影响的、对于成熟期无影响的、当与同一染色体节段的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围的,当与全部基因组中的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围,或者是赋予农作物植物农艺学适度的等位基因或者在繁殖种群或一组种质中存在具有可被认为是其高价值证据的50%或更高频率的单倍型,或者这些的任何组合。
本发明期望通过转化添加转基因,或者通过杂交含有不同T型基因组区的亲本植物或品种,或者这些的任意组合,T型基因组区累积或堆积到植物或品种中。可通过复合T值评估累积的或堆积的T型基因组区的价值,其依靠结合转基因性状的值和与转基因连锁的单倍型的值。本发明进一步期望可通过修改堆积的转基因之一或其中每一个的表达组分,改进复合T值。另外,本发明期望可通过选择至少一个与转基因之一或其中的任何一个连锁的,具有有利的育种值的受体单倍型,或者通过选择经转化或经育种或者通过这些的任意组合的用于堆积转基因的植物或品种,将另外的值成为复合T值的组成部分。
可用于本发明方法的转基因农作物包括,但不限于除草剂耐性作物,例如,Roundup Ready棉花1445和88913;Roundup Ready棉花GA21、nk603、MON802、MON809;Roundup Ready甜菜GTSB77和H7-1;Roundup Ready油菜(Canola)RT73和GT200;油菜籽油菜(oil seed rape)ZSR500、Roundup Ready大豆40-3-2、含MON89788的大豆、Roundup Ready翦股颖(Bentgrass)ASR368、HCN1O、HCN28和HCN92油菜、MS1和RF1油菜、OXY-235油菜、PHY14、PHY35和PHY36油菜、RM3-3、RM3-4和RM3-6菊苣、A2704-12、A2704-21、A5547-35、A5547-127大豆、GU262大豆、W62和W98大豆、19-51A棉花、31807和31808棉花、BXN棉花、FP967亚麻、LLRICE06和LLRICE62水稻、MON71800小麦、676和678和680玉米、B16玉米、Bt11玉米、CBH-351玉米、DAS-06275-8玉米、DBT418玉米、MS3和MS6玉米、T14和T25玉米、H177玉米和TC1507玉米。已被证明并可被本发明应用的转基因植物耐受的除草剂,包括但不限于:草甘膦、草铵膦、磺酰脲、咪唑啉酮、溴苯腈、茅草枯、麦草畏、2,4-D、cyclohezanedione、原卟啉原氧化酶抑制剂和异噁氟草除草剂。编码除草剂耐性中所涉及的蛋白质的多核苷酸分子在现有技术中是已知的,并且包括,但不限于在美国专利5,627,061、美国专利5,633,435、美国专利6,040,497,和在美国专利5,094,945中所述的编码草甘膦耐性的5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS)的多核苷酸分子,所有这些在此引入作为参考;编码草甘膦氧化还原酶、草甘膦-N-乙酰转移酶,或者草甘膦脱羧酶的多核苷酸(GOX,美国专利5,463,175;GAT,美国专利公开20030083480和20050246798,草甘膦脱羧酶,美国专利公开20060021093;20060021094;20040177399,在此以其整体引入作为参考);在美国专利4,810,648中所述的编码溴苯腈耐性的溴苯腈腈水解酶(Bxn)的多核苷酸分子,在此引入作为参考;在Misawa等,(1993)和Misawa等,(1994)中所述的编码达草灭(norflurazon)耐性的八氢番茄红素不饱和酶(crtI)的多核苷酸分子,Sathasiivan等(1990)中所述的编码磺酰脲除草剂耐性的乙酰羟酸合酶(AHAS,aka ALS)的多核苷酸分子;以及在DeBlock等(1987)中所述的草铵膦和双丙磷耐性的bar基因;抗性羟苯基丙酮酸脱氢酶(HPPD,美国专利6,768,044)。本发明的转基因的启动子可表达编码膦丝菌素乙酰转移酶、草甘膦抗性EPSPS、氨基糖苷磷酸转移酶、羟苯基丙酮酸脱氢酶、潮霉素磷酸转移酶、新霉素磷酸转移酶、茅草枯脱卤素酶、溴苯腈抗性腈水解酶、麦草畏单加氧酶、邻氨基苯甲酸合酶、草甘膦氧化还原酶、草甘膦-N-乙酰转移酶或草甘膦脱羧酶的基因。
可应用于本发明方法的转基因作物包括,但不限于,昆虫抗作物,例如,棉花事件,如MON15985、281-24-236、3006-210-23、MON531、MON757、MON1076和COT102;或者玉米事件,如MIR604、BT176、BT11、CBH-351、DAS-06275-8、DBT418、MON80100、MON810、MON863、TC1507、MIR152V、3210M和3243M。昆虫抗性转基因作物可提供对昆虫害虫摄食损害的耐性并且已经证明有效抗某种鳞翅目和鞘翅类植物害虫,其它转基因作物也可能提供对植物害虫的抗性,如半翅目、同翅目、异翅目、直翅目、缨翅目,和植物寄生线虫的某种成员。疾病抗性的转基因作物,例如,病毒抗性番木瓜55-1/63-1,以及病毒抗性南瓜CZW-3和ZW20。雄性不育转基因作物,例如,PHY14、PHY35和PHY36油菜和玉米事件676、678、680、MS3和MS6。另外的转基因作物植物也可能提供对导致植物疾病的真菌和细菌生物体的抗性。
本发明考虑上述所列举的包括用于T型基因组区的育种和堆积的T型基因组区,或者通过间接作图法鉴定的单倍型,或者这些的任意组合的转基因作物和种质,以便如在本发明方法中所述增加T值或者提高总的种质质量。
单倍型
“单倍型”是生物体基因组中的DNA片段,当不同个体中的一个或多个基因座上状态相同的知识相同时,则认为由于遗传其对于不同个体是相同的,并且在物理或遗传图谱上该片段附近连锁不平衡的局部量很高。可通过种群追踪单倍型并且可分析其与给定性状的统计学联系。因此,单倍型群从研究允许阐述祖先携带者单倍型的频率和类型。“群从研究”是一种遗传实验,其中检验一个或多个标记基因座上等位基因的分离和一个或多个性状的单个表型值之间背离随机性的水平。群从研究可在定量或绝对性状上进行,说明或不说明种群结构和/或分层。
单倍型分析很重要,因为其增加涉及单个双等位基因标记的分析的统计学能力。在单倍型频率分析的第一个阶段,基于所鉴定的本发明双等位基因标记的多种组合,测定可能的单倍型的频率。然后比较不同种群和作图种群的单倍型频率。通常,由于在前的种质改良,因此种质集合的种群中单倍型频率越高,其对种质的价值就越大,如与农作物植物的农艺学适度有关的等位基因所述(美国专利5,437,697,在此以其整体引入作为参考)。基于其在一组种质中的频率,可选择有利的单倍型,通常50%或更高的频率将表明该单倍型在种质中有价值。以高频率发生的单倍型对于用转基因的靶向或者T型的选择将是有利的,其中认为在种质中具有高频率的单倍型是有利的。可将在种质中以任意频率发生的单倍型与性状联系起来,并且可根据单一性状或性状的组合得到单倍型的价值。有利的单倍型将向种质提供一个或多个有利性状。通常,可使用本领域的任何已知方法以检验性状和基因型是否显示统计学显著相关。用于测定表型和基因型之间相关的统计学显著性的方法,在本情况中单倍型,可通过任何本领域已知并且具有所需统计学显著性的任何公认阈值的统计学检验进行测定。具体方法和显著性阈值的应用是本领域普通实践者公知的。
在植物繁殖群中,连锁不平衡(LD),其是种群中两个或多个基因座之间背离随机联系的水平,通常持续到大量染色体节。尽管可能关心片段中每个基因的单独效果,但是对于实际的植物育种目的,通常重要的是当在品种、杂种或变种中存在时该区域对目的性状的平均影响是什么。绘制配对LD的量与对照种质集的标记之间距离的图(使用r2统计),其中距离以厘摩(cM,百分之一摩,每次减数分裂平均一次重组,重组是减数分裂时配对的同源染色体之间染色单体片段交换的结果,其通常通过后代中不同祖父母的连锁的基因座上的等位基因的组合进行观察)表示,例如,791大豆原种美国品种和1211SNP基因座的集合具有高于5%频率的稀有等位基因。200个数据点移动平均数曲线甚至是存在间隔10cM的基因座的LD的指标。因此,当预测染色体节的平均效应时,应当认为片段有几厘摩长,并且这是对单倍型区的认可(acception),其是几厘摩长的染色体节,持续到繁殖的多代并且其被一个或多个育种品种携带。可鉴定该片段含有多个连锁的标记基因座,并且在两个品种中这些基因座上常见的单倍型同一性通过由这些品种所携带的全部下面的(subjacent)染色体节的遗传产生高置信度的同一性。
应当指定有利的单倍型是什么并且它们在种质中的频率是多少。因此,将获得或产生正在考虑的用于转基因事件的育种和/或繁殖的种质的分子标记的测量。该标记测量将产生每个品种的指纹。假设这些标记的大致的遗传图位置是已知的。为了简化下游分析,可能需要在该阶段实现质量保证和漏失数据估计步骤以产生全部的和准确的数据矩阵(品种的标记基因型)。误差检测和漏失数据估计可能需要使用亲本-后代检验,标记基因座之间的LD、区间映射、重新基因型分型等。
然后将标记根据它们的接近度进行分类。这种分类可能是任意的(例如,“在开始下一个片段前,从染色体的一端开始并且包括片段中所包含的第一个标记的10cM内的所有标记”)或者基于某些统计学分析(例如,“基于相邻基因座之间LD模式定义片段断裂点”)。
当考虑大量品种集合,并且多个品种在标记基因座上具有同样的等位基因时,需要确定标记基因座上的状态相同(IBS)是否是标记基因座周围染色体区处来源相同(IBD)的优良预报者。“来源相同”(IBD)特征是由两个或多个个体携带的两个基因座/DNA片段,并且所有都来自同一祖先。“状态相同”(IBS)特征是由两个或多个个体携带的两个基因座/DNA片段,并且在看得见的基因座上具有相同的等位基因。片段中多个标记基因座足以表征片段的IBD的良好指标是,它们可预测在片段内的其它标记基因座上存在等位基因。
为了估计单倍型的频率,必需定义基本参考种质(原种自交品种的集合,随机杂交个体种群等)和必需对代表性样品(或者所有种群)进行基因型分型。如果考虑自交个体组,可通过简单的计数测定单倍型频率。如果经基因型分型的个体在片段的一个以上的基因座上是杂合的并且连锁相是未知的,那么将需要使用象期望值/最大化算法的计算技术的估计法(Excoffier和Slatkin,1995)。优选地,使用基于期望值-最大化(EM)算法(Dempster等1977)的方法,其在Hardy-Weinberg比例(随机杂交)的假设下导致单倍型频率的最大概似法估计(Excoffier和Slatkin,1995)。随着单倍型评估,以及每个候选宿主品种的每个染色体节的鉴定,可进一步根据它们引起位于高价值单倍型的事件的可能性对品种进行排列。根据所获的关于基因组中每个区段的物理大小以及转基因插入的随机性或随后方式模式知识的程度,可使用将定位于染色体节中的事件的几种概率分布。可使用可供选择的方法以进行群从研究:宽基因组群从研究、候选区域群从研究和候选基因群从研究。为了进行宽基因组群从研究,可将本发明的双等位基因标记整合到植物基因组的遗传标记的任何图谱中。
本发明包括用于检测单倍型和有利的性状或多性状指数之间相关的方法。多性状指数(MTI)是一个数字实体,其通过结合公式中单个性状值计算获得。最常计算成性状或性状的标准化衍生物的线性组合,其也可以是更复杂计算的结果(例如,使用性状之间比)。然后可在遗传分析中使用该MTI,好像其是性状。当通过标记法或表型法选择时,有利的单倍型向亲本植物和向亲本的后代提供有利的性状。本发明的方法提供选择有利的单倍型和有利的单倍型在繁殖种群中的累积,例如,在本发明中所鉴定的一个或多个单倍型。本发明的具体实施方案,将转基因与有利的单倍型相连以产生其它有利的单倍型一同累积的T型以提高种质。
T型基因组区和有利单倍型的累积
本发明的另一个实施方案是用于提高种质中一个或多个单倍型的累积的方法。用转基因转化植物细胞意味着已将转基因DNA插入到植物的基因组DNA区内。定义为单倍型区的基因组区包括遗传信息并且向植物提供表型性状。遗传信息中的变化导致表型性状的变化并且可测量表型的价值。单倍型区的遗传作图和转基因插入事件的遗传作图允许测定转基因插入与单倍型的连锁。具有在转化植物的基因组中是新的DNA序列的任何转基因,在本质上可作为转基因和其所插入的基因组区的遗传标记。例如,在本发明中,插入到大豆植物基因组内的转基因提供表达草甘膦抗性5-烯醇式丙酮酰莽草酸-3-磷酸合酶,其具有在美国专利6,660,911中公开的SEQ ID NO:28和在美国专利5,633,435中公开的SEQ ID NO:9中公开的DNA编码序列,两者在此引入作为参考,可从其中选择DNA引物或探针分子以便作为基因组中转基因的遗传标记。
另外,转基因可提供一种选择具有插入片段和连锁的单倍型区的植物的方法。选择可能是由于对所应用的植物毒性剂,如除草剂或抗生素的耐性。选择可能是由于检测转基因的产物,例如,mRNA或蛋白质产物。可通过检测插入到植物基因组内的转基因DNA进行选择。转基因也可提供表型选择方法,如,很容易观察的形态学表型,这可以是种子颜色、种子发芽特征、幼苗生长特征、叶的外貌、植物结构、植物高度,以及花和果实形态,或者选择基于农艺学表型,如,产量、除草剂耐性、疾病耐性、昆虫耐性、提高的饲料品质、干旱耐性、冷耐性,或者由转基因所提供的任何其它农艺学性状。
在通过转基因的插入改良农作物植物的发展期间,通常产生许许多多具有不同转基因插入位置的植物。这些插入事件发生于植物的整个基因组中并且被整合到具有许多不同单倍型区的紧密连锁内。本发明提供用于筛选具有转基因插入到具有有利的单倍型区的紧密连锁内的转基因事件,以及选择这些事件用于育种程序以提高有利的单倍型区的累积的方法。该方法包括;a)使用以前所描述的和植物生物技术领域中已知的植物转化和再生方法,将转基因插入到植物细胞的基因组内并且使植物细胞再生成完整的转化植物;和b)使用转基因和连锁的基因组区的DNA标记,测定转化植物的基因组中转基因的图谱位置;和c)将图谱位置和紧密连锁的单倍型联系起来,其中转基因和单倍型包括转化植物中的T型基因组区;和d)将转化的植物与第二种植物杂交,其中第二种植物也可能被转化以含有至少一个与第一个转化植物T型基因组区不同的T型基因组区,或者第二种植物可含有通过遗传标记鉴定的不同于第一种转化植物的有利的单倍型区;和e)通过检测第一种植物的转基因的表达,选择至少一个后代植物或者通过与转基因相关的标记的存在进行选择,其中后代植物在其基因组中包括至少一部分第一种植物的T型基因组区和第二种植物的至少一个T型基因组区或有利的基因型;和f)在与种质改良有关的活动中使用后代植物,活动选自使用植物进行育种杂交,植物的进一步检验、植物通过自体受精的进化、使用植物或其部分用于转化、使用植物或其部分用于诱变,以及使用植物或其部分用于耕作(例如,McCallum等,2000)。
使用本方法,本发明预期优选的T型基因组区选自大量T型基因组区,优选的T型基因组区在农作物植物的种质中具有提高的T值。另外,可在所述育种方法中使用优选的T型基因组区以累积其它有益的T型基因组区和有利的单倍型区并且在繁殖种群中维持这些以提高农作物植物的全部种质。考虑用于本发明的农作物植物包括但不限于,玉米、大豆、棉花、小麦、水稻、油菜、油菜籽油菜、甜菜、高粱、黍、苜蓿、蔬菜作物、森林作物和水果作物。
T型基因组区的基因组作图
本发明的另一个实施方案是用于对至少一个T型基因组区进行间接作图的方法。植物基因组中T型基因组区的作图提供用于选择包括T型基因组区的有利的单倍型区的方法。本发明提供用于绘制转基因插入事件和其与基因组区的关系以及在植物基因组图上的位置的方法。该方法可包括下列步骤:
(a)获得转基因插入事件侧翼基因组的DNA序列;
(b)当获得两个或多个高同源序列时,比较该DNA序列层析谱以消除旁系同源序列;
(c)在序列数据库中搜索DNA序列以证实插入事件是否中断内源基因;
(d)在转基因插入侧翼的基因组区的5’和3’端之一或两者上设计一对或多对DNA引物。当设计多对引物时,其可从每个基因组侧翼区中获得重叠的PCR产物以确保相关基因组DNA的基本覆盖的这样一种方式进行;
(e)使用作图种群的亲本品种作为PCR的模板;
(f)对从这些引物/品种组合中所获得的PCR产物进行测序;
(g)在至少作图种群之一的亲本之间鉴定SNP,或者其它多态性特征如插入/缺失或SSR;
(h)在另外的侧翼序列上通过(g)重复步骤(d),在5’和3’方向上从插入的位点滑过,直到发现多态位点,或者获得另外的一个;
(i)设计试验以便对作图种群的后代植物进行评分;
(j)进行连锁分析以确定这些多态性的图谱位置以及随后确定事件的位置;
(k)将图谱位置与单倍型的位置联系起来。
转基因插入事件侧翼的基因组可包括一个DNA片段,其长度从几百到几万个核苷酸碱基对,或者其长度足以鉴定多态性。基因组侧翼区可以从转基因插入位置的5’或3’端延伸到插入位点的基因组内。“聚合酶链反应”(PCR)是一种靶DNA片段的体外几何扩增的方法,通过使用耐热DNA聚合酶和温度的循环变化以允许模板DNA的重复变性、引物退火和扩增。“旁系同源序列”是两个具有高度相似性但属于基因组上不同基因座的DNA序列。“作图种群”是一组个体,其中在标记基因座上和可能地在一个或多个数量性状基因座(QTL)上的等位基因是分离的,在某种程度上连锁不平衡的存在可作为染色体上邻近的证据并且邻近和不平衡之间存在正相关。作图种群是同一植物物种或者证明同线性或共线性的植物物种。这些种群可用于评估它们之间或者这些和QTL之间标记基因座的相对位置。通常,作图种群是分离的种群。该方法可应用于任何农作物物种,特别重要的农作物物种是,例如,谷物、大豆、棉花、小麦、水稻、油菜、油菜籽油菜、甜菜、高粱、黍、苜蓿、蔬菜作物、森林作物和水果作物。对于这些作物中的一种或多种,有本领域技术人员可利用的图谱,通过实例的方式,参考玉米(Lee等,2002)、大豆(Ferreira等,2000)、棉花(Lacape等,2003)和油菜(Cheung等,1997)的遗传图谱。也可从头产生任何目的作物的作图种群,所产生的遗传图谱可在本发明中用于对转基因插入处的单倍型区进行作图。
所克隆的基因组DNA区的鉴定例如,可用所开发的DNA标记探测Bac文库中所含有的那些以鉴定与转基因插入连锁的单倍型。可通过测量Bac克隆的序列并检查序列中的多态性,发展另外的DNA标记。可从可用作转基因的Bac克隆中分离目的基因以改良同一作物物种或不同作物物种的行为表现。
重组载体和转基因
制备重组载体的方法是现有技术公知的。制备特别适于植物转化的重组载体的方法包括,不限于,在美国专利Nos.4,971,908、4,940,835、4,769,061和4,757,011中所述的那些。也已经回顾了载体的这些类型(Rodriguez等,1988;Glick等,1993)。
在更高等植物中对核酸的表达有用的典型载体是本领域公知的,并且包括从根癌土壤杆菌的肿瘤诱导型(Ti)质粒衍生的载体(Rogers等,1987)。也已经描述了对植物转化有用的其它重组载体,包括pCaMVCN转移控制载体(Fromm等,1985)。已经转化了许多作物物种使其含有一个或多个农艺学重要的转基因,这些转基因它们本身向植物提供有利的性状。一个实例是赋予农作物植物除草剂耐性的转基因。已转化并在植物中表达的编码除草剂耐性蛋白质的转基因包括,例如,赋予草甘膦抗性的5-烯醇式丙酮酰莽草酸-3-磷酸合酶(EPSPS)蛋白和赋予其它除草剂,如草铵膦或溴苯腈抗性的蛋白质(Comai等,1985;Gordon-Kamm等,1990;Stalker等,1988;Eichholtz等,1987;Shah等,1986;Charest等,1990)。进一步的实例包括酶的表达,如二氢叶酸还原酶和乙酰乳酸合酶,赋予对咪唑啉酮或磺酰脲除草剂抗性的突变体ALS和AHAS酶(Lee等,1988和Miki等,1990),赋予膦丝菌素抗性的膦丝菌素-乙酰基-转移酶(欧洲申请No.0242246),赋予对苯氧基proprionic酸和cycloshexone抗性的蛋白质,如sethoxydim和盖草能(haloxyfop)(Marshall等,1992);以及赋予三嗪(psbA和gs+基因)和苄腈(腈水解酶编码基因,Przibila等(1991))抗性的蛋白质。
本发明的植物也可包括赋予对昆虫、害虫、病毒或细菌攻击抗性的转基因。例如,赋予对害虫,如在PCT申请WO96/30517和PCT申请WO93/19181中所描述的大豆包囊线虫抗性的转基因。Jones等(1994)描述对番茄叶霉菌(Cladosporium fulvum)抗性的番茄Cf-9基因的克隆;Martin等(1993)描述了对丁香假单胞菌致病变种(Pseudomonas syringaepv.)抗性的番茄Pto基因以及Mindrinos等(1994)描述了对丁香假单胞菌(Pseudomonas syringae)抗性的拟南芥(Arabidopsis)RS2基因。苏云金杆菌内毒素也可用于昆虫抗性,例如,Geiser等(1986)。
已知在转化的植物细胞中表达作为转基因的病毒外壳蛋白赋予对外壳蛋白基因来源的病毒以及相关病毒感染和/或受该病毒感染所引发疾病的抗性(Beachy等,1990)。
也可以使用赋予增加营养价值或其它增加价值的性状的转基因。一个实例是修饰的脂肪酸代谢,例如,通过用硬脂酰-ACP去饱和酶的反义基因转化植物以增加植物的硬脂酸含量,(Knutzon等,1992)。也可引入有义去饱和酶基因以改变脂肪酸含量。可通过引入肌醇六磷酸酶编码基因修饰肌醇六磷酸含量以提高肌醇六磷酸的分解,向转化的植物中添加更多的游离磷酸。也可影响修饰的碳水化合物组成,例如,通过用编码改变淀粉分支模式的酶的基因转化植物(Shiroza等,1988,链球菌(Streptococcus)突变体果糖基转移酶基因的核苷酸序列);Steinmetz等,(1985)(枯草杆菌(Bacillus subtilis)果聚糖蔗糖转移酶(levansucrase)基因的核苷酸序列);Pen等(1992),生产表达地衣芽孢杆菌(Bacillus lichenifonnis)α-淀粉酶的转基因植物;Elliot(1993),番茄转化酶基因的核苷酸序列);Sogaard等(1993),大麦α-淀粉酶基因的定点诱变;以及Fisher等(1993),玉米胚乳淀粉分支酶II。
也可使用转基因改变蛋白质代谢。例如,美国专利No.5,545,545描述赖氨酸不敏感的玉米二氢吡啶酸合成酶(DHPS),其基本上抗抑制天然DHPS的活性的L-赖氨酸的浓缩。类似地,EP 0640141描述编码能导致高于苏氨酸正常产量的赖氨酸不敏感的天冬氨酸激酶(AK)的序列,以及编码用于提高赖氨酸的反义赖氨酸α-酮戊二酸还原酶的亚片段。
可使用改变植物碳水化合物代谢的转基因。例如,果糖激酶基因是已知的用于在转基因植物和它们的果实中进行果糖激酶基因表达的代谢工程(美国专利6,031,154)。可使用的转基因的另外实例是改变谷粒产量的基因。例如,美国专利6,486,383描述用腺苷二磷酸葡糖焦磷酸化酶(“ADPG PPase”)的亚单位蛋白质修饰植物中的淀粉含量。在EP0797673中,论述了转基因,其中特定DNA分子的引入和表达导致在液泡外面形成很容易动员的磷酸盐池以及提高的生物量产量和/或改变的开花行为。进一步已知的是用于改变植物成熟期的基因。美国专利6,774,284描述编码植物脂肪酶的DNA和用其在植物中控制衰老的方法。美国专利6,140,085提供用于改变开花特征的FCA基因,特别是花期。美国专利5,637,785论述具有调整花发育的遗传修饰的植物,如具有早期花分生组织发育并且在其基因组中包括编码LEAFY蛋白的结构基因。
用于改变植物形态特征的基因也是已知的并且可根据本发明使用。美国专利6,184,440论述由于表达细胞壁调制转基因,显示改变的结构或形态学的基因工程植物。细胞壁调制转基因的实例包括纤维素结合域、纤维素结合蛋白,或者细胞壁修饰蛋白或者酶如内木葡聚糖(endoxyloglucan)转移酶、木葡聚糖内转糖苷酶、扩展蛋白、纤维素合成酶或者新分离的内-1,4-β-葡聚糖酶。
可将提供有利性状的转基因与植物形态学、生理学、生长和发育、产量、营养增强、疾病或害虫抗性,或者环境或化学制剂耐性联系起来。向作物植物提供有益农艺学性状的转基因可以是,例如,包括但不限于遗传元件的下列实例,包括除草剂耐性(美国专利5,633,435和美国专利5,463,175)、增加产量(美国专利5,716,837)、昆虫控制(美国专利6,063,597;美国专利6,063,756;美国专利6,093,695;美国专利5,942,664;和美国专利6,110,464)、真菌疾病抗性(美国专利5,516,671;美国专利5,773,696;美国专利6,121,436;美国专利6,316,407和美国专利6,506,962)、病毒抗性(美国专利5,304,730和美国专利6,013,864)、线虫抗性(美国专利6,228,992)、细菌疾病抗性(美国专利5,516,671)、淀粉生产(美国专利5,750,876和美国专利6,476,295)、改进的油生产(美国专利6,444,876)、大量油生产(美国专利5,608,149和美国专利6,476,295)、大量蛋白质生产(美国专利6,380,466)、果实成熟(美国专利5,512,466)、提高动物和人营养(美国专利5,985,605和美国专利6,171,640)、生物聚合物(美国专利5,958,745和美国公开US20030028917)、环境胁迫抗性(美国专利6,072,103)、药物肽(美国专利6,080,560)、改良的加工性状(美国专利6,476,295)、改良的消化性(美国专利6,531,648)、低棉子糖(美国专利6,166,292)、工业酶生产(美国专利5,543,576)、改良的风味(美国专利6,011,199)、固氮作用(美国专利5,229,114)、杂种种子生产(美国专利5,689,041)和生物燃料生产(美国专利5,998,700),上述专利中所述的遗传元件、方法和转基因在此引入作为参考。
可选择地,可转录的多核苷酸分子可通过编码导致定向抑制内源基因表达的RNA分子影响上述植物特征或者表型,例如经反义、抑制性RNA(RNAi),或者共抑制介导的机制。RNA也可以是改造的用于切割期望的内源性mRNA产物的催化性RNA分子(即,核糖核酸酶)。也可在植物细胞中表达某些RNA分子,其在非植物生物体中抑制靶,例如,以植物细胞为食并摄取抑制性RNA的昆虫,或者植物细胞为食并摄取抑制性RNA的线虫。因此,编码影响目的表型或形态学改变的转录RNA分子的任何可转录的多核苷酸分子可能对于本发明的实施是有用的。
育种和标记
育种技术利用植物的授粉方法。有两种普通授粉法:自花授粉,其在如果将一朵花的花粉转移到同一种植物的同一朵花或另一朵花时发生,以及异花授粉,其在如果将花粉转移到不同植物上的花时发生。已经自花授粉并根据类型选择了许多代的植物在几乎所有基因座上都变成纯合的并产生一致的纯种后代群,纯合植物。
在适当变种的发育中,可使用谱系育种。特定性状的谱系育种法涉及将两个基因型进行杂交。每个基因型可具有在另一个中缺乏的一个或多个期望的特征;或者,每个基因型可补充另一个。如果两个原始亲本基因型不提供所有期望的特征,那么可在繁殖种群中包括其它基因型。将是这些杂交产物的高等植物自交并在每个连续世代中再次提高。由于自花授粉和选择,每个连续世代都变得更纯合。一般地,该育种法涉及五代或更多代的自交和选择:S1→S2;S2→S3;S3→S4;S4→S5,等。可认为自交世代(S)是一类子代(F)并可命名为F。至少五代后,认为自交植物在遗传上是纯的。
每个育种程序应当包括育种方法效率的周期性、客观性评价。评价标准视目的和目标而改变。彻底检验有希望的高级育种品种并且与商业目标面积的典型环境中的适当标准进行比较,通常持续三年或更长。可通过混淆植物性状或环境因素掩盖由于基因型值而在遗传上优良的个体的鉴定。鉴定优良植物的一个方法是观察其相对于其它实验植物和相对于一个或多个广泛生长的标准变种的行为表现。尽管重复的观察资料提供对遗传价值的更好评估,但是单独的观察资料可能是非决定的。
可使用大量和轮回选择以改良自花或者异花授粉作物的种群。通过几个不同亲本的互交,鉴定或产生杂合个体的遗传上可变的种群。基于个体优势、突出的后代或者极好的配合力,选择最好的植物。将所选择的植物互交以生产新种群,其中连续进行多轮选择。可在几本参考书之一中发现通常用于不同性状和作物的其它育种方法的描述(Allard,1960;Simmonds,1979;Sneep和Hendriksen,1979;Fehr,1987;Fehr,1987)。
育种程序中选择具有提高的目的性状(例如,有利的性状如所收获的植物产品的产量,例如谷粒、种子、果实、纤维、饲料的产量;或者农艺学性状,例如,害虫抗性如疾病抗性,昆虫抗性、线虫抗性,或者改良的生长速度,以及胁迫耐性;或者植物的改良的加工产物,例如,脂肪酸分布图、氨基酸分布图、营养含量、纤维质量)的基因型的效率将取决于:1)种群中单个植物的目的性状中变异性的程度是遗传因素的结果并且因此被传递到所选基因型的后代;和2)植物中目的性状中有多少变异性是由于不同基因型生长的环境所致。性状遗传的范围从由一个其表达不受环境影响的主要基因控制的性状(即,质量特征)到其效应很受环境影响的许多基因控制的性状(即,数量性状)。通过下列事实进一步表征数量性状如产量的育种:1)由每个基因的效应所致的差距很小,使得很难或者不可能单独鉴定它们;2)对性状起作用的基因的数量很大,所以很少获得确切的分离比率,即使曾经获得过;和3)可根据环境变化以不同方式表达基因的效应。因此,准确鉴定超亲分离或者具有目的性状的高级基因型非常困难,并且其成功依靠植物育种者将影响种群中数量特征表达的环境变化减到最小的能力。
随着结合到一个基因型内的性状数量的增加,大大降低鉴定超亲分离的可能性。因此,除了所选择的基因,所有育种者可能通常希望的是获得第一复合特征的有利的基因分配与第二特征的有利的基因分配结合形成一个基因型。
将含有转基因的一组基因组区中特定基因组区渐渗到植物种质内定义为回交转化方法的结果。可将新DNA序列已渐渗到其内的植物种质称作回交转化的基因型、品种、自交或杂种。另外,可通过正向育种方法进行特定基因组区或转基因的渐渗。同样地,可将缺乏期望DNA序列的植物基因型称作未转化的基因型、品种、自交、或杂交。在育种期间,可使用与T型基因组区连锁的遗传标记以帮助用于生产具有提高的产量和转基因性状的大豆植物目的的育种。特别地可用本发明的回交和标记帮助的选择,或者正向育种和标记帮助的选择,通过该变体的转化将T型基因组区引入到任何变种中。
在本发明的另一个实施方案中,提供遗传连锁的标记序列并可用于随后大豆或玉米单倍型的选择。通过Lief Technologies现在是Invitrogen的植物DNAzol试剂”(Invitrogen Life Technologies,Carlsbad,CA),从不同玉米或大豆品种中分离基因组DNA,制备多个玉米或大豆品种的基因组文库。将基因组DNA用Pst 1核酸内切酶限制酶消化,在1%琼脂糖凝胶上进行大小分离并连于质粒载体中,用于通过在Sambrook等中所述的标准分子生物学技术进行测序。在ABI Prism377DNA测序仪上使用商业上可获得的试剂(Applied Biosystems,Foster City,CA),通过标准方法对这些文库进行测序。将所有序列集合起来以通过可从DoubleTwist Inc.,Oakland,CA获得的Pangea聚类和对比工具鉴定非丰余序列。将多个玉米或大豆品种的序列聚集起来形成具有一个或多个多态性的基因座,如SNPs和/或插入/缺失。通过下列参数候证明选多态性适合:
(a)共有序列匹配的重叠群或单碱基差异的最小长度是200个碱基。
(b)候选SNP的每侧15个碱基的区域中所观察碱基的同一性百分数是至少75%。
(c)多态性位点处每个重叠群中最小Phred质量是35。
(d)多态性位点每侧15个碱基区中最小Phred质量是20。
由于多核苷酸分子中核苷酸的特性和许多其它原因,从自动测序仪中读出的数据在质量中显著不同(Ewing等,1998)。发展了许多算法来解决准确碱基对判定的问题(Giddings等,1993;Berno,1996;Lawrence和Solovyev,1994)。最广泛使用的算法计算等式q=-10XloglO(p)中作为“q”的序列的质量,其中p是碱基判定的估计的误差概率(Ewing和Green,1998)。因此指定特定序列中具有1/1000不正确的可能性的碱基判定为30的质量评分。质量评分也称作“Phred评分”。使用标记辅助的选择选择植物
发展作物物种中分子标记的最初动机是可能通过标记辅助选择(MAS)提高植物育种中的效率。使用遗传标记等位基因(“等位基因”是基因座上可选择的序列)鉴定在多个基因座上含有期望基因型的植物,和预期将期望的基因型,连同期望的表型转移到它们后代的植物。可使用遗传标记等位基因鉴定在一个标记基因座,几个基因座,或者单倍型上含有期望的基因型的植物,和将预期将期望的基因型,连同期望的表型转移到它们的后代。
标记辅助的选择包括表型性状的作图并且依靠检测个体之间遗传差异的能力。“遗传图谱”是沿染色体所表征的基因座(可鉴定等位基因的DNA标记或者任何其它基因座)的相对位置的图。距离的测量与减数分裂时姐妹染色单体之间交换事件的频率有关。然后使用统计方法将遗传差异,或者“遗传标记”与表型变异联系起来。在优选的实例中,编码负责表型性状的蛋白质的单基因可通过导致表型变异的突变直接检测。更常见地,多个遗传基因座的每一个都对所观察的表型起作用。
通过上述使用标记的任何一种方法使植物基因组中显示有利表型性状的特定遗传标记等位基因存在和/或缺乏,例如,DNA标记是限制性片段长度多态性(RFLP)、扩增片段长度多态性(AFLP)、简单序列重复(SSR)、单核苷酸多态性(SNP)、插入/缺失多态性(Indels)、可变数目串联重复(VNTR)和随机扩增多态DNA(RAPD),以及本领域技术人员已知的其它标记。如果植物的核酸对于期望的遗传标记是阳性的,那么可将植物自交以产生具有同一基因型的纯育种品种,或者可将其与具有同一标记或具有其它期望特征的植物杂交以产生有性杂交的杂种世代。提供使用多种遗传标记的标记辅助的选择方法(MAS)。提供通过使用该方法的MAS所选择的植物。
标记辅助的渐渗涉及由一个或多个标记所限定的染色体区从一个种质转移到第二个种质。在该方法中最初的步骤是通过基因作图定位基因组区或转基因,这是通过连锁分析确定基因或基因组区相对于其它基因和遗传标记的位置的过程。连锁作图的基本原则是染色体上两个基因靠的越近,那么它们更有可能被一起遗传。简要地,通常在两个相对于正在研究的性状在遗传上兼容但分歧的亲本之间进行杂交。然后可使用遗传标记以便接着在杂交的后代中分离正在研究的性状,常常是回交(BC1)、F2,或者重组体自交群。
适当的回归亲本的选择对于成功的回交方法是一个很重要的步骤。回交方案的目的是改变或取代最初自交中的性状或特征。为了完成这个目的,修饰或用非回归(供体)亲本的期望基因取代回归自交的一个或多个基因座,而基本上保留最初自交的所有剩余期望的遗传的,以及因而期望的生理和形态学构成。特定供体亲本的选择将取决于回交的目的。确切的回交方案将取决于将改变的特征或性状以便确定适当的检验方案。可能有必要引入确定期望的特征是否已经成功地转移到后代的试验。在本发明的情况中,可在回交程序期间检验所产生的后代品种以及使用这里所描述的标记系统以便根据标记而不是视觉性状选择品种,标记表示优选的T型基因组区或包括有利的单倍型的基因组区。
转化的植物和植物细胞
如这里所使用的,术语“转化的”指已引入外源多核苷酸分子如构建体的细胞、组织、器官或生物体。可将所引入的多核苷酸分子整合到受体细胞、组织、器官或生物体的基因组DNA内,使得通过随后的后代遗传所引入的多核苷酸分子。“转基因的”或“转化的”细胞或生物体也包括细胞或生物体的后代以及从育种程序中所产生的后代,其中育种程序在杂交中使用这种转基因植物作为亲本并且显示由于存在外源多核苷酸分子所导致的改变的表型。可通过任何植物转化方法将含有本发明的多核苷酸分子的植物转化构建体引入到植物内。在本发明的实践中,用于通过将植物表达构建体引入到植物基因组内转化植物的方法和材料可包括任何公知的并且已证明的方法,这些方法包括如在美国专利5,384,253中所阐述的电穿孔;如在美国专利5,015,580;美国专利5,550,318;美国专利5,538,880;美国专利6,160,208;美国专利6,399,861中所阐述的微粒轰击;和如在美国专利5,824,877;美国专利5,591,616;美国专利5,981,840;和美国专利6,384,301中所阐述的土壤杆菌介导的转化;以及在美国专利5,508,184中所阐述的原生质体转化,所有这些都在此引入作为参考。
具体转化双子叶植物的方法是本领域技术人员公知的。已经描述了使用这些方法用于许多作物的转化和植物再生,这些作物包括,但不限于,棉花(Gossypium hirsutum)、大豆(Glycine max)、花生(Arachishypogaea)、苜蓿(Medicago sativa),以及芸苔(Brassica)属的成员。
用于转化单子叶植物的方法是本领域技术人员公知的。已描述了使用这些方法用于许多作物的转化和植物再生,这些作物包括,但不限于,大麦(Hordeum vulgarae);玉米(Zea mays);燕麦(Avena sativa);果园草(Dactylis glomerata);水稻(Oryza sativa,包括籼稻和粳稻变种);高粱(Sorghum bicolor);甘蔗(Saccharum sp);高羊茅(Festucaarundinacea);草坪草种(例如,种:葡萄翦股颖(Agrostis stolonifera)、草地早熟禾(Poapratensis)、钝叶草(Stenotaphrum secundatum));以及小麦(Triticum aestivum)。对本领域技术人员来说显而易见的是,可使用并修饰许多转化方法用于从许多靶目的作物中生产稳定的转基因植物。引入转基因的方法是本领域公知的并且包括生物的和物理的植物转化方案。见,例如,Miki等(1993)。一旦将转基因引入到变种内,可很容易地通过杂交进行转移。通过使用回交,除了经回交技术转移到变体内的基因座外,基本上恢复变体的所有期望的形态学和生理学特征。可使用本发明的回交和正向育种方法以改良特征或将特征引入到植物内(Poehlman和Sleper,1995;Fehr,1987a,b;Sprague和Dudley,1988)。
转基因的位点特异性整合
已经发展了许多位点特异性重组介导的方法用于将转基因整合到植物基因组内,以及用于从植物和动物细胞中删除不必要的遗传元件。例如,使用由Abremski等(1983);Sternberg等(1981)和其它人所述的噬菌体P1的cre-lox重组系统在多种细胞类型中促进重组。cre-lox系统在与其识别的DNA序列(称作lox位点)的连接中利用从噬菌体P1中分离的cre重组酶。该重组系统已在植物细胞(美国专利5,658,772)、动物细胞(美国专利4,959,317和美国专利5,801,030)和在病毒载体(Hardy等,1997)中有效获得重组。可通过使用分子重组方法完成植物基因组中转基因序列插入或除去的靶向和控制(美国专利6,573,425)。所引入的包括整合到单倍型区内的异源重组位点的多核苷酸分子在本发明的范围内。
Wahl等(美国专利5,654,182)使用酿酒酵母(Saccharomycescerevisiae)的位点特异性FLP重组酶系统以便在真核细胞中缺失DNA序列。设计缺失以便通过使期望的DNA片段彼此相连完成基因的失活或者基因的活化。已在植物中证明FLP重组酶的活性(Lyznik等,1996;Luo等,2000)。
当转座酶存在于同一基因组中时,其它人使用转座子,或者可动的遗传元件从辅助序列中分离靶基因。Yoder等(美国专利5,482,852和美国专利5,792,924,两者都在此引入作为参考)使用含有转座酶序列和转座酶识别序列的构建体以提供用于遗传改变植物的方法,其中改植物含有无载体和/或标记序列的期望基因。在美国20020132350和EP 1308516(两者都在此引入作为参考)中描述使用引导噬菌体重组酶或转座酶靶向特异区的DNA序列的其它方法。可特异性设计锌指核酸内切酶以识别DNA序列并可靶定基因组中的特异DNA序列以产生对转基因的插入有用的重组位点(Wright等,2005;美国专利7,030,215;美国20050208489;美国20050064474,在此以其整体引入作为参考),例如,靶向包括本发明的序列表中所列的DNA序列的单倍型并且包含在本发明人考虑的玉米或大豆植物的基因组中。
当其是优选的T型基因组区的成分时,含有另外的重组位点的转基因提供向T型基因组区添加另外转基因的机会,因此增加种质中该区的价值。本发明考虑T型基因组区也是除去或者向基因组区添加新遗传材料的特异性重组活动的位点。
包括下列实施例用于证明本发明的优选实施方案。本领域技术人员应当意识到,实施例中所公开的技术代表本发明人所发现的在本发明的实践中能更好地起作用的技术,并且因此可被认为构成其实践的优选模式。然而,根据本发明的公开,本领域技术人员应当意识到,不偏离本发明的精神和范围,可在公开的具体实施方案中进行许多改变并且仍然可获得同样的或相似的结果。
实施例1
单倍型的鉴定
本实施例举例说明鉴定在实施本发明方法的数据库中有用的大豆单倍型。通过根据大量标记的可遗传性将大豆的染色体分成几个单倍型。对在表1中所列的4个单倍型的集合鉴定了单倍型的等位基因型。参考表1,通过对照鉴定对基因组位置进行作图的单倍型,例如,C8W6H5指8号染色体,该染色体中的窗口6和该窗口中的单倍型5(基因组区);SEQ_ID涉及所列的序列并且标记ID号是与标记基因座有关的DNA扩增子的任意识别名;START_POS指DNA扩增子中标记的起始位置;HAP等位基因指起始位置上SNP/Indel标记的核苷酸,其中*表示Indel的缺失;“其它标记状态”鉴别窗口中标记的另一个核苷酸等位基因。
表1.用于表征与草甘膦耐性大豆事件有关的四个大豆单倍型的标记基因座的概要信息,包括序列鉴定(SEQ ID和标记ID号)和用于表征这些序列中等位基因(HAP ALLELE)的多态性的位置(START POS)。
实施例2
具有农艺学性状和单倍型的数据库的制备
本实施例举例说明在本发明的方法中有用的数据库的制备。参考表2,该数据库包括农艺学性状的计算值,例如,育种品种集合中特异性等位基因的大豆单倍型的产量、成熟期、植物高度和倒伏,以及该单倍型频率。可测量其它性状,例如,谷粒、种子、果实、纤维、饲料、油的产量;或者农艺学性状,例如,害虫抗性如抗病性、昆虫抗性、线虫抗性,或者改良的生长速度,以及胁迫耐性;或者植物的改良的加工产物,例如,脂肪酸分布图、氨基酸分布图、营养含量、纤维质量以及用于编辑这些其它性状的每个单倍型的价值的数据库。如果在种质中固定单倍型,那么这些单倍型的农艺学性状值代表所列性状的均值中预测的种群改变,其它每件事情同样。“产量”的值是每英亩大豆的蒲式耳。“成熟期”的值是天数(大豆品种的成熟期是与确定成熟期的标准对照的集合相比,该品种的相对开花时间)。“植物高度”的值是成熟时从土壤表面到最高植物组织的顶端所测量的高度的英寸数。“倒伏”的值是与标准对照的集合相比,植物的百分数(倒伏是农作物植物的主干在垂直方向上移动了巨大角度的现象,有时候到植物躺到地面的点)。
使用单倍型中每一个的育种值选择与转基因联合的单倍型,对于农作物种质的改良将是最有益的。育种值是所测量的性状和这些性状将如何影响种质改良的评估的组合。测量与草甘膦耐性的转基因事件有关的大豆单倍型,结果显示于表2中。对于其对产量的影响,单倍型C8W6H5将是有利的单倍型,由于其在种质中具有非常高的频率(94%),单倍型C18W3H8将是有利的单倍型,表明由于该染色体节,靶大豆种质中几乎不存在变异性,使得在其中的转基因事件的扩散过程是中性的。单倍型C19W3H6对于产量通常是中性的。
表2.所述的对于产量、成熟期、植物高度和倒伏的四个单倍型的经计算的育种价值。从365个大豆品种的样本中估计大豆种质中单倍型的频率。
对于四个新草甘膦耐性大豆事件中的每一个测定单倍型区。17194与单倍型C16W8H43连锁,17426与单倍型C18W3H8连锁,19703与单倍型C19W3H6连锁,以及19788与单倍型C8W6H5连锁。如表2所示测量这些单倍型的相对效应。如果在种质中固定单倍型,那么这代表在所列性状的均值中预测的种群改变,其它每件事情也同样。19788和所联合的C8W6H5的T型是所测量的四个T型中最有利的。这个结果证明,评价转基因事件和连锁的单倍型区两者以继续提高农作物生产力在通过转基因方法改良农作物行为表现的方法中很重要。
在重复的田间试验中将新的草甘膦耐性事件与40-3-2的回交转化到A3244种质内进行比较。这在重复的田间试验中进行证明,包括从美国的十七个地点中所收集的产量数据。A3244(美国专利5,659,114、ATCC号97549)是Asgrow(Monsanto,St Louis,MO)的原种大豆种质,其用作用于转化的亲本品种以产生新的草甘膦耐性大豆事件17194、17426、19703和19788。产量研究的结果显示40-3-2A3244回交产生平均65.7蒲式耳/英亩、19788产生65.6蒲式耳/英亩、19703产生65.7蒲式耳/英亩、17426产生65.3蒲式耳/英亩、以及17194产生65.8蒲式耳/英亩。四个新品种的产量比具有渐渗的40-3-2T型基因组区的同一基因型高大约5蒲式耳/英亩。当考虑每个事件的单倍型时,最有利的事件是19788。
这些分析证明,测定每个正在发展的转基因事件的T型作为商品的价值。不考虑转基因已渐渗入其中的单倍型区的农艺学效应,可能导致将低行为表现事件引入到农作物的种质内。
实施例3
育种价值的用途
对于昆虫耐性基因插入到大豆植物的基因组内的四个单倍型区,测定每个的单倍型区和育种价值。在表3中显示每个单倍型区的相对育种值,测量法的定义与实施例2中所述相同。该表是用于测定插入昆虫耐性基因的单倍型(T型)及其育种值的数据库。使用该数据库信息选择包括T型的转基因事件。特殊的事件,GM 19459,含有与C6W4H1单倍型有关的昆虫耐性基因的T型,其中C6W4H1单倍型对于成熟期是有利的单倍型。
表3.昆虫耐性大豆事件的四种单倍型的产量、成熟期、植物高度和倒伏的计算的育种价值。从2589种大豆品种中估计种质中单倍型的频率。
对于与如表4中所列的转基因昆虫耐性大豆有关的4个单倍型的集合,鉴定单倍型的等位基因型。参考表4,通过对照鉴定对基因组位置作图的单倍型,例如C1W1H2指1号染色体,该染色体中的窗口1和该窗口中的单倍型2(基因组区);SEQ_ID涉及所列的序列并且标记ID号是与标记基因座有关的DNA扩增子的任意识别名;START_POS指DNA扩增子中标记的起始位置;HAP等位基因指起始位置上SNP/Indel标记的核苷酸,其中*表示Indel的缺失;“其它标记状态”鉴别窗口中标记的另一个核苷酸等位基因;“NA”表示另一个标记等位基因不存在。
表4.用于表征与昆虫耐性大豆事件有关的四个大豆单倍型的标记基因座的概要信息,包括序列鉴定(SEQ ID和标记ID号)和用于表征这些序列中等位基因(HAP ALLELE)的多态性的位置(START POS)。
实施例4
应用于玉米育种
本实施例举例说明对于昆虫耐性基因插入到玉米植物(LH172)的基因组内的四种单倍型区测定单倍型区和育种值。在表5中显示每种单倍型区的相对育种值,测量的定义与实施例2中所述相同。该表是用于测定插入昆虫耐性基因的单倍型(T型)及其育种值的数据库。使用该数据库信息选择包括T型的转基因事件。特定的事件含有与C1W36H2单倍型有关的昆虫耐性基因的T型。
表5.昆虫耐性玉米事件的四种单倍型产量的计算的育种值。从6335种玉米品种中估计种质中单倍型的频率。
对于如表6中所列的转基因昆虫抗性玉米的4个单倍型的集合鉴定单倍型的等位基因型。参考表6,通过对照鉴定对基因组位置作图的单倍型,例如,C1W19H14指1号染色体,该染色体中的窗口19和在该窗口中的单倍型14(基因组区);SEQ_ID涉及所列的序列并且标记ID号是与标记基因座有关的DNA扩增子的任意识别名;START_POS指DNA扩增子中标记的起始位置;HAP等位基因指起始位置上SNP/Indel标记的核苷酸,其中*表示Indel的缺失;“其它标记状态”鉴别窗口中标记的另一个核苷酸等位基因。
表6.用于表征与昆虫耐性玉米事件有关的四种玉米单倍型的标记基因座的概要信息,包括序列标识(SEQ ID和标记ID号)和用于表征这些序列中的等位基因(HAP ALLELE)的多态性的位置(START POS)。
实施例5
T型基因组区的间接作图
在转基因插入侧翼的基因组区中鉴定DNA标记以提供通过DNA标记与作图种群的比较,鉴定转基因的基因组位置的方法。可通过基因组区的分离,该区域的测序,农作物植物的作图种群中同一区域的分离,以及测定相对于作图种群中已知标记的位置,发展任何转基因事件的DNA标记。可测定转基因与作图表型的联系、包括单倍型基因组区的数量性状基因座。
例如,对于MON89788,从延伸到转基因插入位点的5’基因组内(SEQ ID NO:55和56)和延伸到相对于转基因插入位点的3’基因组区内(SEQ ID NO:57-58)的DNA序列中选择DNA引物对。使用DNA扩增法生产包括从转基因插入位点的5’到3’区的部分大豆基因组的DNA产物。对这些DNA产物进行测序。使用同一引物对从是四个作图种群的亲本的七个大豆品种(507354、Minsoy、Noir、HSl、PIC、88788、A3244)中扩增DNA。当比较不同品种的序列时,在3’侧翼序列的119位上鉴定单核苷酸多态性(SNP)(SNP119,SEQ ID NO:59)。表7显示在所检验的8个品种上该位置处的等位基因的组成。
表7.在包括MON89788的不同大豆品种中侧翼序列上的多态性。
根据生产商所提供的说明书,从SNPl19中发展Taqman(PEApplied Biosystems,Foster City,CA)端点分析。在表8中给出引物和探针序列。为了对SNPl19多态性作图,使用从HSl x PI407305(PIC)之间的杂交所衍生的,由140个个体组成的F2群。通过使等位基因评分与存在的使用MapMaker的等位基因数据集对比,测定SNPl19的图谱位置(Lincoln和Lander,1990)。在连锁群Dla+Q上发现了SNPl19(Song,Q.J.,等,2004)。因此,将MON89788间接地对该同一位置进行作图。
表8.用于对单倍型进行作图的Taqman测定的引物和探针分子
已经根据布达佩斯条约对上述公开的和在权利要求中所引用的包括事件MON89788的Monsanto Technology LLC的大豆种子保藏在美国典型培养物中心(ATCC),10801 University Boulevard,Manassas,Va.20110中。该ATCC保藏号是PTA-6708,保藏于2005年5月11日。该保藏将在贮藏库中保持30年,或者在最后一次请求后5年,或者在本专利的有效期间一直保持,无论哪一个更长,并且如果必要的话,在该期间将可被替换。可从所保藏材料的基因组中分离本发明的DNA分子并且如必要的话,修正序列,可从所保藏的材料中分离另外的DNA分子,用作这里所公开的单倍型区的探针或引物。
所有出版物、专利和专利申请都在此以同样的程度引入作为参考,如同每个单个出版物或专利申请被明确和单独地说明被引用作为参考一样。
可根据本发明的公开,在不需要过多实验的情况下,制备和实施这里所公开的和所要求的所有组合物和方法。尽管根据优选的实施方案已经描述了本发明的组合物和方法,但是在不偏离本发明的概念、精神和范围的情况下,可对方法和步骤或者这里所述方法步骤的顺序进行改变,这对于本领域技术人员是显而易见的。更具体地,当将可能获得相同或类似结果的时候,可用既在化学上也在生理学上相关的某些试剂替换这里所述的试剂,这将是显而易见的。认为对本领域技术人员显而易见的所有这种类似的取代和修饰都在所附权利要求所定义的本发明的精神、范围和概念内。
参考文献
下列参考文献明确地在此引入作为参考,在某种程度上它们提供补充这里所阐述的示范性的程序上的或者其它细节。
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序列表
<110>Bull,Jason
Butruille,David
Eathington,Samuel R
Edwards,Marlin D
Gupta,Anju
Johnson,Richard
Kennard,Wayne
Rinehart,Jennifer
Wu,Kunsheng
<120>提高植物育种的方法和组合物
<130>MONS:115US
<140>UNKNOWN
<141>2006-05-26
<150>60/685,584
<151>2005-05-27
<160>63
<170>3.3
<210>1
<211>664
<212>DNA
<213>大豆(Glycine max)
<400>1
attcagaagg ctgttaaaac cccctcaggt caccaccact aaacaagaca acaagacaat 60
aacagataaa agcctagttt gtcttgcatg cacttacatg cacctttcat ttttttttct 120
tgcatgcatc atggtcccct actaatacca tttatcttca actattcccc cctctcccaa 180
aatcattcct tgcccttcaa cttttcataa ttgtcttaat taaatgtttg gattaaagtc 240
tataaaagta tcacaaggct tactttttca aaactggata tctaggtaaa attttactct 300
caaacatagt tttgggagta accaaacatt accctaaact gattttaatt tcaaacatat 360
acttttaaac cctcccactg gaaatccgaa cacatcctaa gatgacttat tatatttgca 420
tctatctaat aataataata aaagtaaatg atttttatat tgatacttaa ttatgagttg 480
ttttatgata tgtttattga cttttatagt gattagtatc tactttaaaa atcatatcta 540
ttttggacta gctgagagtg tttatattga caatacatag aaattaaatt ttaagaataa 600
gaaaatgata atcatatttt aggatattgg ttaagaataa aataaaaagt tttattgaaa 660
aaat 664
<210>2
<211>1156
<212>DNA
<213>大豆
<400>2
aaagccaatc agatgctact gagtaaaatc agaaaaaatg tgaactaaga gcaatgacaa 60
gagtcaagaa tgctatacct gctgctcagt catttgaaga aaagcaatag tatagtacac 120
caattctctg atattagcca caaccaccta tagaaatgca aaccatagat caaagtaatg 180
atacaattga acacaatcag cagaaaaata agatttcaag aatgttctga accaaacctt 240
tcccagtctt ggattaccca caatggtcaa catgagttca aacagctgta aataacaact 300
tagataaaaa tgataattca aaaattatta caagaaatga aggaaacaca aacaaaaaac 360
catgcaaacc tgatttacgc aagttttaga cagatctcag ccagtttatt atttaaaatc 420
ctataattat aattttttat catttccata caataatcca gcaaaactat acgacactaa 480
taaaaggtat agatcaaaca aagaggttcc tttcaatagt aagctgatga agccatcatt 540
gcagtgacta cagtatcccc tgtgaattcc agtttgacta atttgaaatt taggactgtc 600
ttccttgcca aataacaagt taaaggatat cattctagca gctgattcag tttgacactt 660
catagtgacc tttctctctc tggaatactg acaactatgt acagtttaaa ttaaacaatc 720
aagttaacag cagttttctt tggggcagta tgcagatgat acggtttcct ttgggacagc 780
aacattggca aatgtaagag caatcaaggt gatgttgagg agctttgagt tggtatcggg 840
attaaagata aactttgcca acatctgttt ttgggcaatt gggatgactg agcaatggat 900
gaacagtgct gtcagataag ttgcagaatg atgtcagtac tcttttctta tttgggtatc 960
tctattgggg caaactcaag acgtagtgag atttgggatc taatagtcaa gaaatgtgag 1020
agaaaattgt caaaatgaaa acaaaaatat ctttctttcg gggaagggtg actttgatta 1080
agtcagtcct aaactcgatc cctaaaattt ttttcttttt tcaaggctcc aaagaatgtg 1140
gtggataggt aggtga 1156
<210>3
<211>657
<212>DNA
<213>大豆
<400>3
atagttcact aggtttgtac tcatgccata atatgccaat ctttcacagc attcatttcc 60
tgacataatg tttttagcac cttagtgcga ttttaatact gaaatatgca taaaagatgg 120
tatcgaaatg aaagaagaaa aaaaatccaa taccaagtat gaagcggcat gctttccaat 180
ttccagtttt tttcttgatg gcaggctttt tgtaaatatc aactgtccca tcttctgtgt 240
ataaactatc ttctcccaca tcatgtgatt ttttgacatc tcccatggtt tcacagcaga 300
tttcctattt gctgcttctt gttctctttg atatctacta caaatgtctg tttgcagggt 360
gctggaaatg agtaaacaaa ataatcggca aaaggtacac gaaaattaaa cagattgcta 420
tcatgaattt catattataa atacttgatt tgagggtgtt tatgaagtag gaatagcaaa 480
gagaggttca gcaaagcaat gaatatgtta gctcatgaag ctccaatcac aatccttcgc 540
aaacacattt gatggcaaca ttgtgatttg ggattattag tggtacaaag tgatagttat 600
aatcacaaag aattaatgta gaactgcagg tgacttagga ggtccgggtt cgacccg 657
<210>4
<211>659
<212>DNA
<213>大豆
<400>4
atttgggcgg cctacacttt tttgaaaatt aaaatatact tttgtgtagc taatttccgt 60
tttgcatgtg tgtgtgtgtg taatggagag agagatagag agaaagagtt agtttggttc 120
ctagtggcac tgaaattact accaaatctc caaaagtagc tatggagtta tttaggatgt 180
ctaatgagtt gagtttatgg tcttatttat atgtggaaat aatgatttat taatccagag 240
cagatgagtt aaaagtttct ctagagaatg gtagtttcta aatgaataaa taggatagaa 300
tctctagcac tcaaagaatg aaagaatgtt tgcattttat tatcacctga gccaatttca 360
gatatctcga ttatttcctc ttaatatccc atggcaacat tcattgcgtt aagccaacat 420
tttaaatgaa agtatctgtg atctccaagt ctttgatatt catttgtcta ttccaaattt 480
tggttccaac tggcttcgaa agctttgatc ctcctccctg ctttcagcat gatttcctca 540
ctcttcttga actttccata ctgaggaagt ctgagacaca atggtaaaac tatcatggtt 600
atggaatcca tgaaacaaac atcattattt tctattaagc tctgaattgt agaaataca 659
<210>5
<211>372
<212>DNA
<213>大豆
<400>5
cattcatcag aagtgctcaa agcatttaat ttcagtgcca aagcactgca acttttcagc 60
tcagacaaaa caaacttgct agcttccttg gtaactgtgg aggatttgat agacttgaaa 120
accttctcaa cagattcttg agcacatctg cgtacctaaa aagcaaacaa accacaacac 180
aatttaaaca aacaaaaaat ccataagctc agagtaatga aatgattaat aggcatggac 240
caatgtctcg aaatcaaagg tcatatcata accaaatgct gattgcattg taaggtaaaa 300
ccatcaccaa atctatacat aattggatat aaacaaggtt taaaactaaa gttgcagaca 360
cattagcagc ac 372
<210>6
<211>1448
<212>DNA
<213>大豆
<400>6
aacagaactc tatggccgca agacaagata gaccaaagag gaaggacccg gcatattaaa 60
agcagtgaca aagtaggaaa attgcactca tttacgatca acccaggttc ttgtagatct 120
agagtactag taaggttctc taatcactta tggctcttaa tgttgaatag ccagaagtga 180
taaaatcaaa tcaaataacc ccctagggtc ggcctagtga cggggttttt ggtagcatgc 240
acaaagtctt agattgtaat cttgttgagt cattgtacac caaataaata aataaataaa 300
attaaaatta atgtaaaata tgataaatgc aagtggaatt tatttccaac taatttatgc 360
tcgttctcaa cataaaaaat caagagattt gttgtgcata actctttctt aagccatata 420
tcatgactct tacctgctta gctgtcgcaa aattcagtag cgcttcacat tcatcaagca 480
tggctttttc cctagctgat acaacagcaa cttcagacat taaactattc atgccctcca 540
cctattccag acacacacac aaaaaaaaaa aatggtgtca gccttaaggc tttagggatt 600
taccattgaa tcaaaaagga aaatcattag gaagaaaaaa catacagtta gtagaagaaa 660
aaaagtttga tactaaatgt gtaggcctag aaaatagcaa atgctagtgt gatattgtga 720
gtcaaaccag tagaactcct aaaaaagtaa cacaccccgt gacagcaaag ggcagatagc 780
agatcccatt gcttgcatgg catcaacagc tgaatagata gcatgcttca aatgttcaat 840
atctgcctga aaaagtgtaa acacagtaat gatgttagtg tctctttgca cgcatcgaac 900
caaaagaaca gaaaccacca tacatacctt tgcccctcca gttagaggaa gacgaagagt 960
acttgcctcc aagtcttcta cagccccgga taaggcatca atatgatcac tttcaagtac 1020
agcccagtca tcaaggtagg ccatctgtgt cattaattgt aaaaggacta caatttaaaa 1080
aagtatcaaa aaaaagttga atcatttaaa taagaaaatg gtttcatata tgacttgtaa 1140
tcatatccac cattaataat atgagttatg aataccatgt tatgacagac tagcataaac 1200
aattaaacat aacttttcaa tgtgcagggc caacatcttg ctgagtatat tttcctcatt 1260
tataaacttc acaataaata tctctagtta aattaccaaa aatgaaaatc gggaaaaaaa 1320
aaaaagaaag aaagaaaaag taattgtaat gtatcatcaa caataatatc gcacatagaa 1380
tgataaatat ttcaggcaag agagaagtat tacttgatca ttcaaaatag aattcagctt 1440
cagctcaa 1448
<210>7
<211>922
<212>DNA
<213>大豆
<400>7
agtttgctag gaagtgggtg ccattctgca agaaattttc tatagaacct agagcaccag 60
agatgtactt cagtgagaaa attgattacc tcaaggacaa ggtgcagccc acctttgtta 120
aggatcgtcg agctatgaag gtctgtatca tatctatcag actagtactt gaacacatgg 180
gagtttaaaa gttagtttaa agcttattca gtgttaattg ggtgtttgac agagagaata 240
tgaagagttt aaggttagga tcaatgcact tgtggcaaaa gctcagaagg ttcctcaagg 300
aggatggatt atgcaggatg ggacaccatg gcctggaaat aatactaagg atcatcctgg 360
tatgattcaa gtctttcttg gtcacagtgg aggtcatgat actgaaggaa acgagcttcc 420
tcgtcttgtt tatgtttccc gagagaaaag gcctggattc caacaccaca agaaagctgg 480
tgctatgaat gctttggtag atttttttga gcagtttttg ttgttcctat gatgtccatt 540
cacctttata tgagacacaa ttccttgaca cttccaatta ttgctgtgat ttgcagattc 600
gggtttccgc tgtgctcaca aatgctcctt tcatgctgaa cttggattgt gatcattatg 660
tcaataacag caaggctgct cgagaggcca tgtgcttttt aatggacccc caaactggga 720
agaaggtctg ctatgtccaa tttcctcaaa gatttgatgg cattgatagg catgatcgtt 780
atgctaatag aaacacggtt ttctttgatg taagtcactg caagaaacac agcatcagca 840
tagcatggcc ttttctttga agcatttgac tatttttttt tggtagtgta agctaatact 900
aactatttct tcttctttgt ct 922
<210>8
<211>730
<212>DNA
<213>大豆
<400>8
tccctattat tcactgaagt aatgaataag tcgttgaaga aagttgggca tgtcattatg 60
tcaaaatgct tctgacttct gagggtcaaa agtttacacc tcttttctat tttcgtaaaa 120
ttcctgagga acatttttct tctgacatgt aaagtgaaat tttatagctc attgctgtac 180
tgccgtttaa tatctgacaa tcattgaagt taattaaact atctcataaa agttgttggt 240
gatgaatgtc tggaggtgta agcgcaaaat ttgcgaccag ttaatgaatg tcttatcaac 300
gaaaatacgt gtactactaa tcaaccaaca tatgtggctt aaacaatcct agtttgccag 360
tagtataaat gctggggtta cattatcagt agatgttttt attagagaac caggtcatga 420
tcttcagttg aatattgcca caagtatgac atgtgttatg cttgtttttt ccatcagaat 480
agagtagtgg aaaaaaatgc taatctgtga caaatttagg ttgtgtaagt tgaagtagtt 540
gcatggaatg cgcttcatca tgatccttgt gtcagtttct aattttcaat gttattttgg 600
cgtaaacagg ttgaggaata tagccttcgg acgcatctca tgcagatcaa gcaaataagt 660
aaccaatctc ggaaattcaa acacgcatga ccctgacctg cttctacgct aacaagaagt 720
cttttgcagc 730
<210>9
<211>717
<212>DNA
<213>大豆
<400>9
tcttatttga tttctccaat gatatgcata tagtggtgtt taaccatgtt tcttgacatt 60
tatttgtgtt ctatttgatt atgtgactgc tccagaatag agtatttatg caatatcttg 120
gtaatggaaa ctaacaaagt ggaattaatt aactcattgg agccattcat tatgattgtt 180
tctttcaaat tgtctattga gttcaatcat ttgttgcttc tattgatttt atttaatatt 240
ttagtaggca tgatcggtcc aggggctgta gaaatatagt gaaggatttg aaagtctttg 300
acacattcaa tacttggttg cattatgaat atgttgaaga caaattatag aagataaatc 360
taaggagcaa ttttatatat caacaagcag caagggaata tgttcgaaca gatggtgggc 420
acataggttt ggagctgtca tgagcttaat tcatttgagg atgacccata ttttaaccgt 480
caaaagcaaa acatagaata aaaggaatat tgattctgtt tgcattttgt ttggggtact 540
ggctagacta gatacgtttt cctggtccaa tggaaacctt tggatcgttg gttgatttga 600
agttagtaat atgctgaagg aggaagctac aagagaagtt tgcatttcac gatcattttt 660
ccttatgtat aggttgtttt ctattgttta ctcacatttt cagctgcagg catgcaa 717
<210>10
<211>807
<212>DNA
<213>大豆
<400>10
tgcataatca accaactgat atgacatttt ctgtggaatg gacagaaccg ttattatcat 60
gatgttaatc agtagatcat ttgccatctg gcttccagat gttaatctct tagtagaatt 120
tttcattgcc tagtattgag aataaaacag atttgagact taaggttctg tatgcaatac 180
aatgaattgt ttattagcat tgtctacttc ttgatactga tggttgtcat tacagtaata 240
tgtcgaaatc tataataact aataatcact taagcaacaa gttaaattct gtttttggta 300
ttttgtcatg ggtgtcttta atgcaagttt atatctttga tgcttttttg gttttatttt 360
tacaaaatag tagatgaagt tcattaagat gtttttcctt attgattgtg aaatggaatg 420
catgataata tttggtgttc tgtctacctc tcctgaatta gaccagtcag tttaattctg 480
ttgctctctc tgttttattt tactctcaat ctttgtgagt ttttcggttc acttgagttg 540
tactgtctct agaaggtcct attactttat tggtcaagaa aaatatagaa ggatattaat 600
ccaaccttgt gatttgtgtg cattacactc atacacattt tatgttcata tagtcatctc 660
aatctaatta gttgttctat gcaaactttg ttgtggaatt gaactgcttc ctgctgtgca 720
tttaacttgc cttgcttact gttctccttc tgtgtgtctc aggttagaaa ttctttgaat 780
gctcagcctt ggatccataa taatctg 807
<210>11
<211>839
<212>DNA
<213>大豆
<400>11
caaacttgca tgcctgcaga ctatatggca ccgcccctat agtgcccctt ccaacgatta 60
cctagaattt aatttgatct tctgaaggta gatgaataaa taaataaacg tgtgaaaata 120
aaacagtaag tacatgccag tacgtaataa tgtgaactag tttgtataca tgaatttagg 180
tccaatgctg caaaagacct agttagactt ggaacataaa aggatatatt taaatgactc 240
tcaagattaa ctaaataata cacagacaaa tcagataatt aaactgcacg gccactaagg 300
gatcagcata tgtgaaagtc tcagagagca gacatgtcgc tagttatata taaatcaagc 360
tgattttatt atctatatgg gaatcaaata caagcttaat tctcttttgc tagcttcaat 420
ttggatacac ataattccaa cctccaccaa ttgataacaa atactagtaa tgtacacatg 480
ctattgtgcc cccgggtagc ttaactcttg aaaaacacat tctcgtggca tctcttgacg 540
cacaccctcg taattcgaag caacaagagg aggataaatc agagaactgg tttcacccca 600
atcagtactt tgtccacaac ttgaagaagc acaagcacca cccattatct tatcggtaat 660
catttcctcc ccatagaaga actccaacga cccacctaat aacctttgag aataatcatt 720
agcagaaacc atttccccaa ctgtagtggt agtgaaactg ctttgtcctt gcaccaactc 780
agcacggtac tcattgctct gaccaaactg tacttggttg ttgccaatag agttcatgc 839
<210>12
<211>751
<212>DNA
<213>大豆
<400>12
aaacactggc ttcggattta tcacttgtaa gtagagtttg ctaactaaaa tgctttgtca 60
ctctttattt tcaggttttg ttctgatatc caaaatcctg gctatgttga ttgccattca 120
aattgtaata agtctacatc tcaagcgtct ttgtttttgt gttccgactc caacagtaga 180
agaaatggtg tttttggtag accactttgt gtgaacccct ctggcaggag aaacctagtt 240
ggtccagctt tttattctct ggagactagt gcttatgacg tggctgcttt agaatctcct 300
tcccgtgttg cagaagaaaa agttggtgtg ctgcttctca atctaggagg accagagaca 360
ttgagtgacg tgcaaccttt tctgtttaat ctttttgcag atcctgtatg ttagtttgta 420
tttgtgcttt ttctactgtt gatttttctt tttcctgttt atgtaaattc cattagcatt 480
agtacatgtt catatgattt gtatgctaat gtgtttcttg tattgacata ggatatcatt 540
cgtcttccaa ggttgtttcg gtttctccag cgaccattgg caaaattgat ttctgtactt 600
cgggctccta aatccaagga agggtatgct gctattggtg gtggctctcc tttacgaaaa 660
attacagatg accaggtgga gtttaaattt tttggttttc ccattatctg ctttgtggag 720
cttttatctt tctgcaacat gaatcttttt t 751
<210>13
<211>663
<212>DNA
<213>大豆
<400>13
aatatgaaga agtacctgct ttaccattca acttagtgac tgtaagtatt caatactaga 60
gccaagttca cttttctatt tagctacata ctaggggggg ttctcttggt aaaaagaaac 120
tatctatact tatatgttat ggaattacat gactttcatg atacaaatca catgaatatc 180
aatagttgca agtagttctt taatgattta tatttcttag gaacatgact tgtgcataac 240
ttctttgagg tcaatccacg gcttagagta attctgggaa cccgtttgca tcattgtaaa 300
caggcatttc acactttcga atgcatcaaa tgaagcaaca tttttttata attggcattc 360
aatgtccatt tggatggttt gaactgataa ccatttggat ggtttttaca attggcatct 420
gtgtcttcag gaaggggact tgagcaggac agcttccttt gcagatgatg gagaggtttt 480
agatggaata attactcgaa gccggggtga ggttagacgt gtttgcagtc caaaggtgat 540
gaaatccact ccaaacctat cccaagagtt aacaagtcca aggctcacag ataaagtata 600
cagccctcgg ataagccatc tcagaggaaa tcaaagccct cgaggtgttg ggagaggatc 660
att 663
<210>14
<211>713
<212>DNA
<213>大豆
<400>14
cttaagtctg aaaacaattt gtctacttgt acataatctt tatcatggac aaagtatcaa 60
gaacagaaaa tattttatat attgtctact cttgcctcat tcttacacat ctttatttta 120
tttattttgt ttcaagttat ttgttattga aaaagataaa agtgttaact gtttttttaa 180
taatattcta atttaaataa attcaaacct atatttgagc tcttttttta atgaggataa 240
tttaatattt tttatgttat aacttgtgta attaatattt ttttgagaga actcagcaaa 300
aaataaataa atttttgaga gaaaaataat atttttttta aagaagtgtg tattatttta 360
aaaaataaat aatatgagat ggaggcaaca tgtgatttta acaatgactt gtaacatcta 420
taagctcaaa atttttgaaa aatgaactgg cgtaggataa aattaaacta cctggataaa 480
gcaaaggttc ttcccaattg gttatttaaa gcaatcttct ttgtataatg gataccataa 540
cttcaatctc ttaactacca tgatttgatt gaagcgatcg atctcacaaa gatgttcctt 600
tcaatattct taaactcaag tacaattttc cctcaaggac ccactatgtc tatattccat 660
tggattacat agtaaaagca aaccaataat ttctctacct ttagctgcat ttt 713
<210>15
<211>534
<212>DNA
<213>大豆
<400>15
cttgcatgcc tgcaggagac tttgagaaag cacacttcag ttgtttaccc gatataggag 60
agatacaagt taaagggtta atggtaagta ctttactttc tgtttagtat ctatgcatcc 120
ttttatgaat ttctgcacca atgagttttt gctcaagtta ctgcacattc tcctaggtga 180
agcgaaaatc atgcttctac tacctgcctg attcgttccc tatacagtat gctttccctg 240
caatcagagg aacatggttt cttcatgtgg aagtgaagca tttaaagcgt ttgcgcattc 300
cgtgtccacc tggtgatgca gctgttcttt caaaacataa ggacttaaag acctgcaatg 360
gtgaggataa ggcaaaatgc aacagtgagg aaaataaaat ggaagggttc caaccccgtt 420
catgttttgc agaagagcat gaaactacta atcatgtttc aaagaagctg aacaaaaaga 480
gaatttctaa tgaaaaccac acgcagaatg aagccactgg aatgccagaa agat 534
<210>16
<211>433
<212>DNA
<213>大豆
<400>16
ttgccaatgc agctgctggc ttgagtgcag ccatggcagc tcagcttgtg tggacccctg 60
ttgatgtcgt gagccagagg ctgatggttc aaggtgtttg tgattcggga aatcctaagg 120
cttcagctct tcggtacatc aacgggattg atgccttcag gaagatcttg agcagtgatg 180
gtcttagggg cttgtatagg ggttttggga tatcaatttt gacctatgcc ccttcaaatg 240
cagtttggtg ggcttcatat tctgttgcac aaaggatggt ttggggtgga gttgggtact 300
acttgtgcaa gggaaatgat agtgcactga agcctgatac aaagactgtg atggcagttc 360
agggagtcag tgcagcagtg gctggtggca tgtctgcttt gatcaccatg ccactggata 420
ccatcaagac aag 433
<210>17
<211>554
<212>DNA
<213>大豆
<400>17
aaaaaaaagg acaatcatta aacacgtatc taaaatgcat ttcatcaaaa tgaaaaatta 60
tgcaatactg aaaatccatg cgtgttataa aggcaaacaa aatgaacttg gagagcaatg 120
caacaaagta ctttttacag tcaatgtgca ctttaaaaaa tagtatattt catacttaca 180
taaaagagct gaatgagtgc aagacgtacg aaagaataaa atttcaaagt gccacctaag 240
tcacagagtt tatgagaaac aaactgtgag ctttggtcag gtaatatcca ccacaatgca 300
gggatgacaa ccgagtttag gacgaatata ctgcacaaaa atttaaaaga tgttgaaatc 360
attaaacacg tagattttag attcatgatt tgttcaggac aatcaatcca tggatgacaa 420
aaatatgtac aatcagattc cttcgagtca ttatgtcaaa agtatacata atccaatttc 480
tttgccacaa aatttcattc actgtgttga aataaattga agctagtttc acttctcctt 540
ctgcaggtcg actt 554
<210>18
<211>810
<212>DNA
<213>大豆
<400>18
tgagtttaat catgtatctt ctttttcaat gcttttggtt ggacattaaa gccatatttg 60
tttggatttt gtgccatata ctatcaatcc aattttatta agaactaaga cctactagtt 120
tttcaaacaa ggtcagcaat actcaaaaat aaattgccaa gttggaccct gtagttttgt 180
aaatgtatcc caacaattat aattaaaaag tagttgtact gtataatatc tagcaaattc 240
aaaattctaa agtcaatttt ttactgtcta atccaaatgg acgctaagat actagactat 300
tgatactcac agaacattat ctgtagttaa catgaaaaat gtagtttgtg gttttgatgc 360
ttcctttttt attttattta agtgacttag tttgtagatt ttactttgca gggagactat 420
catgttgaca gtgaattttg tggtacggac agtgtacagc tgaaaggatc tgagattact 480
gctgaactta agtatctctt aaacttgttg acattgtgtt ggcacttttc gaagaagccc 540
tttcccttgt ttttagaaga aactggctac agtgaagaaa acgttctcct tcgagaagcc 600
aaagcaggag taagtcttgt ttgaatttta ggaaaaaatg ataataattc aatatctgta 660
ctgtgttgac taagtcattg atagttatta acacacattc tcttttgagg aaggatggaa 720
ggctgaaagc acaagagatg ttgttttatt tagactgata aaatatggga taaaaaattg 780
atgatagatg ccttcttttt gcttcacttt 810
<210>19
<211>1222
<212>DNA
<213>大豆
<400>19
aggtgcagct gcccttttgt actcacattg aatcggaaat tgcagactca cgatgcaatc 60
gacgaagatg gtgaagaaaa tgggagtgat acgcccactg atacgccatt aggtattggc 120
cgtgtttctc atcggttaat ccaagcccct gcaacatggt tggagacaat ttcaacattg 180
tcagagactc tcaggttcac gtattcggag acacttggga aatggccaat tggggatttg 240
gcgtttggca tcagctttct tctaaagcgg caggtaatga caacgagtag attttggttc 300
tttattgttg ccctgatttg aagcaactga aaatgccgga aagctgtgtc gtttttttat 360
ctatctgtaa ctttggacac atttaagtag taagtagaat atagaatcag tatttagtgt 420
ggaagccagg tgcatatttt tcaggtagaa ctataattga tctgaaatgg tagttgcaac 480
ctgcacttaa tgtgcaactc acataattca cctaaaggat tgcccgtcac tgacattgat 540
gaatgaaaga gagagaaata tagagaaagt aaaatggaca atggtcatgg aagttatgcg 600
agttaggtga actttttcat gtgtaaataa aattgttgat gattaaatgg ttggaagacc 660
aatttaatgc tttccatctc aataaaaaaa attatggtcg ttaaagaaat aatcccacat 720
ttggagagca gttataattt atattaactc ttaagtgttc cttacatgtg ataggatctt 780
tttttcatgg gtgttgtttt tgttttgctt tttaagcctt ctcaacatca cagcagtggt 840
ttgggatcat gacttgcaat atttgaactt cttttgttac ttgttaatca tgatcacttt 900
gaagttgaca tattagatta ttcttggatt ttatgattta catgatatga tttctgttta 960
tactttctag gagagtaata tggctaaggt agcttagaaa tcagactatt ctctacaaaa 1020
tgaatcctca caagattgtt cacatgacct gtgctacttt atatatttga ttttgattta 1080
aatcatatat taagcatttt ttaaggtaga tcagtttcat gacatcctgg actacttaat 1140
ttcttcatct cagctcaaca taaatagatg agaagttgct ctgtaatatt ggttttgtgc 1200
cagtttaatt tgttcatatt aa 1222
<210>20
<211>682
<212>DNA
<213>大豆
<400>20
gcaacaaatg gatgtacaca gtccgagccc tgcataattg gagcaaattg catattccaa 60
cccttaatga gaatacagaa atcaaaactt gataaataaa atacttcaaa tttgcccaca 120
ttggttgcta atagctattg cagagtatac acaattattc cagtaataca aacttgcata 180
ttaccacaga actcttaatc ataccaacat taaaatagtc tctgtagcca ggctttgagg 240
gcacaagaaa gtgcaacaaa tagttgaaaa aacactgtat gttgtggttc actaatctct 300
tatgaaacag gttatgtaga aaggcttcag ttgtcaccta ctaacatcag ttcaccttac 360
acttgtaatc gtagctacat cttgcttccg gaaacaaagc tatgtatcta atcactaata 420
atgacatcaa agtatgaagt aagatatacc ttttcttcag aggtactctt gacgaaacac 480
aggaatgcct ctgagaactg agagccactg gaaccggttc gcaagtcagc aacgcaaggg 540
cattcaaggg ctttctgagt catttcctcc acagactgaa aaatccaaaa tttccaattg 600
ttttcattcg ttcaacctgc caagttgaaa gcaaaagatc aaagcaattc aattcaaacc 660
tcagtgttct gatttccata tt 682
<210>21
<211>681
<212>DNA
<213>大豆
<400>21
actgaaacat tcgaaattcc ttacataatt tattcttatt aaaaataaca gtaatctttt 60
gacttgaatt ggtacagaag tacaattatt ggttgaggct tttatttcac gcataccaca 120
atgaaacaca tttcaatttt tcttacccct ggttaattta atgtaccgaa tttatacatc 180
aaagagaaga taactttcga agtaaaaatg attatcctaa acaccgtatg ataaagtgta 240
taagattgtt caccattact taggtttttg gaaatgtcaa accttagcac tatggtaagt 300
ttgttgcctt gtaacttgga ggtcatgggt tcaaatcctg caaacagcct ctccttaggt 360
aggaacctca tgcattggac tgccattttt gttgctttgg tgtgagcata gatactgctg 420
aattctttga gatgccactt ctcacttatc ccatttttaa cttgcaatct taacttatgc 480
ttgtaactac gttcctgaag gcctagaaat ggtgggataa ggatttatgt gttgtttctt 540
gatggatgtt ttgcagacct tcatgctggg tggatcacat gtcactggac aatgaaacag 600
gattggatcc accaggcata agagttaggc ctgtctctgg acttgtagct gctgattact 660
ttgctgcagg tcgactctag a 681
<210>22
<211>1002
<212>DNA
<213>大豆
<400>22
tgagagcttc cattcagaac tactatcaag tactgacagt tagcttcaat acttcattta 60
taataaacag aataatcgct taaatgaaat tggtttagtt tcattcacat taatttcagg 120
cacagtgctt tagatgtaat caattcaggg agctgagaaa gaaattccac aaaccctcag 180
attttaaaag ttgaacatcc tcagcttgct gcatcaatta acagtaaaaa agaaaggaaa 240
tgagaaaaaa tgagatttaa gattttatag caatttcatg tgagatatta gagcagatat 300
gagagttgta actctgaaat ttcaactcac tatccaattt tcttccacag tattatcatt 360
gactccatgt aggttattaa ctgagttcag ctgaatcctg tcagttggat ttgagaatga 420
ataatgatgt tgatatttat gtttttatgt tccaaaaggc cacctttggg caaagggaat 480
acaaacaatt acaatgaaac aagtaattat atacagaaaa ctgagaaaag aaaaaaatca 540
acaaatacct gctttctcca tctgaaaatg agcaactcag ggcagggagt tcagatgata 600
actgcttagc atgatcttta gaaccagagc gacataaatc cacaccagaa cttgactgag 660
ataactctga gtgattctgt gcatgaaagt aaattaaata ttaagctgaa tttaggaaat 720
aacgtaatta ctttatcagg aaggaggaag gagaaagcaa aagcatggat aaaaaggaac 780
ttctgcttat gttgctttgg acacaattta taaattttgt aatatgttta gatgttaaag 840
ctgaactaac ttcaaaaaca gaatgagact caacatttag cacactttca agcaaaaatt 900
gttcatgaaa atatcatcag ttcatcactt ttgaagttaa tcaaatgttg cctgcttact 960
ggtaatattt taccaatact atcagcacaa gtagttttat cc 1002
<210>23
<211>785
<212>DNA
<213>大豆
<400>23
cgacttcgta gcctgcagca agcaaaggcc caactccgtc cagggacggt ccagtacggt 60
ccaagatgat caagctcacc actatgcaca tttctttcgc ctcccaaacc tgaacttgct 120
gcaacagttt taaaagaata ttaatattaa tattaatatt aaagtttcct acagtaagtt 180
attatttaga ataaacataa aaaaaattta tatgaatatt tatttttaat aaataaatag 240
tattcataaa aatgctaaaa tcaagcaagt aattttccta caattttaaa atttgcaaga 300
aaattacata caaatttaaa atccacaaga aagataaact gtgtatttta aattcctgaa 360
aaattaaata caaatttaaa ttccgaagga aaattactag caaatttaaa ttctaccaga 420
aaattatttg caattcaccg aaaaattact tgcaaataaa ttatctgtga aatttctagc 480
agattctttt agtaaaactt tatttataga cacaccactt tttatgtaaa acattttgcc 540
gcagaaattg ttgtatttgt tctagaaaaa ttagcaagaa attttctatg agtttcaaaa 600
ttttcaaaaa attaattatc tactaaggta ttatttagga acccaagtat tggaaattca 660
caggtaatta gtaataagaa aaattctata agatatcgta aaaatataga tcacaataaa 720
gcaagataaa cgtacgggga aaaaaaaatg taaaagggaa tctatcttcg tataaactaa 780
cgtat 785
<210>24
<211>805
<212>DNA
<213>大豆
<400>24
tattaggtca gccattatga caacatcgga tatattcgac aatactaagg aactcatcaa 60
ggacattgct gatgattaca aaccagcctc tcctttagcc ttgggatctg gtcatgtcaa 120
ccccaacaaa gcccttgacc ctggacttgt ttacgatgta ggagttcaag attatgtcaa 180
tcttctctgt gcaatgagct ccactcaaca gaacatctca atcatcacta gatcgtctac 240
taataattgc tccaatcctt ccttggatct caactaccct tctttcattg gtttcttcag 300
tagcaatggt tcttctaatg aatcaagggt agcttgggca tttcagagaa cagtgaccaa 360
tgttggggag aaacaaacaa tctattctgc taacgttaca cccatcaaag ggtttaatgt 420
tagtgttgtt ccaagcaagt tggtgttcaa ggagaagaac gagaagctaa gttataagtt 480
aaggatagaa ggtccaatgg tcgaaggctt tgggtatctg acttggacgg acatgaagca 540
tgcggtgagg agccctattg tggtcaccaa tcaggcaccc tcaaattcaa tttccatata 600
gatcaatttt gtgatggata aatgtttttc atatgtttga agttaaaaat atatattaat 660
agaggaaatg ttcgtacatg aatgattatc atttctgata ataataataa ttttttttgg 720
aaaagtttta acaccaattt taattttttt tttcttatca cgcacaccaa ttttaattgt 780
tacgtactga aataatacgt tagtt 805
<210>25
<211>1222
<212>DNA
<213>大豆
<400>25
tagatctgca ctcgtgaatg ataacattgt tgaattaagg attttgatgc ttgatgcttg 60
atgcttgact atgagagaga atactattga aaattgaagt gaatacttag aagaagttca 120
tggccttgga atggaatgat catgtgaacc tcattacctg ccgacttggc actgcatata 180
tggatctaat tcaagtcctt ttcatcctcc taaatgcctg tcccttcttc tttagttctg 240
atcctcaact tatccacatt agctttcttt ttctagtatt tacaaggatt gctaaaatta 300
attttatttg taataataaa aatgtttatt attgttgtct ataattatta ataaatacaa 360
ttactcgttt tagtgtacat atttcttatt tctatatacc ctttaatata ttaattattt 420
tcttcataaa ccttcaagat gtaactgttc taattttttt ctaaaaaaac tgttatcaat 480
actttcttta attgtttccc ttttttaaaa taaagataga agcatgaagt gtctcatttt 540
caattattta aataaacaat actttagtta gacacaagtt cgaactataa gtttcccata 600
attttgctcc attatatcct acaatttttg tgaaatatat atattcttac aagataatat 660
tacgcacaac ttttcatcaa aatgttacaa acaactcgag cattttagga catttttttt 720
caagtaaatc ccaggccgaa taatcatcaa cctatgttac attcaccccc aacataaaaa 780
ctaacggggg aagatatcta ttgttagtct gtacatttgt tagtgcctga tctctctcgc 840
ctacacagtc gcttgttctt ttaaaaaaaa ccagttagtc accgtttatt ggtcttctcc 900
ttgcctgcaa acaagtttgc cttgtgtcag aattaagcat tactatagag aagcataatt 960
ttcttaaata agattactca ccaaatatag ttgattttaa aggaaatcga attgatgaac 1020
ccttaaatct cagctcccga ttatgcttgt ttctattttg tttctcaata gcactggaac 1080
tattgctagt ttctccggtc agaaagtttg ccactttact taccttttca tggtacacag 1140
caggtggggc aagcttcaat ggaggcaagt tttctatttg cataaatctc tgattcttct 1200
gcaagctgct caagatctgg aa 1222
<210>26
<211>1177
<212>DNA
<213>大豆
<400>26
agtatttttt aaagtacagt gagaaaatgt aaaataaata aataaataaa taaattatct 60
tagctatcat attattgccg ataaaaaaaa atgtcttggc tatcaaagct cttaaagctt 120
accatttagt acggatcctt ccgtggcatc tttatacgcc catttacatg catctatggt 180
actttcagat gcgtatctaa aaaaaaaatt acccaagtta agtatgtata tatgctttga 240
ataataatca gagacaacta aagaagctgg tttctttcat aaaaaaaaaa gaagctggtt 300
tctgttgttt ttctagttat gggtttttgg gatttaaata aagaactcat ttttaagcat 360
gtgataggat ggatatgcca ctattttcaa catcagagaa ggatattata tttttatatt 420
ctaaaggatt attttaatac tattatatgt attgtattta aattatttat aataaaaatc 480
ttaccaagaa aattgaaaga tataaacgtg aaactcgcaa aagaaacatt atagaaataa 540
ttggatttgg gtaaatgata tattaattat attattaata ataatgggac atacgtagct 600
gggcatggaa ggtcattatc accgcagttc tcccattctt ctatctgatc ggcccatact 660
ttctgtttat ttagataaaa ataaataaaa aattgaagat atacaacctc aaacttcaca 720
acccaaatcc ttatttagat tgatgattaa aacaaaattg catacaatac cgtaatattc 780
tgttgaagtg catcgatgaa ttcgttcatg tcagagtcat aaaaattgtc gacttctgtc 840
tgaaggatgg tactatccca aacctgtatg tatatgaagg taaaattaaa catatcatat 900
tgtcgtatat atagtatgtg aagacaagaa atggcaagtt ttaatgcatt ccttcatcca 960
gtttctaatt taaggactca tatttttatc tcaatacatc agattttaaa atgcacacat 1020
tcgagattta aaccatctga tcttcaccta acggtgtcga tctatgaatc catgaagaaa 1080
aaattgactt acatggtgaa gattttgctt ccttttatac cagcgaacag taattgcatt 1140
gcccccatgt ctgagagaaa gccacaatgt agaggct 1177
<210>27
<211>685
<212>DNA
<213>大豆
<400>27
agtttgcatg cctgcagcca agctctcgtg gatttggtgg tgttgttcgg gtaaattttt 60
cataatttta tattaaattc ttatgtttct tgatgtgttt tgcaccaaat tcacctattt 120
tgggcataac agacacccac cattgcctgt tcctgctgtc tgtgaaagtc agctgcttta 180
cagctgatgc cgtgggctgt tgtagctctg tcaaactcat ggcctcttaa aaaaaacata 240
ccccagtgtc ataaggctct tcactatgcg aaagtatggg agagggtcat tgtatgtagc 300
cttgtccttg ctgatgcaag gaggttgctt ccgaattcaa acccatgacc aactggttag 360
gcacaacttt actgttattc caggactcgc cctctagcca aaatgacctt aacaaaaaat 420
agtctctagc taaatgaatt gtgtcaatgg tgttatttta aaggttaaac aaatgtgtat 480
agtccatcag agacaaaaga gtttacacac taaaactgat agcataaatt gtcacaggct 540
gctattatgg atatacaagt tgttccccat ggttttctta catgcggtgg ggatggaatt 600
gtaaagctgg tacggctgga aaataacttg cttggccatg gaattgagtt atgatacttc 660
tgagatcctt tgggttgatg acaaa 685
<210>28
<211>1343
<212>DNA
<213>大豆
<400>28
cttgcatgcc tgcagaaaat tataaaattc ataaaacgct tctacagaaa attcaaaaag 60
attgatttgc tatatatcct atacacttgg acattttagt cgaaacctct atggatagag 120
actttaagga cacaatatta tgaaaaatat tcagctcaaa tattataaaa tgttaaaaaa 180
caatgcatct caatattttt ttgaaaggtg tacttttaac atgtttatga attgtacttt 240
cttggctaat gagtgttttc cctggttaat gttatggatt gtactttgta ggtgtactga 300
tatatttttt ttcattaaat actaatgttg attattcaat tttagaacag tgtacgcata 360
gtatatgact gttattgata aggtatttgt tatcgataag gtgcagttaa tataagcaga 420
gaaagaaact aaaaggttaa ctacatgatt aagcttaagt gatgaaatgg ataatccctt 480
aaaagtcatc catgatttgt atatttggtg ctttgcaaaa acaatcattt aagttcgtct 540
tcaaaatcta gtaacagatt acttaaccat tcttttagat cactacaaat atagtatttg 600
tttttttaga aggaaaaaat tgggtctgtg gcatcttaat ttttgtgatc acatttttgg 660
ttctagtgat actaacttct tagttcttac aatgtatgta tattttttct ttttacaaat 720
gtcactcttc tcagctggat tcatggtttg aaaactcttt cctcataatg tcaacaggtg 780
gctccctata atacatttta ctcccaattg gaaaagcata tgaatgaagt tggaattgtg 840
cccacagtta accgatggga tgagcctcta gcattgggca tggttgatcc ccatgattca 900
ttatctcatc cagcaggtgt ctctgatgtt caagctgagt ctgctacacg ggtggaccct 960
gatcagttca ctgattttgt ggtatgaatg tttctttaac attgacttgt aaggaaagta 1020
aaatagtgga tatatgtgtg cacacatgtg tatgcgccag tagatggtat ctttaacatt 1080
catatatgct ttttctctgt ctgtattgtt gtcatgcaga ttccaaactg gtttggagga 1140
gagtccactg gggctacaaa aggcaaccca ttcacgttac cagatgccta tatggtatct 1200
cagcataaaa atgtatgtgt ggtattaatt gatttgtata ttaattaacg attggatttc 1260
caaatctttt ggtgatcaaa ttttcaaaaa actttatttt aagcgaaata tgttttaact 1320
acaatgaaat tgtatcttct tct 1343
<210>29
<211>1062
<212>DNA
<213>大豆
<400>29
aacttgtgat tcttaatagc cttctcacgc tttttgttgt caaaggttaa ttgatgcagc 60
tttccatata gcagataagc actatataat tacgtatttc aaactacaca ttagtaatta 120
tgtcaggact tttgattatt tctgttggtc aaatttagaa tatggtacta agttaatcta 180
tagtaaatta aactaaccct ttttgagatt agatataact ctctactttt ttttaatatt 240
acattgacat ccttatacag ttatatatat atatatatat atatttaaaa taattaagga 300
agatttatat gtataaaggt gtcaatgtaa actatatatt tttaaatata acaaaaaaga 360
atagtgtagg ggtatataat aagaaaaagg taacgggtaa atttgatgaa atttcaaggg 420
gttaatataa tttaatggca ttcaactggg aaagcaatga gggatgatat tgattggtgc 480
gttgttggct tctaatgtgt ccaaagatgt gttacggaaa attgagcacc aaaagtaccc 540
atagtggttt agagaagtta ctgaaaatga aagcatgtgg tccactctgt ttgatcgatc 600
tccatttctt taaagaattg aatcaaactc tattattaac atactttctg gttccagaat 660
gaatagatat aactagactt gttttatctg acaacaaata ttattccatt tgataaggac 720
gaaacttatg ttcaaattct actttgttag ttaatgtaag aattttattg atagacgatt 780
tgaatgtatt tgagtacgaa ttttttgaat tgagactcaa attaaaatat gtctaacctg 840
atcagtgaat tattgagatc taatttacct atatattttt ttataaaaaa agaattattt 900
tatctaaacc ttttgaaaaa ttaactactt atacatactt tttcaacaac tactcctacc 960
ttagtattct gacctagggc ggctcaattt tccttttttt atttatcagt atgacatatt 1020
aacaaactcg gctgcaggac atgcaaggct ggcggtaaag ga 1062
<210>30
<211>1095
<212>DNA
<213>大豆
<400>30
aaaagttagt agaatttcgc cttaggtggt ttgggaatgc atgaagaaga cctaaagaag 60
cctcgataag aagagcagat catatggagg gcaatctatt tccttgtatc tctgtattgt 120
atagagaggt gcagtattca actcactctc tcaagttagt agttggcata ctgtgtcagt 180
ttgtaaagtt agttattgac agttgtcata actaactaac tgtttcctaa ctatctaact 240
tcataactct ataaatagag tgttgtaact caggattcat taacctccat aatattttct 300
cattccattt atcttctttc ttttctcctt tttctatgat ctaaacagag ttctaatgtg 360
atctattagt tttctattat ggtatctaga gcttggtgag atcttcaatg gctgcgaaca 420
gcaccacatt cctttccgct tcttcttttt cccaattcca tatcacataa acttgatgat 480
tcaagctttc ttctatgtcg tcaacaattt gagcctgcta tcaaaccaca caaacttaac 540
gattcgttgc taatcctcag attccacttc gatttctctc tgaagaagat caagaagttg 600
gacgtgaaaa tccagcttac gaagcatggg aaaagcaaga tcaggtgtta ttagcctgac 660
acaaatcaag gcacttgctg atgctcttgc ttcagtagga agccctataa tgattcaaga 720
gcacattgat tcaattgttg aaggtctttc tccagattat cacccgataa tcgagataat 780
ttagagtaag tttgaaaccg ttccaatcac gcaagttaaa gcacttcttc tagctcatga 840
gtcttgtctg aataacttca acgattaatt acactcatgc acaacacaga gcgaattcgc 900
attcctaaaa ttacactttg ccaaaaaagt caagttctca atcggatcct gaaagttttt 960
tctggttttc gcggtggttc tgcgtgcggt agctataata ggggtggcag cagcggcggt 1020
agtggtggcc attgtcacac tggtgcaggt caatttgcct atttccaatg ccaagtctgc 1080
tttaaatttg gtcat 1095
<210>31
<211>508
<212>DNA
<213>大豆
<400>31
ttgctcaaca tacaaaacct accacgcatt gttaatttgc aacttaatct ttcgttattc 60
tatcttgtag gcccatccat gtttgtccaa gaatgcctcg accgcttcct taatggccaa 120
gtttggcact aactgtgatg gatcaagggg ttcccgtgtg attgggtcga atttacccac 180
ctgtttaaga agttgaatta ttgcacactg acttcattga agtggaaagc attcaacaga 240
gaaggtgaaa catgcaaatc aggttacctt ctgaagatgc tcaagaatca ctgctctctc 300
atatgtaagt ccacttggag tgattacagg atcatggaaa atgtcgagtg taattctaca 360
gcacaaataa tctggcacct gtagactcaa caagacaatt tgtgtaatta ataagatggt 420
aatttccagg gcaccactga aaaaagattt gatgaatata gaattaacaa aacaagccat 480
gtacacacct cagtaggtgt gtcagctt 508
<210>32
<211>580
<212>DNA
<213>大豆
<400>32
gcgtgcctgc aggtaaaaat ttatgctatt aacgaagaag atgttaatgg caggtacgtt 60
tgttattcag acatatcatg caaaatataa cttgcttagc agcttcacag aacccaatag 120
ggcatgaata atatttccgt ttaactggtt accttaccag cagacgcaaa aaacctcatg 180
ttagaccaca accaacacat gtggccagaa tcaataagca tagtaatttt actaacagag 240
aaaattttta accatgttca gtgtggcaaa tatatgctta tggggtacag taaataatga 300
taactatgct accaaagttt catgcattgg gggaggataa atgaagggaa ttgtttgaga 360
ttattttaag gagatacaaa taagagatct ctagttaaca taacaaaatc cttcaattaa 420
tgagcattta ctttttttga gctctccact tcggagtatt ttgtaagcta aattacttta 480
tacactttct ggtgcttgtc aaaattgaat tttaacattt attagaagag cagaaattta 540
taaaaacatg tcatatttgt ttttttatta aatcctttat 580
<210>33
<211>815
<212>DNA
<213>玉米(zea mays)
<400>33
agccccctga aacattgagc tattaaaaat tagaaaacga cacaccttca gatactttct 60
gattctaaat ctaaatatga aacctcttgg tccttacttg caagtaaaag ctacatatta 120
tacaagtaat ttgatgacaa tgagctatgc agtcagcatg gaaatgtcaa aacctatctg 180
tgagaaaata tcaggagacc tagcattagg attcttttgt tttatttttc tactgattga 240
attcatgcat gacttaccta gtgcaatcta gatgaaaagg tctaacattt tccccatctt 300
aattaagcct gctacaattc acaactgggg agatagaaac tcaaactatg gcagaggcat 360
aagtgtgtat tagagtcaat ccatctacaa tgaaaagtat gctatattca taggtattgg 420
catttcaatt aatcagacaa acagtagtta ctgcacgata aaacaactga acatcaactt 480
tctagcattt tgctgagata ggacccccgc agggtacata ttacaagaca ggatatgaag 540
gatggagtag catagcatta ttctagcata attatattaa cataacatga ttaggtcagg 600
tcatactcat ccggaagttg tgtttcgtca ccatagtaac tttggcagca aaacaaacta 660
gacagaacta agtagtgaaa aagaagcaca gatcgcagag agcagttcac atattttact 720
actaccaagt agcaagaacc atgtttcatc accaccctac ctttggtagc aaaacaatct 780
acatagaact aattattagt ataaaagaag ttgaa 815
<210>34
<211>763
<212>DNA
<213>玉米
<400>34
tgtaagttat ctacttattt gttccctatt ttcatttatt tatttaaagt tgagatttat 60
ccaaagtatt gttagtgtaa tgttttttct tctgccacat taggtttttg tcaatgcacg 120
gcttcatgtc tcaggagggg ctcttggggg tggtcgcatg gtagaagact cctcaagcgt 180
tgcaggttcc atattttaaa ctttctttga tgatcacatt tttgtagtat tcttttttta 240
cgtaaaacaa ttctgtggta ttcaatagca aattatacac ttcttacaag tcaacatata 300
gatttcacta tctcagtttc tttggaggtt actagcagaa aagtaattta gaaatgatta 360
atatatttta ctgaggttct caacgttgtg tttttcggtt gctggtaaat tgctcatttg 420
ctgctatagt tgattagtaa atatagcagt atttatgtca ttactggtta cttgtaatgc 480
aaaccttttt cattgaagta catgttctgt aaaatactag aacatggtca gtactttcag 540
cattggtcac taacttttat gttttatgcg agtaataata tttctatttc catgtttatc 600
tacttagttt ccatgccatg ccgccttttc agtattggac actgctgctg gagtttggtg 660
tgataccaaa tcagtagtta caactccaag gacaggaagg tatagtgcag atgcagcagg 720
tggtgatgct tctgtagagc ttacacggcg gtgcaggcac gca 763
<210>35
<211>750
<212>DNA
<213>玉米
<400>35
tgaggcaatc agtgctactt tagctgctgt aaaggctagg caagttaacg gtgagatgga 60
gcattcacct gacagggaac aatctccaga tgctgcacca agtgccaagc aaaattcaag 120
ccttataaaa ccagatcctg ctcttatgaa caattcaaca ccaccacctg gggttcggtt 180
gcaccataga gcagtgagtt gaaaaaatag ttcattttgc tgcttgttgt ttaaatttag 240
ttattctatt cttatttaga cattcagtct gtttaactta gaagtcatca catttacatg 300
aaaaatgctc ttatttgttt tgatgccagt tacatatttt ggccttgtag gttgtggtag 360
cagcagaaac tggaggtgcc ttaggtggca tggttagaca gctctcgatt gaccagtttg 420
agaatgaagg tagaagggtc atttatggca cccctgagaa tgcaactgcg gcaaggaaat 480
tgctggatcg acaaatgtct attaatagcg tgcccaaaaa ggtaatctac atttttctac 540
tattgtaaga ttactgacaa aagcaacaca tgctagaaaa ctgaaagagt tattatcata 600
atggcttctg ctaaaaaaac aagcacttca tatgatgaca ttttctctaa gatgtagatt 660
tctatattga tttgttataa ttataatctg tggcccaata attcaggtaa ttgcttctct 720
gctgaaacct cgtggttgga gccccctgtg 750
<210>36
<211>607
<212>DNA
<213>玉米
<400>36
cagtcactac tgtgcttttg actggaactt gtgtcgtctt atggacatca gagaagaatg 60
atggtagcag gccatctcgt gccatggcca tcaatattct tggctgaagg aagtatcaaa 120
gtggaagtat gaataccaat agcagcataa actgaagttg tgcaatgcat atgcttgttg 180
cgagaacaga taaagcagaa aactgatgat atatatccag attatatgcc agtattcttc 240
agatgttact cattttaaaa ccatgcccac ttggctgatg actcatattt tccatcaatt 300
tgaatcacag aagaaatttg atgatacatt ggttaagata tgcttatacc tgtggcaata 360
tagaccccat caaggttgag cagagagcaa gaacagcacc agttgttaca agatacctaa 420
aacaagataa gcacatgaaa acaatctcag tcagacgcac accagatcat ccatgaaaaa 480
aatgagaaga agtccttaca ttgcccagtg catcccatgt ttggcaaagg cagatgaaat 540
aggggtgtct gggtccatag caaagtatgg taccagacca acaataacaa ctgaaaccaa 600
catgtac 607
<210>37
<211>607
<212>DNA
<213>玉米
<400>37
cagtcactac tgtgcttttg actggaactt gtgtcgtctt atggacatca gagaagaatg 60
atggtagcag gccatctcgt gccatggcca tcaatattct tggctgaagg aagtatcaaa 120
gtggaagtat gaataccaat agcagcataa actgaagttg tgcaatgcat atgcttgttg 180
cgagaacaga taaagcagaa aactgatgat atatatccag attatatgcc agtattcttc 240
agatgttact cattttaaaa ccatgcccac ttggctgatg actcatattt tccatcaatt 300
tgaatcacag aagaaatttg atgatacatt ggttaagata tgcttatacc tgtggcaata 360
tagaccccat caaggttgag cagagagcaa gaacagcacc agttgttaca agatacctaa 420
aacaagataa gcacatgaaa acaatctcag tcagacgcac accagatcat ccatgaaaaa 480
aatgagaaga agtccttaca ttgcccagtg catcccatgt ttggcaaagg cagatgaaat 540
aggggtgtct gggtccatag caaagtatgg taccagacca acaataacaa ctgaaaccaa 600
catgtac 607
<210>38
<211>1025
<212>DNA
<213>玉米
<400>38
ctctagagga tccccctggt ggttgagagg tactaccagc aagtgacgat gtactgaggc 60
cagtagattg ggctggggag ctgaacccaa aaaggttgtt tccagagctc ggaaatgtaa 120
atgaaggggt tgaagcagac tgtaaacctg gtgcagtgga agtactagat ggtgcagcca 180
cagatattcc agcagcatca cttgaaggtg caactgcggt gaaagtaggc gtagatgtgg 240
cgggggaact tgaaaagctt gcaatacttg atgacacagg tgaaataaaa agttccgatg 300
atgccttgct gcttgcatct ggtgcaacag atttcacttc agcttttgta ccccctatac 360
caaatgataa ggtgctagtg ccatcagatt tagctgatgt taccaatgac aatccaaccg 420
gagcactgga agaaaaacta aatcccgtaa atgcaggaga cgatgaaata gctggactgc 480
tactggatac ggcaaatatg gaagtggaaa caggggcttc atggattcag tttgatgagc 540
ccactcttgt cctcgacctt gattctgaca aattggctgc attctctgct gcatacgcag 600
aacttgaatc tgtactttct ggattgaatg tgcttgttga gacttacttt gctgatgttc 660
ctgctgagtc ctacaagtat gttatttatg ttgctagctc acccttttct cagccattgc 720
tcctcctaga cccttttgtg atgacatcat acttgctgtt cttttgaatg caggacccta 780
acatctctga gcagtgtgac tgcttatggt tttgatcttg tccgtggaac ccaaactctt 840
gggcttgtca cgagtgctgg tttccctgct ggaaagtacc tctttgctgg tgttgtggat 900
ggacgcaaca tctgggctga tgatcttgct acatctctca gcactctcca gtctcttgag 960
gctgttgttg ggaagggtaa tcatgcttgc acttattgtc tgccataaaa ttggatttag 1020
ttaca 1025
<210>39
<211>450
<212>DNA
<213>玉米
<400>39
tgaaacgata gctttattta tatcactatt ggtacagtta gatagaaaag tttcaggcct 60
caatcctaag taaccgaccc cttacatatt tcgaacttct atttacaggc ctaggcaaca 120
acaagctacc tatagtgcac cggcagagcc catgctcgcc gttgcacggg ctgccgtccc 180
taacggcggc agatgtcttg cgtcgtgaca cctcccgcat ccggcgccgc ttcgcctcac 240
aatcgtccgt ggtggcttct tttgcgtcgg ccccggcccc ggcgccggca gccaccataa 300
tccccctaga cggctcaccg gacgcgggcg cgctggacta caccgtgaac gtcggctatg 360
gcacgccgga gcagcagttc ccgatgttcc tggacaccat cttcggcgtg tccctggtct 420
tgtgcaagcc gtgcgcccca ggttccagca 450
<210>40
<211>373
<212>DNA
<213>玉米
<400>40
tcacaaattc atctttaaat tggccctaca tatgatataa ctcacactga gtaactgttg 60
atatcatatt ctaaatgact aaaagatttc agttattagc atattatgat atcacacacc 120
tttccaacaa cctcaaacgt ccattgtttc aaccggaagg ccactgcgtt tagatgatta 180
tttggcatgg aggcccagtg tgtatcacca tttaaactct gaaaaggtta gactttccct 240
gatgaaacct tctaattaag tgggtaagca aagcactatt cagtaattgt atcacctcct 300
gttctagcag gaatctccaa aatctcttca ccaggccaaa ctccgtgggg aacgtgttac 360
tcactttgtg ctt 373
<210>41
<211>1106
<212>DNA
<213>玉米
<400>41
gcggctccac ggccaggcac aacaagcccg gtgtctccga ccaccgagca agggtcgctc 60
ccgttctccc gcgtcccagc gcaccagccc ccgggagtcg gcacgtccgc gccgcggccc 120
aggatctcct cgtctatcct gtccagcacc gggtcgtcct cgtggaactt gcgggcgaac 180
ggcgcgtcgc tggcgaccat gcggtccagg tcctccgccg tcaagtagtg cgggtgctgc 240
ttcggagggt tgtcccacga gatgtagtgc aggtcgtggt tcaccgtcgt gttcttgaac 300
tcctccgcgt tgcacacaac ggtgtggaag tatccttccg gcgacgagat gaagtttgag 360
tagtacatga gcactgtgcg aggtaggttg tcccagcccc atatgcagta ttccacaaag 420
ggcctggaca gtgccatcca ggcagaacct gcgatgtcat aggctcatca ggtgcttcta 480
tatctgaatc agtaactgac atatatagat gcctggttat ctatatgagc tgtctggacc 540
tagagtagtg tttatatcta agctgtagtg tctgtttaag aaattggaat caattaattt 600
cctgcctaca cagagaagtg caaacctcct ttggggggag atggctaagc atggataatg 660
gatgtagaac ctgaacacca tatttatatt aatgaaaaaa cctgattttt gtgggaaaag 720
ttcattgaca cggtttcatt aatatataaa ttagtaactg acacataaaa caaggatagc 780
ctttcaaaaa ttaatggcag ctaacataaa tggatcaggg gagtaatcga gccaggaatg 840
ccttggttgg tttccagaac aggaggtcat ttttacatgc aaataaatgt gttgccatac 900
aaatgttctc tactacaggc aacaaacaaa tagaatcaca aatgtttgtt ccagcaataa 960
gatcaaacaa aataagatta tacagatgga aaggtaattt tttttaccaa ggagaatttc 1020
aggcagcgat atactacact gtacaaaaaa aaaagttaaa aaaataccac agctgtatgc 1080
actattttga agaagaccta aggtaa 1106
<210>42
<211>958
<212>DNA
<213>玉米
<400>42
catcttgcat gcctgcagtt tctttggtag acataggtgg gcatgaaggt ggttagttct 60
ttaagattta agatgctaat ctttatgttg agttatactg tgattaaaat gaagataaac 120
atatagtagt gtggtctgtg gtgtcaggat ttgttttcat aaaaattctt tctgcatgct 180
taggtctgtt tgttaaggta tatgctagtg taaaagatgg tgctctcaga catatccatg 240
attcaaaatt aaattagtgg ttgtgacgaa gaattttgag ctgggaaggc agtgtccaca 300
ggcaattgct tagagccctg ccctgctgtg tatccttaaa attgacatga atttaggacc 360
cttgtgattt atacatctat gccaaattgc caagtgctac ttttctctat gtggaaggag 420
atacatgcat ggaaagtttt gcgcgagcct tgagtgtatc acagactatc acagctgaga 480
gggatatctg aaggatatat catgattcat gattaagtta atgtgaagat ctgactaaaa 540
ttgtgcttgc agtaaacaaa acagttctgg ttgggaacca cagatctctt aggaacattg 600
tattctgatc tgactaaaat tgtgcttgaa atgaacaaaa cagttattgg caacctcaga 660
tgcttagata tattaggaac aaatgtattc cgatctgact aaaaccgtgc ttgcagtgag 720
caaaacagtt cttgttggga acctcagatc tcttaggaac aaatgtgatc tgatctgact 780
aatattgtgc ttgtagtgat aaaaaagctc ttgttgggaa catcagatat cttaattttt 840
ttgaaaaacg caggagagct gcgcatattt atagataaga tagaagaaag ggtcttacaa 900
gagaggtaca ggttagggac acctgcaccc acacacacgc actatcaact gaaacaaa 958
<210>43
<211>683
<212>DNA
<213>玉米
<400>43
tggtatgctc tctgaacttt tgctctgtaa ctgtgaccct caataaaaaa aattcagtta 60
aaggaatagg tcccgttgac cgagctcttc gattctctct gaatgcagat agctacactg 120
gtagctgtat acgcggactg ggggttcact tcgatcgaag gcattggatg gggctgggct 180
ggtgtggtgt ggctctacaa cctcgtcttc tacttcccgc tcgacctcct caagttcctc 240
atccgatacg ctctgagtgg caaagcgtgg gatcttgtca ttgagcaaag ggtgatcaat 300
ataaactgct cgttttgtca tgcacagcaa agcacagcac agcacctgtt tgagtgaatt 360
ccatgcacgc gcggtcggtg tgtcgctaat cgccggggtt ttgcagattg cgtttacaag 420
gaagaaggac ttcgggaagg aggagagggc gctcaagtgg gcacacgcgc agaggacgct 480
ccacgggctg cagccaccgg atgccaagct gttccctgac agggtgaacg agctgaatca 540
gatggccgaa gaggccaaac ggagggccga gattgcaagg taagatgttg aagtccgtgg 600
agatggtatc gcttgagggg aaagaaaggg caccgatgtc agcgtttccc atgatctctc 660
catatgcttt gggatatcta taa 683
<210>44
<211>772
<212>DNA
<213>玉米
<400>44
cacccctacc aaatcgagca cagctcagaa gaggcagggg aggatcactc tcctaccaac 60
caaaagtcac ctcgacactg gacgtgatac aagagacaca aaacatgggc cagaagacac 120
catgcatcag acagttctga gaacttattc aacagcaaaa gtcatcttcg gatttgttgg 180
cttcctcgtt ccaccatcct ttttggcatc ttttcctgta gaacagacca cgaatccaac 240
caaaagaagc aagcataaat cacatcagta gggtcataaa agaccttgtt gttaaagcac 300
acatcattcc taaacttcca aatggcccag agaatggctc ctatgcctag aacaatcaaa 360
ttcctcgtgg ttttcttata aatgttcatc cagtcctcaa aaagagaatc caggttttga 420
ggacatctct tcacatccag ggcaacctga agaactctcc acacaaaagt ggccactggg 480
caattgagaa aaaggtgatt ggtggattcc aaaacaccac agaaacaaca atcagtcaac 540
ccaggccaat tccttttttc aggttttctt tggagataat ttttaatctt cagaacaagc 600
cacaggaaaa cttaaatttt tgaggcattc ttattttcca aagaaactta ggaactttag 660
tatgacgtca aatagatata cttggctaat aattttgaag aatttaatga ttattatttg 720
gtatcataaa tgctattatt tgattatata aaaattggtc aaacttatga tg 772
<210>45
<211>544
<212>DNA
<213>玉米
<400>45
atacaatgag atggttggca tgcaaagtat tgtgttgaaa ctgataaaac attatcttat 60
atctcaaagt tctcaattgt ttacaagaag gtaaagccct gtataatttt atggcagata 120
actgcaactc ccatagaaaa tccaatgccg gaggttccca catgagtagg gtctgggaag 180
agaaaaacca aggcaagcct tcccccgcag atgaggggag gctacaaatc tcatcaacag 240
acaaaactca tccatcggcg ttggtagcgc ccagatgcca catctgggtc tggcactgga 300
cgcagcatcc accatccatt tgtcctatcc tttgatccac agttcattta atttagtcca 360
caaggaataa catatctact tctaattaat ccaagtgaaa tgggactatc ttcgtgcatt 420
cctttacctc cacatctgcc tacctcttgc accatcgcta tttctctcgt gcggaattcc 480
aaggctgggt taaaaaaaac acggaacacc cctcacccct gtcggcctgt tcttccactg 540
acgc 544
<210>46
<211>452
<212>DNA
<213>玉米
<400>46
cttccatcca cgaactcccg ttctccattt ccagccatgg cggtgtcgat cgagctcacc 60
aaggagtacg gctacgtcgt gctggtgctg gtggcctatg tcttcctcaa cctttggatg 120
ggcttccagg tcggcaaggc ccgcagaaag taagctctcc gaaatctgaa tcgctcgtcg 180
ccattgttgt cttcgtttgt ctccgcccaa cttatttcat caacggacat aataatatga 240
tggtttccgc tgtgattctt ctttaggtac aaggtgttct accccaccat gtacgccatc 300
gagtcggaga acaaggacgc caagctcttc aactgcgtgc aggtgcgccc aagattctga 360
catcctctcc cctcccccgt gattaattaa ttgctcttgt gaggggttgg gactttggga 420
ggcatctaaa tttccgctgg ttcttgtggt tg 452
<210>47
<211>1064
<212>DNA
<213>玉米
<400>47
tagctttaga gcatgtggaa atttcagctt ctggacaggc tactaacttc cctacttgca 60
cgcaagcata aggtatggtt ttaatgaaca tgttacccaa gtttgtgttt ttttagtatt 120
tcttaactaa ctttagatca actgatatat gtttgtaggt tctaatattc tcacaatgga 180
caaaagtttt ggacattctt gagtattacc tagattcaaa aggccttggg gtttgcagaa 240
ttgatggtag tgttaatttg gaagagaggc ggcgacaggt aacatgctag cctgtgccaa 300
tatattactc ctttcattcc aaattataaa atattgactt ttctagatac attacttttg 360
ctatgtatct agatatacac tcatgtggga gcctccaaca ctggatctgc cctagataca 420
cactaagtct atatgcataa caaaaagtga tgtatctaga aaagccaaaa cgtcttgtaa 480
tttgggacgt gtactatttt tctaaagaac atctaaactc tgaggatttg cactgtagtt 540
agatatttgg gtacatgtat aaatttattt cacaaaaaaa atcttttgtt actgttttct 600
catgcagata gcagagttga atgatttgaa tagcagtctg aatgtcttta ttctgagcac 660
acgggctggc ggacttggta tcaaccttac ttctgctgat acatgtatcc tttatgacag 720
tgactgggta attctctgtc aagactacta tactcatcag aaaaatgttt acaagaaagc 780
cttttttttt gctggctact ttctttggct gctgattgac ttattatgct agttctaata 840
tggtgctcgt ttactcgaac ctccagaatc ctcagatgga tcagcaggcc atggatcgat 900
gccaccggat tggtcaaaca cgcccagtac atgtatatag gctggctacc tcatattctg 960
ttgaggtatg cttcagtgat ccgtgttttc agacttgtca ctttggctat tgtctcaggt 1020
ttactcagct tttcaccttt gcaggaacgg atcatcaaga aagc 1064
<210>48
<211>1588
<212>DNA
<213>玉米
<400>48
gggcgccggc gggtgagatc ggcaggggca tgccgccgtt ggccccggcg gcggtagaga 60
atcctgatga tgctgcagag tggtgcaacg gcacgtgagg ggagccctgc gcttgccatg 120
cccatgaggg gaatggctgg gcgcccagct caggccttcg ctccaccggc ctttcctgga 180
cctgcacgat atcgcagcag cacgagcatg agctttgttt gtcaggtttt gaccacagtg 240
aacgtttcca cctttaagtc gttcacagga aacagagcga acaaacaacc aagattggat 300
atcggcgagc atgattaatc tttcatgatt ctgttttaat taattgattg attttaacgt 360
atgtgcagca caatgaagac ttgatgctag ctctatttcc cgtaaaaatt cagagaatcc 420
tcaggactac agctgcagca cgtgatccct gatcagcgta ctaaaagata catcccatgt 480
tatgtcaaga aactgcgacc caacaaccgc gactgcggtg tttcaaaaga tggaaagtgg 540
cagaacggcc tggtcgaaac accaaccagt ccacacacca aacagtacct gccattatag 600
taaggagtac cattgccccc cacccccccc aaaaaaaaca gaaaaagaag tgctgtatag 660
tatttttcca aggagcaaga cgtttcataa gaaatttcta aacaagctaa cagtaacagg 720
ttggtaggga atcaatctac aagtaaatgt ctatctagct gctctcagtc aaaaaggtta 780
gacatgtcgc acatatatat tcggcttgtt tctttcccat tatttccttt ttggaacaca 840
ggtcatgttc agctaaccat cctggattct acgatagttt ggaataactg atggagtatg 900
tagtaacaga catttgcatg tgtaagtgta acaaagggat cttggcgata cctggtggtt 960
cggaaagaag ccatgacaag ctgacgagta caagcgctga tggtgagatg acactgctgg 1020
gttttggtga tacccgggcc aaggggatga tgagctcatt ggctacagca gcagcaggtg 1080
aaaggagcaa acactgtgag atcaataata gttgtaggac ctggaccacg aattaatatc 1140
gatctccccc aaccatatgc caatcaaata gtcctatgaa tctttgtagg ctaggagtta 1200
ctagtaggaa ctagtatcag aggtccaaga ttctacaaag acaattcaac cgacagttgc 1260
ttccatctta agattttaat tttttttcta gatacctctt taggcaccat aagtaaaaag 1320
ctatatactg gaaaattgaa cgggtggttt tacctgagaa gccatcgtat ttgaagattc 1380
aggggaatca catgttggct ggaggccagc taaggtattg tcccttttgg ggtcttgcac 1440
attaggctgt gaaatccgca aatccaaatc aatggcatca ccatcatcaa ttgctgacaa 1500
cgtagagaga accatgagat acacctcaga tactatatat attaaaaaaa agaacggatg 1560
acaaggccga gtgtaatacc ctcggttt 1588
<210>49
<211>784
<212>DNA
<213>玉米
<400>49
aacataattt catgtactgt tcgtacagag attatcttta gagagaatag taagtactac 60
cttcgttctt gaatatttat catctgctag tttaatttta aactaaaacg tgataaataa 120
aaaaaacgaa gagggtatct tctatcttgt aataccaacc acgtgaagac cttcactcca 180
cggttgtttt gcctttttag attatattaa aactttgccc atacaaaaca gtttcttagg 240
gaaatttcca gatttatctc actcgcaatt accaactggc catcgttatt ttttagaacg 300
atatcttgca atcttctaag ctgactctgt aaatcttcac gtagccatct cctaaaaatg 360
aggctctagt ttttatattt catggcttca actgaacaat cacggtcctc gtttttttta 420
aaaaaaatga gaaaagtgtg tctttaaagt aacctatagc cacaccatct agtatgccaa 480
aaaaatggtg gatttttcat tggccgacac cgtagacctg ctgcttaagt aaatatatct 540
ggtttggggt taattagaag tatgccactc caaatttaaa gagttataaa gtagtttatt 600
ttcatgtcgc tcaatgacat caaaatgaat tacctatcta attttataaa aacagggtga 660
caagtatgta attaatcttt tttttcttag ctaggtttag tgatcttagt ccatctaatc 720
aaatgatatt tccctcttcc aaaaaaaacg ctttctagca ctatccatct ttccaataga 780
tgtc 784
<210>50
<211>802
<212>DNA
<213>玉米
<400>50
tgctgcgtcg tcgtgttgtt acgatcgtcc tttttttttt gaaaatttat tgaaggcccg 60
tgcttcttct tctcttcttc ttttccttct ccactgtttt cctcgcatgc tacatgacag 120
tctctctctc tcgttaactc tgttggtgtt cttattattt gatccggatg caagtaatac 180
tatacaaaca ggaatcggga atgcacgctc ccaatttttg acgcctccat gcatgtagtg 240
gcggagttcc gtaaaattaa cgtccgagat gtactattct atcttttgat tttttttttc 300
gtgttttatt taaaaataaa cagcggatga taaatattta ataacggaga gtaacattta 360
aaacaatcca gttaaactat gacaagtagg gaaagtggat tagatattcg ggaacaatta 420
cttacaatag aggagaatca cgagcacatg gcagcagaca gtcaacactc aggacacaac 480
gttcgccgct caggtggccg ttgttcattg gccaaggagg cactctgcac tcgcctatga 540
ataacaaaaa aagataatca agttgagaaa gttatatagc ttggaagaag atgtaatgac 600
aggggtggat ttgggcggtg gcatggcgcc agcccccgcg cgcgacactg ctatagggtc 660
gcagggaggc tggaggcgga tggtggaggc cgatgccaga ctggcggtgg tggatgcccc 720
gagtcaagcc cgccccgacc gcctcggagg gttgctggtt tgttcctcat tgccgaattc 780
cccggaagcc cttaaggctt tt 802
<210>51
<211>793
<212>DNA
<213>玉米
<220>
<221>不确定
<222>(1)..(793)
<223>n=a,g,c,或t
<400>51
aaagtaagca aaactaagct gaaatctgca agaagcagta ttgagattac caaaccaaca 60
agatcctgca gttaagttca acagaaccaa gatcagcatt cagcacaaaa tgaactgatt 120
cataatcact ctgtggacag taacagtggc agagaaagac ctgatgccca actggtttcg 180
aattgtgcaa gatatgagaa aatgggtaaa acaaatctct cctgtctttt agtgtcctta 240
agctattact attgagaact ccaccagtga tccttttgcg cattagagca tccttaatcc 300
tggactgact tagttgagta tcagcgagca tgttctgtgt gctgcatagt ataataataa 360
gccatcataa ataataatga tgataaacga aatcaataac ataaaacaca taataactca 420
cgtgttctgt gtgctgcgta gtataataat aagccatcat aaataataat gatgataaac 480
gaaatcaata acataaaaca cataataact cacggtgtgt gttgctctat ttttactcta 540
gggcttccat caggttcggt aagtgcagct tctccttcaa cagcataaac aagaagacca 600
agttggcagt accctaatgc tgtagcttgg aaccaggcaa gatcaacacg gataccattc 660
tcagctaaag agggatttgc atgagattca agccagttga tgaccggaag aaacgttact 720
ttcagatttc caccacggac caaacgcagg gggatnctnt agagtcgaac ctgcaggnnc 780
agcaagtcat agg 793
<210>52
<211>748
<212>DNA
<213>玉米
<400>52
atcatttttc aaccaagtaa tggagatcgc tttttatttt tagtatgtcg catcgttggc 60
tagagctttg atctaccttc atggcaagca tgttatccat cgggacatta aaccagagaa 120
tcttttagtt ggagttcagg tacttgcatt gtgttctttt ttggtatccc tctggcaccg 180
ggtctgctct gcctcatctg gaagactgga actatatgct tgctttacat cgtcgtctgt 240
ttccttggaa cagggcgaga tcaaaattgc cgactttggc tggtctgtgc acaccttcaa 300
cagaagacgg actatgtgcg gaactctgga ttacctgcca cctgaaatgg gtactttgct 360
agccatttac ctccagttat gaactagttc aatgggtttt gagaattccc acagcagtac 420
ctagctgctt tccttggctc tgaaacctgt tgtggtttat ccagtggaga aggcagaaca 480
tgattaccat gttgatatat ggagccttgg tgttctgtgc tatgagttcc tttacggggt 540
cccacctttc gaagctaagg agcactcaga aacctacaga aggtaactcc acaactctgg 600
gatcttagta tgtcgtccct aacttgccga tatcttgccc tagattattt cctgtggctt 660
ttgacttttg agctatgctc tgataactgt gaggaaactt ttgaagttgc atatagtgct 720
agtgtagcaa agagagagca ctatattg 748
<210>53
<211>610
<212>DNA
<213>玉米
<400>53
atattcaaaa gggaaacgaa gatggcttgt ttattagttc agttgcctct agctcaaatc 60
tgtgggcttt aattatggat gctggcactg gctttacatc tcaagtatac gaactttcta 120
attactttct tcacaaggta agcaatcaat tctgttgact tcaaagatct gtaagggtcc 180
ttcccctttt ttcttcttaa tataatgata ttcagctctc ctgcttattt gagagaaaaa 240
aaccttcaaa gatatgtaag ggttcttcct ctttgtaata gggtctttac ccttcctttt 300
cttcttctta atataatgat acacaatttc tcctgcttat tcgagaaaaa aattgagaga 360
aaaaaccttc aaaaatttac tgttctttgt ttatgattgt acgccaacac ttcactgatt 420
atacatcctc aatgatgtct cattgtcatt cgcatgcttg tactgtgtta ggaatggata 480
atggaacagt gggagagaaa tttctatatc actgcactgg ctggggcgaa taatggaagc 540
tctttggtga ttatgtcaag aggtaattaa tgtaaatgtt tgagcttgat gcttagactg 600
caggcatgca 610
<210>54
<211>883
<212>DNA
<213>玉米
<400>54
atttgaatag gatacatatg aaaaaagaga aattaaaggc ctgtttggtt cactacctca 60
gttgccacaa tttgcctaac ttttctgcct gaggttagtt attcaattcg aacgactaac 120
cttaggcaaa gtgtggcaca tttagccaca aaccaaacag gccctaagtg tttgctcagc 180
caaacatcgt gtatcagctt gaaatccaaa atatgtttgg caaaacatag cacatttatc 240
aagaaatcat agaaggcaaa atgcaatatg ctaatggaaa aggctcacag gtgactacga 300
tatctctcaa caggatatac aatgcttgag atggagttcg ccatactcag aaatttgttt 360
gacatgtgtc attttataat tttattttag aagctaggat tcagacttca actggagtag 420
atcaagtcaa tacataaaca gtattctttc atactaagaa atatcacctt gtaagattcc 480
tcaagcctgt ccttgtattt ttcaaccaac agctctttac tcaatgccag atctctttcc 540
actactgcct ctgtctcaaa ctattaagag acaagagaac acattactct atctattcaa 600
aacaattcct gtaatcaagt gataatataa ctataaccaa ctaaacatca tgaaaaaatt 660
gcagtgcaat acctgaataa caatgtgagg atacaatgtt tgtcctttac ggattggtgg 720
atcaagagtg ataacaacaa atgtatgagg attgtttgac tgcaaaggtc aaccatgcaa 780
tgagtttagt tacatgtcac aatgtaatac tatgtacaat aataaagcac aagcctcaac 840
catatgatac caaggaacag aaaccttttg gcaaaaagga aag 883
<210>55
<211>24
<212>DNA
<213>大豆
<400>55
tgcttgcttt ggacctacac aaaa 24
<210>56
<211>24
<212>DNA
<213>大豆
<400>56
aaaagcccaa aaggaagagt ggag 24
<210>57
<211>24
<212>DNA
<213>大豆
<400>57
gcgatgacct tgtatggggt agac 24
<210>58
<211>24
<212>DNA
<213>大豆
<400>58
ccatgccctg attcattcat cata 24
<210>59
<211>1249
<212>DNA
<213>大豆
<400>59
cagactctag tgactaccac cttcactctc ctcaagcatt tcagcctctt ccccgctcag 60
actccttagc tttgggagcc aaattatccc ttacgttctc gacttcaacc atatgtgata 120
gctgcctatg ataccatggc tacttcccgt tagttcttta tctttccttt ccgctttatt 180
ccatgcctta ccgatcctct gaagtgtctt tgcattagct tcattgaaac ctcacgcgat 240
gaaaggtgtg atggtctcct ccgatggcgc acttctcata gggtaaccta attgtcttac 300
gaccaacata ggattataat taatacaacc cctcgtccct ataaaaggga catttggaaa 360
tccttcacat aagcataaca ctcctacccc tctttctttc cactgtggga accaactaat 420
ggacgctcct atcatgcctg ccaagagttc ttcccaattt gcctcgtcct ttcctgagca 480
catgcgatga ccttgtatgg ggtagacaga tctactttca tgattgaaga cgtgggatac 540
caaccacaca taaagagcag gcgcacaaca gaaaatcctc gtagtgctct tcttgcatct 600
taagtcaaat gtatcataca cttatgctaa aacaacaatg atcgggcttt ccttgctatg 660
gtgataagca agaaaagcat cgattgctac tagatccacc aactcgtcta cattcgaaaa 720
tagtactatc ccaaacacta gcagtgctaa tacgtcgatg aatgatgccc actctccttg 780
gctggccaga gtttccgcct tctcctccaa tcacttcctt ggtattcccc ctaccctatt 840
cctactttgc ttcactcagt ctaattctca tttcgagatc ttgacaactc ctgctattct 900
cgccatagaa ggatagtacc cagaaaaaag gtatggcttc cttcctccta tcgggcatcc 960
taagatccct tcgaactcct ctatggttgg tgctaactga aagtccccaa aagtgaagca 1020
tctgagtgat tggtcatagt attgggtgag agatgcgatg gcttcaacga acacttctat 1080
catcaccaga tcccaaatct tcccatatac cttgttgaag gactgacgtt gagctcgatc 1140
catccgatgc cccagttttc gcaagatgac tacttctaga ttcttgagtt cgacacgata 1200
gaaccttttc ttaaaagaca gtgcttgtct gaccccatct catcagact 1249
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Claims (37)
1. 一种繁殖转基因植物的方法,包括下列步骤:
(a)提供用于在一组种质的基因组的至少两个不同单倍型中鉴定至少一种农艺学性状值的数据库;
(b)用重组DNA转化亲本植物以产生至少两个转基因事件,其中将重组DNA插入到所述的亲本植物基因组中与至少两个不同单倍型连锁;
(c)使与不同单倍型连锁的事件与所述农艺学性状值数据库相参照;和
(d)选择植物用于育种,所述植物包括具有更高参照值的单倍型的转基因事件。
2. 权利要求1的方法,其中重组DNA选自编码选择标记的DNA、编码评分标记的DNA、DNA重组位点、编码提供农艺学增强的蛋白质的DNA以及用于基因抑制的DNA。
3. 权利要求1的方法,其中所述至少一种农艺学性状是产量或多特性指数。
4. 权利要求1的方法,其中选择用于育种的所述转基因事件具有与下述单倍型连锁的重组DNA,其中所述单倍型选自对于产量不产生负影响的、对于成熟期不产生正影响的、对于成熟期无影响的、当与同一染色体节段的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围的,当与全部基因组中的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围的单倍型。
5. 权利要求1的方法,其中通过标记辅助的选择法选择用于育种的植物的后代植物。
6. 权利要求1的方法,其中通过检测转基因的表达或者转基因农艺学性状的表达,选择用于育种的植物的后代植物。
7. 权利要求1的方法,其中所述的植物是农作物植物,其选自饲料作物、油料种子作物、谷类作物、水果作物、蔬菜作物、纤维作物、香料作物、坚果作物、草坪作物、糖类作物、饮料作物和森林作物。
8. 权利要求7的方法,其中油料作物选自大豆、油菜、油菜籽油菜、油椰子、向日葵、橄榄、玉米、棉籽、花生、亚麻子、红花和椰子。
9. 权利要求8的方法,其中所述的大豆在其基因组中具有至少一个遗传标记,其中该遗传标记通常与选自C8W6H5、C 18W3H8、C19W3H6、C16W8H43、C1W1H2、C1W2H1、C14W7H2和C6W4H1的单倍型连锁;所述单倍型进一步包括遗传连锁的转基因。
10. 权利要求9的方法,其中所述的遗传标记是选自SEQ ID NO:1-32的DNA标记。
11. 权利要求6的方法,进一步包括将后代植物与第三种大豆植物杂交以产生另外的后代植物的步骤。
12. 权利要求7的方法,其中所述谷类作物是玉米并且在其基因组中具有至少一个遗传标记,其中该遗传标记通常与选自C1W19H14、C1W30H4、C1W36H2和C8W4H5的单倍型遗传连锁;所述单倍型进一步包括连锁的转基因。
13. 权利要求12的方法,其中所述遗传标记是选自SEQ ID NO:33-54的DNA标记。
14. 权利要求1的方法,其中所述的重组DNA和单倍型在0到大约10cM内的遗传距离连接。
15. 权利要求1的方法,其中所述的重组DNA和单倍型在0到大约5cM内的遗传距离连接。
16. 权利要求4的方法,其中所述的单倍型等位基因与农艺学适合度有关或者在繁殖种群或者一组种质中以50%或更高的频率发生。
17. 权利要求2的方法,其中所述的农艺学增强选自除草剂耐性、抗病性、昆虫或害虫抗性、改变的脂肪酸、蛋白质或者碳水化合物代谢、增加谷粒产量、增加油量、改变的植物成熟期、提高胁迫耐性和改变的形态特征。
18. 权利要求17的方法,其中除草剂耐性选自草甘膦、草铵膦、磺酰脲、咪唑啉酮、溴苯腈、茅草枯、麦草畏、2,4-D、cyclohezanedione、原卟啉原氧化酶抑制剂和异噁氟草耐性。
19. 权利要求6的方法,其中后代植物含有选择用于育种的植物的单倍型的至少一部分,其中该部分选自至少10cM、至少5cM或至少1cM。
20. 权利要求19的方法,其中在与种质改良有关的活动中使用后代植物,其中该活动选自使用植物用于育种杂交,植物的进一步检验、植物通过自体受精的进化、植物或其部分用于诱变的用途,以及植物或其部分用于耕作的用途。
21. 一种将转基因插入到植物单倍型内的方法,包括:
(a)将包括至少第一重组位点的靶位点整合为与单倍型遗传连锁;和
(b)将包括至少第一重组位点的转基因表达盒引入到植物细胞内,其中所述表达盒中的该第一重组位点两侧是包括目的转基因的多核苷酸;和
(c)提供识别并实现表达盒在第一重组位点重组的重组酶,由此产生优选的T型基因组区,其中优选的T型基因组区具有经估算的T型值,其中优选以前不含有转基因的单倍型,或者对于以前含有转基因的单倍型更优选的单倍型,其中单倍型选自对于产量不产生负影响的、对于成熟期不产生正影响的、对于成熟期无影响的、当与同一染色体节段的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围的,当与全部基因组中的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围的单倍型。
22. 权利要求21的方法,其中重组位点选自FRT、突变的FRT、LOX、突变的LOX位点,和锌指核酸酶修饰的位点。
23. 权利要求21的方法,其中单倍型等位基因与农艺学适应度有关或者在繁殖种群或一组种质中以50%或更高的频率发生。
24. 权利要求21的方法,其中重组酶是cre或flp。
25. 一种对至少一种T型转基因事件进行作图的方法,包括:
(a)转化的植物或品系中的至少第一个转基因事件周围的侧翼序列中,鉴定作图种群的亲本品系之间的至少第一个多态性,其中转化的植物或品系可能与作图种群的亲本品系不同;和
(b)分析作图种群后代植物的多态性,
(c)进行连锁分析以确定多态性的图谱位置以及因此确定转基因事件的图谱位点;和
(d)将图谱位点与转化植物的单倍型联系起来。
26. 一种用于提高种质中一种或多种T型基因组区的累积的方法,包括:
(a)将转基因插入到第一种植物的基因组内;和
(b)测定基因组中转基因的图谱位置;和
(c)将图谱位置与单倍型联系起来,其中转基因和单倍型包括T型基因组区;和
(d)将第一种植物与第二种植物杂交,其中第二种植物含有至少一个T型基因组区或者与第一种植物T型基因组区不同的单倍型;
(e)通过检测第一种植物的转基因的表达,选择至少一种后代植物,其中后代植物在其基因组中包括第一种植物T型基因组区的至少一部分,以及第二种植物的至少一种T型基因组区或者单倍型;
(f)在与种质改良有关的活动中使用后代植物,其中活动选自使用植物用于育种杂交、植物的进一步检验、植物通过自体受精的进化、使用植物或其部分用于转化的用途、使用植物或其部分用于诱变的用途,以及使用植物或其部分用于耕作的用途。
27. 包括优选的T型基因组区的农作物植物,其中将T型基因组区的转基因进一步定义为赋予选自除草剂耐性、抗病性、昆虫或害虫耐性、改变的脂肪酸、蛋白质或碳水化合物代谢、增加谷粒产量、增加油量、改变的植物成熟期、提高胁迫耐性,以及改变的形态特征的性状;T型基因组区的单倍型选自对于产量不产生负影响的、对于成熟期不产生正影响的、对于成熟期无影响的、当与同一染色体节段的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围,当与全部基因组中的任何其它单倍型相比时,相对于农艺学性状或者多性状指数在最佳50%的范围的单倍型。
28. 权利要求27的方法,其中如果一种单倍型在繁殖种群或一组种质中以50%或更高频率存在,那么其具有很高的价值。
29. 权利要求27的农作物,其中优选的T型基因组区包括在0-约10cM的距离内遗传连锁的转基因和单倍型。
30. 权利要求27的农作物,其中优选的T型基因组区包括在0-约5cM的距离内遗传连锁的转基因和单倍型。
31. 权利要求27的农作物,其中所述农作物植物是转基因的除草剂耐性大豆植物并且其中转基因与鉴定为C8W6H5的单倍型遗传连锁。
32. 权利要求31的大豆植物,其中遗传标记选自SEQ ID NO:1、2、3和59。
33. 权利要求27的农作物植物,其中所述的农作物植物是转基因的昆虫耐性的大豆植物,其中转基因与鉴定为C6W4H1的单倍型遗传连锁。
34. 权利要求33的大豆植物,其中遗传标记选自SEQ ID NO:29-32。
35. 权利要求27的农作物植物,其中所述的农作物植物是转基因的昆虫耐性的玉米植物,其中转基因与鉴定为C1W36H2的单倍型遗传连锁。
36. 权利要求35的玉米植物,其中遗传标记选自SEQ ID NO:48-50。
37. 一种提高种质中一种或多种单倍型的累积的方法,包括:
(a)测定基因组中转基因的图谱位置;和
(b)将图谱位置与单倍型联系起来,其中转基因和单倍型包括T型基因组区;和
(c)将第一种植物与第二种植物杂交,其中第二种植物含有至少一个T型基因组区或者与第一种植物T型基因组区不同的单倍型;
(d)通过检测第一种植物的转基因的表达,选择至少一种后代植物,其中后代植物在其基因组中包括第一种植物T型基因组区的至少一部分,以及第二种植物的至少一种T型基因组区或者单倍型;
(e)在与种质改良有关的活动中使用后代植物,其中活动选自使用植物用于育种杂交、植物的进一步检验、植物通过自体受精的进化、使用植物或其部分用于转化的用途、使用植物或其部分用于诱变的用途,以及使用植物或其部分用于耕作的用途。
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US8273961B2 (en) * | 2009-07-02 | 2012-09-25 | Monsanto Technology Llc | Soybean variety A1015499 |
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US8263831B2 (en) * | 2009-07-02 | 2012-09-11 | Monsanto Technology Llc | Soybean variety A1016054 |
US8115069B2 (en) * | 2009-07-02 | 2012-02-14 | Monsanto Technology Llc | Soybean variety A1016124 |
US8207407B2 (en) * | 2009-07-02 | 2012-06-26 | Monsanto Technology Llc | Soybean variety A1016238 |
US8263829B2 (en) * | 2009-07-02 | 2012-09-11 | Monsanto Technology Llc | Soybean variety A1015993 |
US8115071B2 (en) * | 2009-07-02 | 2012-02-14 | Monsanto Technology Llc | Soybean variety A1016137 |
US8115070B2 (en) * | 2009-07-02 | 2012-02-14 | Monsanto Technology Llc | Soybean variety A1016053 |
US8163982B2 (en) * | 2009-07-02 | 2012-04-24 | Monsanto Technology Llc | Soybean variety A1016496 |
US8198516B2 (en) * | 2009-07-02 | 2012-06-12 | Monsanto Technology Llc | Soybean variety A1016523 |
US8193424B2 (en) * | 2009-07-09 | 2012-06-05 | Monsanto Technology Llc | Soybean variety A1016522 |
US8263832B2 (en) * | 2009-07-09 | 2012-09-11 | Monsanto Technology Llc | Soybean variety A1016467 |
US8319031B2 (en) * | 2009-07-09 | 2012-11-27 | Monsanto Technology Llc | Soybean variety A1016242 |
US8319033B2 (en) * | 2009-07-09 | 2012-11-27 | Monsanto Technology Llc | Soybean variety A1016136 |
US8198517B2 (en) * | 2009-07-09 | 2012-06-12 | Monsanto Technology Llc | Soybean variety A1016189 |
US8269071B2 (en) * | 2009-07-09 | 2012-09-18 | Monsanto Technology Llc | Soybean variety A1015669 |
US8319032B2 (en) * | 2009-07-09 | 2012-11-27 | Monsanto Technology Llc | Soybean variety A1015901 |
US8330000B2 (en) * | 2009-07-09 | 2012-12-11 | Monsanto Technology Llc | Soybean variety A1015971 |
US8334433B2 (en) * | 2009-07-09 | 2012-12-18 | Monsanto Technology Llc | Soybean variety A1016109 |
US8269072B2 (en) * | 2009-07-15 | 2012-09-18 | Monsanto Technology Llc | Soybean variety A1016117 |
US8314294B2 (en) * | 2009-07-15 | 2012-11-20 | Monsanto Technology Llc | Soybean variety A1016502 |
US8319035B2 (en) * | 2009-07-15 | 2012-11-27 | Monsanto Technology Llc | Soybean variety A1016167 |
US8212121B2 (en) * | 2009-07-15 | 2012-07-03 | Monsanto Technology Llc | Soybean variety A1016115 |
US8319034B2 (en) * | 2009-07-15 | 2012-11-27 | Monsanto Technology Llc | Soybean variety A1016511 |
US8217230B2 (en) * | 2009-07-15 | 2012-07-10 | Monsanto Technology Llc | Soybean variety A1016122 |
US9155327B2 (en) * | 2009-07-15 | 2015-10-13 | Monsanto Technology Llc | Soybean variety A1016131 |
US8222490B2 (en) * | 2009-07-15 | 2012-07-17 | Monsanto Technology Llc | Soybean variety A1016495 |
US8212122B2 (en) * | 2009-07-16 | 2012-07-03 | Monsanto Technology Llc | Soybean variety A1016268 |
US8273963B2 (en) * | 2009-07-16 | 2012-09-25 | Monsanto Technology Llc | Soybean variety A1016240 |
US8273966B2 (en) * | 2009-07-16 | 2012-09-25 | Monsanto Technology Llc | Soybean variety A1016274 |
US8222491B2 (en) * | 2009-07-16 | 2012-07-17 | Monsanto Technology Llc | Soybean variety A1016190 |
US8273965B2 (en) * | 2009-07-16 | 2012-09-25 | Monsanto Technology Llc | Soybean variety A1015862 |
US8163983B2 (en) * | 2009-07-16 | 2012-04-24 | Monsanto Technology Llc | Soybean variety A1016008 |
US8334434B2 (en) * | 2009-07-16 | 2012-12-18 | Monsanto Technology Llc | Soybean variety A1016082 |
US8278511B2 (en) * | 2009-07-16 | 2012-10-02 | Monsanto Technology Llc | Soybean variety A1016184 |
US8115072B2 (en) * | 2009-07-16 | 2012-02-14 | Monsanto Technology Llc | Soybean variety A1015645 |
US8278512B2 (en) * | 2009-07-16 | 2012-10-02 | Monsanto Technology Llc | Soybean variety A1015631 |
US8115075B2 (en) * | 2009-07-17 | 2012-02-14 | Monsanto Technology Llc | Soybean variety A1016273 |
US8119869B2 (en) * | 2009-07-17 | 2012-02-21 | Monsanto Technology Llc | Soybean variety A1016276 |
US8158864B2 (en) * | 2009-07-17 | 2012-04-17 | Monsanto Technology Llc | Soybean variety A1015460 |
US8273964B2 (en) * | 2009-07-17 | 2012-09-25 | Monsanto Technology Llc | Soybean variety A1015646 |
US8334435B2 (en) * | 2009-07-17 | 2012-12-18 | Monsanto Technology Llc | Soybean variety A1016278 |
US8115076B2 (en) * | 2009-07-17 | 2012-02-14 | Monsanto Technology Llc | Soybean variety A1016279 |
US8278513B2 (en) * | 2009-07-17 | 2012-10-02 | Monsanto Technology Llc | Soybean variety A1016281 |
US8115074B2 (en) * | 2009-07-17 | 2012-02-14 | Monsanto Technology Llc | Soybean variety A1015657 |
US8207409B2 (en) * | 2009-07-17 | 2012-06-26 | Monsanto Technology Llc | Soybean variety A1016347 |
US8269073B2 (en) * | 2009-07-20 | 2012-09-18 | Monsanto Technology | Soybean variety A1015632 |
US8115073B2 (en) * | 2009-07-20 | 2012-02-14 | Monsanto Technology Llc | Soybean variety A1015662 |
US8207410B2 (en) * | 2009-07-21 | 2012-06-26 | Monsanto Technology Llc | Soybean variety A1016317 |
US8203040B2 (en) * | 2009-07-21 | 2012-06-19 | Monsanto Technology Llc | Soybean variety A1016332 |
US8158865B2 (en) * | 2009-07-21 | 2012-04-17 | Monsanto Technology Llc | Soybean variety A1015484 |
US8278514B2 (en) * | 2009-07-21 | 2012-10-02 | Monsanto Technology Llc | Soybean variety A1016515 |
US8203041B2 (en) * | 2009-07-21 | 2012-06-19 | Monsanto Technology Llc | Soybean variety A1016334 |
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US8420895B2 (en) * | 2009-09-30 | 2013-04-16 | M.S. Technologies Llc | Soybean cultivar S080137 |
JP5735517B2 (ja) | 2009-09-30 | 2015-06-17 | ビーエーエスエフ ソシエタス・ヨーロピアBasf Se | アニオン性農薬の低揮発性アミン塩 |
US8420896B2 (en) * | 2009-10-23 | 2013-04-16 | M.S. Technologies Llc | Soybean cultivar S080120 |
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PE20121693A1 (es) | 2010-01-22 | 2012-12-01 | Bayer Ip Gmbh | Combinacion de espiromesifeno y abamectina como insecticidas |
CA2792123C (en) | 2010-03-12 | 2018-12-11 | Junhua Zhang | Plant health compositions comprising a water-soluble pesticide and a water-insoluble agrochemical |
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US8436226B2 (en) | 2010-04-09 | 2013-05-07 | Monsanto Technology Llc | Soybean cultivar 1000679 |
US8455721B2 (en) | 2010-04-16 | 2013-06-04 | Monsanto Technology Llc | Soybean cultivar 99401805 |
US8440883B2 (en) | 2010-04-16 | 2013-05-14 | Monsanto Technology Llc | Soybean cultivar S100004 |
US8450565B2 (en) | 2010-04-16 | 2013-05-28 | Monsanto Technology Llc | Soybean cultivar 98179010 |
US8426690B2 (en) * | 2010-04-23 | 2013-04-23 | Monsanto Technology Llc | Soybean cultivar 98164635 |
MX347199B (es) * | 2010-06-04 | 2017-04-18 | Monsanto Technology Llc | Evento de brassica transgénica mon 88302 y métodos para su uso. |
US8283530B2 (en) | 2010-07-08 | 2012-10-09 | Monsanto Technology Llc | Soybean variety A1023171 |
US8309810B2 (en) | 2010-07-08 | 2012-11-13 | Monsanto Technology Llc | Soybean variety A1019993 |
US8283531B2 (en) | 2010-07-08 | 2012-10-09 | Monsanto Technology Llc | Soybean variety A1023484 |
US8389813B2 (en) | 2010-07-08 | 2013-03-05 | Monsanto Technology Llc | Soybean variety A1022840 |
US8383893B2 (en) | 2010-07-08 | 2013-02-26 | Monsanto Technology Llc | Soybean variety A1020078 |
US8383892B2 (en) | 2010-07-08 | 2013-02-26 | Monsanto Technology Llc | Soybean variety A1015895 |
US8324465B2 (en) | 2010-07-08 | 2012-12-04 | Monsanto Technology Llc | Soybean variety A1023169 |
US8309811B2 (en) | 2010-07-08 | 2012-11-13 | Monsanto Technology Llc | Soybean variety A1022841 |
US8426691B2 (en) | 2010-07-08 | 2013-04-23 | Monsanto Technology Llc | Soybean variety A1016011 |
US8383894B2 (en) | 2010-07-08 | 2013-02-26 | Monsanto Technology Llc | Soybean variety A1023199 |
US8293997B2 (en) | 2010-07-15 | 2012-10-23 | Monsanto Technology Llc | Soybean variety A1024318 |
US8618362B2 (en) | 2010-07-15 | 2013-12-31 | Monsanto Technology Llc | Soybean variety A1024337 |
US8299333B2 (en) | 2010-07-15 | 2012-10-30 | Monsanto Technology Llc | Soybean variety A1024289 |
US8304624B2 (en) | 2010-07-15 | 2012-11-06 | Monsanto Technology Llc | Soybean variety A1024320 |
US8507762B2 (en) | 2010-07-15 | 2013-08-13 | Monsanto Technology Llc | Soybean variety A1024315 |
US8399743B2 (en) | 2010-07-15 | 2013-03-19 | Monsanto Technology Llc | Soybean variety A1024305 |
US8395025B2 (en) | 2010-07-15 | 2013-03-12 | Monsanto Technology Llc | Soybean variety A1024249 |
US8395026B2 (en) | 2010-07-15 | 2013-03-12 | Monsanto Technology Llc | Soybean variety A1024300 |
US8304623B2 (en) | 2010-07-15 | 2012-11-06 | Monsanto Technology Llc | Soybean variety A1024238 |
US8404938B2 (en) | 2010-07-15 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1022842 |
US8420897B2 (en) | 2010-07-15 | 2013-04-16 | Monsanto Technology Llc | Soybean variety A1024301 |
US8293998B2 (en) | 2010-07-15 | 2012-10-23 | Monsanto Technology Llc | Soybean variety A1024338 |
US8319038B2 (en) | 2010-07-15 | 2012-11-27 | Monsanto Technology Llc | Soybean variety A1024319 |
US8314302B2 (en) | 2010-07-16 | 2012-11-20 | Monsanto Technology Llc | Soybean variety A1024184 |
US8293999B2 (en) | 2010-07-16 | 2012-10-23 | Monsanto Technology Llc | Soybean variety A1024231 |
US8309814B2 (en) | 2010-07-16 | 2012-11-13 | Monsanto Technology Llc | Soybean variety A1024213 |
US8324466B2 (en) | 2010-07-16 | 2012-12-04 | Monsanto Technology Llc | Soybean variety A1024181 |
US8309813B2 (en) | 2010-07-16 | 2012-11-13 | Monsanto Technology Llc | Soybean variety A1024212 |
US8309812B2 (en) | 2010-07-16 | 2012-11-13 | Monsanto Technology Llc | Soybean variety A1024109 |
US8319040B2 (en) | 2010-07-16 | 2012-11-27 | Monsanto Technology Llc | Soybean variety A1024255 |
US8319039B2 (en) | 2010-07-16 | 2012-11-27 | Monsanto Technology Llc | Soybean variety A1024340 |
US8367897B2 (en) | 2010-07-16 | 2013-02-05 | Monsanto Technology Llc | Soybean variety A1024230 |
US8324468B2 (en) | 2010-07-16 | 2012-12-04 | Monsanto Technology Llc | Soybean variety A1024223 |
US8324467B2 (en) | 2010-07-16 | 2012-12-04 | Monsanto Technology Llc | Soybean variety A1024216 |
US8314303B2 (en) | 2010-07-16 | 2012-11-20 | Monsanto Technology Llc | Soybean variety A1024197 |
US8383895B2 (en) | 2010-07-16 | 2013-02-26 | Monsanto Technology Llc | Soybean variety A1024198 |
US8420898B2 (en) | 2010-07-16 | 2013-04-16 | Monsanto Technology Llc | Soybean variety A1024210 |
US8404939B2 (en) | 2010-07-18 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023485 |
US8450566B2 (en) | 2010-07-19 | 2013-05-28 | Monsanto Technology Llc | Soybean variety A1023611 |
US8399744B2 (en) | 2010-07-19 | 2013-03-19 | Monsanto Technology Llc | Soybean variety A1023486 |
US8455723B2 (en) | 2010-07-19 | 2013-06-04 | Monsanto Technology Llc | Soybean variety A1023586 |
US8502028B2 (en) | 2010-07-19 | 2013-08-06 | Monsanto Technology Llc | Soybean variety A1023487 |
US8404940B2 (en) | 2010-07-19 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023596 |
US8399745B2 (en) | 2010-07-19 | 2013-03-19 | Monsanto Technology Llc | Soybean variety A1023603 |
US8507763B2 (en) | 2010-07-19 | 2013-08-13 | Monsanto Technology Llc | Soybean variety A1023510 |
US8450569B2 (en) | 2010-07-26 | 2013-05-28 | Monsanto Technology Llc | Soybean variety A1024227 |
US8455722B2 (en) | 2010-07-26 | 2013-06-04 | Monsanto Technology Llc | Soybean variety A1023585 |
US8404941B2 (en) | 2010-07-26 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023667 |
US8431776B2 (en) | 2010-07-26 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1023535 |
US8404942B2 (en) | 2010-07-26 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023673 |
US8378182B2 (en) | 2010-07-28 | 2013-02-19 | Monsanto Technology Llc | Soybean variety A1023748 |
US8383897B2 (en) | 2010-07-28 | 2013-02-26 | Monsanto Technology Llc | Soybean variety A1023763 |
US8404943B2 (en) | 2010-07-28 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023694 |
US8389815B2 (en) | 2010-07-28 | 2013-03-05 | Monsanto Technology Llc | Soybean variety A1023761 |
US8410339B2 (en) | 2010-07-28 | 2013-04-02 | Monsanto Technology Llc | Soybean variety A1023771 |
US8378181B2 (en) | 2010-07-28 | 2013-02-19 | Monsanto Technology Llc | Soybean variety A1023729 |
US8378183B2 (en) | 2010-07-28 | 2013-02-19 | Monsanto Technology Llc | Soybean variety A1023750 |
US8450570B2 (en) | 2010-07-28 | 2013-05-28 | Monsanto Technology Llc | Soybean variety A1023833 |
US8507764B2 (en) | 2010-07-28 | 2013-08-13 | Monsanto Technology Llc | Soybean variety A1023705 |
US8404946B2 (en) | 2010-07-29 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023840 |
US8404944B2 (en) | 2010-07-29 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023795 |
US8455726B2 (en) | 2010-07-29 | 2013-06-04 | Monsanto Technology Llc | Soybean variety A1023981 |
US8466349B2 (en) | 2010-07-29 | 2013-06-18 | Monsanto Technology Llc | Soybean variety A1023943 |
US8383898B2 (en) | 2010-07-29 | 2013-02-26 | Monsanto Technology Llc | Soybean variety A1023788 |
US8436228B2 (en) | 2010-07-29 | 2013-05-07 | Monsanto Technology Llc | Soybean variety A1023868 |
US8404945B2 (en) | 2010-07-29 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1023821 |
US8471108B2 (en) | 2010-07-29 | 2013-06-25 | Monsanto Technology Llc | Soybean variety A1023999 |
US8455724B2 (en) | 2010-07-29 | 2013-06-04 | Monsanto Technology Llc | Soybean variety A1023941 |
US8431787B2 (en) | 2010-07-29 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1023844 |
US8455725B2 (en) | 2010-07-29 | 2013-06-04 | Monsanto Technology Llc | Soybean variety A1023969 |
US8404947B2 (en) | 2010-07-29 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1024059 |
US8410340B2 (en) | 2010-07-29 | 2013-04-02 | Monsanto Technology Llc | Soybean variety A1023838 |
US8420900B2 (en) | 2010-07-29 | 2013-04-16 | Monsanto Technology Llc | Soybean variety A1024077 |
US8415533B2 (en) | 2010-07-30 | 2013-04-09 | Monsanto Technology Llc | Soybean variety A1024631 |
US8415530B2 (en) | 2010-07-30 | 2013-04-09 | Monsanto Technology Llc | Soybean variety A1024626 |
US8431779B2 (en) | 2010-07-30 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024638 |
US8420899B2 (en) | 2010-07-30 | 2013-04-16 | Monsanto Technology Llc | Soybean variety A1023692 |
US8420902B2 (en) | 2010-07-30 | 2013-04-16 | Monsanto Technology Llc | Soybean variety A1024640 |
US8450567B2 (en) | 2010-07-30 | 2013-05-28 | Monsanto Technology Llc | Soybean variety A1024104 |
US8415532B2 (en) | 2010-07-30 | 2013-04-09 | Monsanto Technology Llc | Soybean variety A1024630 |
US8404948B2 (en) | 2010-07-30 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1024092 |
US8420901B2 (en) | 2010-07-30 | 2013-04-16 | Monsanto Technology Llc | Soybean variety A1024627 |
US8436227B2 (en) | 2010-07-30 | 2013-05-07 | Monsanto Technology Llc | Soybean variety A1023511 |
US8431778B2 (en) | 2010-07-30 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024341 |
US8415531B2 (en) | 2010-07-30 | 2013-04-09 | Monsanto Technology Llc | Soybean variety A1024629 |
US8399746B2 (en) | 2010-07-30 | 2013-03-19 | Monsanto Technology Llc | Soybean variety A1024350 |
US8404949B2 (en) | 2010-07-30 | 2013-03-26 | Monsanto Technology Llc | Soybean variety A1024103 |
US8450568B2 (en) | 2010-08-01 | 2013-05-28 | Monsanto Technology Llc | Soybean variety A1025240 |
US8426692B2 (en) | 2010-08-01 | 2013-04-23 | Monsanto Technology Llc | Soybean variety A1024702 |
US8455728B2 (en) | 2010-08-01 | 2013-06-04 | Monsanto Technology Llc | Soybean variety A1025241 |
US8431780B2 (en) | 2010-08-01 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024650 |
US8431788B2 (en) | 2010-08-01 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024763 |
US8420903B2 (en) | 2010-08-01 | 2013-04-16 | Monsanto Technology Llc | Soybean variety A1024693 |
US8431781B2 (en) | 2010-08-01 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024666 |
US8455727B2 (en) | 2010-08-01 | 2013-06-04 | Monsanto Technology Llc | Soybean variety A1024755 |
US8445754B2 (en) | 2010-08-01 | 2013-05-21 | Monsanto Technology Llc | Soybean variety A1024705 |
US8431777B2 (en) | 2010-08-02 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1023849 |
US8395027B2 (en) | 2010-08-02 | 2013-03-12 | Monsanto Technology Llc | Soybean variety A1024001 |
US8426693B2 (en) | 2010-08-18 | 2013-04-23 | Monsanto Technology Llc | Soybean variety A1024720 |
US8492617B2 (en) | 2010-08-18 | 2013-07-23 | Monsanto Technology Llc | Soybean variety A1015881 |
US8431782B2 (en) | 2010-08-18 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024747 |
US8431785B2 (en) | 2010-08-18 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024750 |
US8431783B2 (en) | 2010-08-18 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024748 |
US8431784B2 (en) | 2010-08-18 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024749 |
US8455729B2 (en) | 2010-08-18 | 2013-06-04 | Monsanto Technology Llc | Soybean variety A1015644 |
US8431786B2 (en) | 2010-08-18 | 2013-04-30 | Monsanto Technology Llc | Soybean variety A1024751 |
CA2805770A1 (en) * | 2010-08-24 | 2012-03-01 | Basf Se | Agrochemical mixtures for increasing the health of a plant |
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WO2012072489A1 (de) | 2010-11-29 | 2012-06-07 | Bayer Cropscience Ag | Alpha-beta-ungesättigte imine |
WO2012072660A1 (en) | 2010-12-01 | 2012-06-07 | Bayer Cropscience Ag | Use of fluopyram for controlling nematodes in crops and for increasing yield |
EP2460407A1 (de) | 2010-12-01 | 2012-06-06 | Bayer CropScience AG | Wirkstoffkombinationen umfassend Pyridylethylbenzamide und weitere Wirkstoffe |
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CN117144056A (zh) * | 2023-10-31 | 2023-12-01 | 中国热带农业科学院三亚研究院 | 与番木瓜果实果糖积累相关的单倍型分子标记的应用 |
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