CN112852990A - 基于分子标记的高异黄酮大豆品种选育方法 - Google Patents
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Abstract
本发明公开了一种基于分子标记的高异黄酮大豆品种选育方法，旨在解决以传统方法选育高异黄酮大豆品种效率低、准确性不足的技术问题。本发明基于分子标记在早期选择同时携带有Satt249和Satt285基因的且异黄酮含量及产量满足育种要求的杂交后代，再经多轮繁殖直至获得生物性状稳定的高产品种。本发明的主要有益技术效果在于：性状选择准确性，筛选效率高，且操作简便，从众多的大豆种质资源中快速选择出有价值（高产并且高异黄酮含量高）的品种或育种材料；可以快速的将大豆高产表型、高异黄酮表型聚合在一起，选育出品质优、高产、高异黄酮含量的大豆新品种。
Description
技术领域
本发明涉及育种技术领域，具体涉及一种基于分子标记的高异黄酮大豆品种选育方法。
背景技术
大豆及其制品是高营养的植物性食品，是粮食作物中唯一接近全价蛋白的优质蛋白质作物。大豆除了提供优质植物蛋白、油脂和磷脂等营养成分外，还含有多种人体有益的生理活性物质，如大豆异黄酮、大豆皂苷、植酸、低聚糖、多肽等。其中大豆异黄酮(Soybean Isoflavone)是大豆种子中积累较多的一类次生代谢产物，它的组成和存在形式主要包括染料木素、黄豆苷元、大豆苷元、染料木苷和大豆苷等。
许多研究证实大豆异黄酮有弱雌激素活性、抗氧化活性、抗溶血活性和抗真菌活性的生物效能，能有效的预防和抑制白血病、多种癌症、妇女更年期综合症等多种疾病的发生，尤其是对乳腺癌和前列腺癌有良好的预防和治疗作用。
不同大豆品种中大豆异黄酮含量显著不同。鉴于大豆异黄酮对人体保健及疾病防治的重要性，及未来食品保健及医药卫生事业的广阔应用前景，培育高异黄酮含量品质性状的大豆品种成为当务之急。
由于大豆异黄酮数量性状受多个数量基因座位(QTL)和环境因子的共同作用，因而对它的遗传控制非常难。而育种工作者总是希望把多个大豆异黄酮基因导入到同一个优良的大豆异黄酮含量低的品种中，获得高产并且大豆异黄酮含量高的专用品种。而通过传统的方法来选育高含量的大豆异黄酮品种，不仅周期长、效率低，而且会因连锁累赘而导入不良基因。
发明内容
本发明要解决的技术问题是提供一种基于分子标记的高异黄酮大豆品种选育方法，旨在解决以传统方法选育高异黄酮大豆品种效率低、准确性不足的技术问题。
长期以来人们认识到大豆异黄酮数量性状受多个数量基因座位(QTL)和环境因子的共同作用，因而对它的遗传控制非常难。而本发明人在长期的研究中发现，satt249-satt285标记区间与大豆异黄酮含量相关，并且异黄酮总含量最适遗传模型符合MX2-AI-A，即2对加性-上位性主基因×加性多基因混合遗传模型。两对主基因间加性效应分别为-2.531和-9.147；两对基因加性效应值均为负值，表明基因来源于父本；两对基因间加性效应之间的上位性互作效应为3.210，存在着明显上位性互作；主基因遗传率为50.24%，多基因遗传率为49.14%，说明异黄酮总含量存在多基因效应，其中主基因效应和多基因效应基本相等。
基于此，本发明采用如下技术方案以解决所述技术问题：
设计一种基于分子标记的高异黄酮大豆品种选育方法，包括如下步骤：
（1）选择对应的大豆品种作为母本、父本，单株种植于开花期进行杂交，收获杂交种F1；
（2）将所得F1代种子单株播种后自交育种；
（3）将上步所得F2代种子全部单株播种，并于苗期分别利用如下引物进行PCR扩增检测：
satt249：上游引物GCGGCAAATTGTTATTGTGAGAC，下游引物GGCCAGTGTTGAGGGATTTAGA；
satt285：上游引物GCGACATATTGCATTAAAAACATACTT，下游引物GCGGACTAATTCTATTTTACACCAACAAC；
再于田间选取、标记携带同时携带有satt249和satt285基因的单株继续自交育种，种植期间测量记载所选株的株高、结实率、百粒重、单株产量，标记性状满足育种要求的植株，收获其自交种子；
（4）测定上步所得各单株种子中的异黄酮含量，选取同时携带有satt249和satt285基因且异黄酮含量及产量满足要求的种子作为自交育种材料；
（5）将上步所得自交育种材料按照（2）～（4）所述步骤重复进行播种、苗期分子检测、成熟期籽粒鉴定、高异黄酮含量材料筛选，直至大豆植物学性状稳定，则得高异黄酮大豆育种材料。
在所述步骤（1）中，所述母本为大粒高异黄酮晋豆23、鲁黑豆2号、淮豆1号等，所述父本为小粒高异黄酮灰布支黑豆、张家口黑豆等。
选择亲本本身性状好，一般配合力大的做亲本，有利于后代异黄酮含量的提高。例如，豫豆29异黄酮含量高（4300 μg·g -1 ），一般配合力大（8.22）。选择特殊配合力SCA大的组合，高×高组合是优选，有利于后代异黄酮含量的提高；豫豆29是优选亲本。
在所述步骤（2）中，PCR扩增检测的方法如下：
（Ⅰ）DNA提取
①提取液配方： EDTA 终浓度20 mM，Tris-HCl（pH 7.5）终浓度100 mM，PEG8000终浓度1%（W/V），NaCl 终浓度1.4M，CTAB 终浓度2%（W/V），SLS（sodium lauryl sarcosine）终浓度0.3%（W/V）。
②提取步骤：
1) 提前水浴加热提取液至65℃；
2) 取对应单株的叶片在液氮中研磨成粉末，取适量装入离心管，加入提取液迅速混匀，65℃水浴15 min；
3) 加入等体积的氯仿/异戊醇（24:1，V/V），混匀；12,000 r/m 离心10 min；
4) 把上清转移到另外的离心管中，加入等体积的异丙醇并混匀，有絮状的DNA 沉淀出现；
5) 把DNA 沉淀转移到另外的离心管中，加入70%乙醇洗涤2 次；
6) 室温下晾干DNA 沉淀，使乙醇充分挥发从而除尽，用TE 溶液或灭菌超纯水溶解DNA，
此时得到DNA 粗提液，可以用于PCR 检测；
（Ⅱ）PCR扩增反应
反应体系为10 µL，包括10×buffer 1µL，dNTP 0.2µL，1U Taq酶 0.1ul， 20 ng•µL-1基因组DNA 2µL，20 ng•µL-1引物1µL，ddH2O 5.7 µL，在Applied Biosystems GeneAmpPCR System 9700上进行PCR。
PCR扩增程序为：94℃预变性5 min；然后94℃变性40s，50-60℃（依引物而定）退火40 s，72℃延伸1 min 10 s，30个循环；最后72℃延伸10 min。
（Ⅲ）扩增产物检测
PCR扩增产物采用6%非变性聚丙烯酰胺凝胶电泳的方法进行检测，具体程序如下：10 µL扩增产物中加入2.5 µL的指示剂即加样缓冲液（含有50 mM Tris-HCl pH 8.0，50 mMEDTA，1% SDS，0.25%溴酚蓝，0.25%二甲苯青，50%甘油），吸取4-5 µL样品在8%非变性聚丙烯酰胺凝胶上进行电泳检测，缓冲体系为1×TBE，120V电泳4-5 h左右。
8%非变性聚丙烯酰胺凝胶采用快速银染法进行检测，具体步骤是：
1）0.1%硝酸银溶液500 mL染色15-20 min。
2）去离子水快速漂洗20 sec左右。
3）显影液（500 mL去离子水+10 g NaOH+0.2-0.4 g Na2CO3，750 µL甲醛现用现加）显色，不断摇动约10 min，直至DNA条带清晰可见。
4）去离子水漂洗1次。
把染色后的凝胶放入5%的丙三醇溶液中浸泡片刻制成干胶，以便进行带型统计或观察照相。
与现有技术相比，本发明的主要有益技术效果在于：
1. 性状选择准确性，筛选效率高，且操作简便，从众多的大豆种质资源中快速选择出有价值（高产并且高异黄酮含量高）的品种或育种材料。
2. 可以快速的将大豆高产表型、高异黄酮表型聚合在一起，选育出品质优、高产、高异黄酮含量的大豆新品种。
附图说明
图1为Satt249基因电泳图。
图2为Satt285基因电泳图。
图3为本发明方法选育所得郑豆0689的植株样本照片。
图4为本发明方法选育所得郑豆0689籽粒性状照片。
图5为本发明实施例所得郑豆0689的系谱图。
具体实施方式
下面结合附图和实施例来说明本发明的具体实施方式，但以下实施例只是用来详细说明本发明，并不以任何方式限制本发明的范围。
在以下实施例中所涉及的仪器设备如无特别说明，均为常规仪器设备；所涉及的材料如无特别说明，均为市售常规材料；所涉及的检测、试验方法，如无特别说明，均为常规方法。
实施例1：QTL069育种材料的获取
（1）以栽培大豆晋豆23为母本，以山西农家品种大豆灰布支黑豆为父本，单株种植于开花期进行有性杂交，收获杂交种F1（每株收种50~50粒，共收1000～2000粒）；
（2）将上步所收得的F1代种子单株播种后自交育种；
（3）将上步所得F2代种子全部单株播种，并于苗期分别利用如下引物进行PCR扩增检测（具体方法如前述发明内容部分所记载）：
satt249：上游引物GCGGCAAATTGTTATTGTGAGAC，下游引物GGCCAGTGTTGAGGGATTTAGA；
satt285：上游引物GCGACATATTGCATTAAAAACATACTT，下游引物GCGGACTAATTCTATTTTACACCAACAAC。
根据PCR检测结果，于田间选取、标记携带同时携带有Satt249和Satt285基因（即同时具备图1和图2所示基因条带）的单株继续自交育种，种植期间测量记载其株高、结实率、百粒重、单株产量，最终选择各类性状满足大豆育种要求（如高产、稳产、农学性状好等）的植株，收获单株种子；
（4）测定上步所得各单株种子中的异黄酮含量，选取同时携带有satt249和satt285基因且异黄酮含量及产量满足要求的种子作为自交育种材料；
（5）将上步所得自交育种材料按照（2）～（4）所述步骤重复进行播种、苗期分子检测、成熟期籽粒鉴定、高异黄酮含量材料筛选，直至大豆植物学性状稳定，则得高异黄酮大豆育种材料。
本发明研究过程中以栽培大豆晋豆23为母本，以山西农家品种大豆灰布支黑豆为父本杂交衍生获得了447个RIL群体，按上述方法步骤从中获得了一个异黄酮含量高、生物学性状稳定的大豆育种材料，命名为QTL069，其异黄酮含量如表1所示。
表1 大豆异黄酮含量测定结果
实施例2郑豆0689选育
于2007年以实施例1中所得QTL069为母本、豫豆29为父本进行有性杂交，系谱法选择，以选择高产、稳产、优质、抗病、广适为主攻方向，经多年连续南繁加代选育最终得到植物学性状稳定、高产稳产且异黄酮含量高（见表1）的大豆新品种，并于2010年进行品系鉴定、决选品系；2011年参加河南省夏大豆预备试验，系谱命名为郑豆0689，其系谱图如图5所示。
鉴定表明，郑豆0689有限结荚习性，紫花，灰毛，叶形卵圆，叶色绿，荚熟色褐，夏播中早熟品种，不裂荚（见图3）。生育期108.8天，株高83.0cm，有效分枝2.7个，主茎节数17.4个，单株有效荚数52.8个，单株粒数100.1粒，百粒重20.3g。籽粒圆形，种皮黄色（见图4），成熟落叶性好，抗倒伏。
郑豆0689于2012-2013年完成河南省夏大豆区域试验；并于2014年参加河南省大豆生产试验，7试验点汇总，全部增产，单产幅度145.51～290kg/亩，平均亩产218.50kg，比对照豫豆22号增产15.96%，居8个参试品种第1位。
上面结合实施例对本发明作了详细的说明，但是，所属技术领域的技术人员能够理解，在不脱离本发明构思的前提下，还可以对上述实施例中的各个具体参数进行变更，或者是对相关材料及方法进行等同替代，从而形成多个具体的实施例，均为本发明的常见变化范围，在此不再一一详述。
Claims (4)
1.一种基于分子标记的高异黄酮大豆品种选育方法，其特征在于，包括如下步骤：
（1）选择对应的大豆品种作为母本、父本，单株种植后于开花期进行杂交，收获杂交种F1；
（2）将所得F1代种子单株播种后自交育种；
（3）将上步所得F2代种子全部单株播种，并于苗期分别利用如下引物进行PCR扩增检测：
satt249：上游引物GCGGCAAATTGTTATTGTGAGAC，下游引物GGCCAGTGTTGAGGGATTTAGA；
satt285：上游引物GCGACATATTGCATTAAAAACATACTT，下游引物GCGGACTAATTCTATTTTACACCAACAAC；
再于田间选取、标记携带同时携带有satt249和satt285基因的单株继续自交育种，种植期间测量记载所选株的株高、结实率、百粒重、单株产量，标记性状满足育种要求的植株，收获其自交种子；
（4）测定上步所得各单株种子中的异黄酮含量，选取同时携带有satt249和satt285基因且异黄酮含量及产量满足要求的种子作为自交育种材料；
（5）将上步所得自交育种材料按照（2）～（4）所述步骤重复进行播种、苗期分子检测、成熟期籽粒鉴定、高异黄酮含量材料筛选，直至大豆植物学性状稳定，则得高异黄酮大豆育种材料。
2.根据权利要求1所述的基于分子标记的高异黄酮含量大豆品种选育方法，其特征在于，在所述步骤（1）中，所述母本为大粒晋豆23、鲁黑豆2号或淮豆1号等。
3.根据权利要求1或2所述的基于分子标记的高异黄酮含量大豆品种选育方法，其特征在于，在所述步骤（1）中，所述父本为小粒灰布支黑豆或张家口黑豆等。
4.根据权利要求1所述的基于分子标记的高异黄酮含量大豆品种选育方法，其特征在于，在所述步骤（2）中，PCR扩增检测的方法如下：
① 取每株大豆植株片叶，用CTAB法提取DNA；
② PCR扩增：反应体系为10 µL，包括10×buffer 1µL，dNTP 0.2µL，1U Taq酶 0.1ul，20ng/µL基因组DNA 2µL，20ng/µL所述引物1µL，ddH2O 5.7µL，扩增程序为：94℃预变性5min；然后94℃变性40s，50～60℃退火40s，72℃延伸1 min 10s，30个循环；最后72℃延伸10min；
③ 采用6%非变性聚丙烯酰胺凝胶电泳的方法进行PCR扩增产物检测。
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