CN113736848A - 一种早中熟苹果杂种苗耐高温着色早期选择方法 - Google Patents
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Abstract
本发明公开了一种早中熟苹果杂种苗耐高温着色早期选择方法。属于林业种植技术领域。包括：杂种苗田间选择；实验材料选择和处理；筛选样品中代谢物质增加，激素水平上升，褪黑素合成相关基因、MYB转录因子、类黄酮合成途径中的F3H、DFR基因表达上调的植株作为选择对象。利用果实与叶片间的相关性，进行杂种苗的早期选择和预先选择，淘汰不良类型，减少供选植株的数量，提高育种效率。
Description
技术领域
本发明涉及林业种植技术领域，更具体的说是涉及一种早中熟苹果杂种苗耐高温着色早期选择方法。
背景技术
苹果是世界主栽果树之一，我国苹果的产量及栽培面积均占全世界总量的一半以上。苹果栽培过程中常伴随着各种逆境胁迫，对苹果的生长发育和产量品质造成一定影响。
早中熟苹果果实成熟期处于夏季高温期，导致果实着色差、品质低下，困扰着早中熟苹果栽培的发展。
选育耐高温、着色优良的早中熟品种对苹果产业的良性可持续发展具有十分重要的意义。而当前栽培的早中熟苹果品种中成熟期高温着色不良、品质差、不耐贮运、裂果炸果面果等问题。
因此，如何提供一种早中熟苹果杂种苗耐高温着色早期选择方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种早中熟苹果杂种苗耐高温着色早期选择方法。从杂交材料中选取植株叶片，在高温处理前题下，通过全方位、多角度科学严谨的指标检测，包括光合色素、多种抗氧化酶、有机渗透调节物质、植物激素水平、相关表达基因等方面，利用果实与叶片间的相关性，进行杂种苗的早期选择和预先选择，淘汰不良类型，减少供选植株的数量，提高育种效率。
为了实现上述目的，本发明采用如下技术方案：
一种早中熟苹果杂种苗耐高温着色早期选择方法，包括以下步骤：
(1)杂种苗田间选择；
(2)实验材料选择和处理；
(3)检测分析，筛选代谢物质、激素水平和着色相关基因表达水平中至少10项指标上调2倍以上的植株作为选择对象。
优选的：步骤(1)杂种苗田间选择：对田间杂种苗进行生物学形态调查，剔除病害严重、畸形生长幼苗，选择生长健壮植株。
有益效果在于：排除畸形植株，减少工作量。
优选的：步骤(2)实验材料选择：选取一年生枝上着生的功能叶作为试材。
有益效果在于：保证选取材料一致性，减少实验误差。
步骤(2)处理：选取的功能叶片叶柄用无菌棉球包裹，喷水保湿，叶片置于光照培养箱中40℃、5000LX普通光下处理8～12小时，以室温保存为对照。
有益效果在于：实验室模拟大田中高温环境，利于后期检测的准确性。
优选的：步骤(3)代谢物质：叶片光合色素含量、过氧化氢酶CAT，超氧化物歧化酶SOD，过氧化物酶POD、酚类物质总量、脯氨酸、可溶性糖和可溶性蛋白中的多个。
优选的：步骤(3)激素：褪黑素、吲哚乙酸、水杨酸激素中的多个。优选的：步骤(3)着色相关基因：SNAT和HIOMT褪黑素合成基因、MYB转录因子、类黄酮合成途径中的F3H、DFR基因中的多个。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一种早中熟苹果杂种苗耐高温着色早期选择方法，本发明利用果实与叶片间的相关性，进行杂种苗的早期选择和预先选择，淘汰不良类型，减少供选植株的数量，提高育种效率。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明提供的果树图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明实施例公开了一种早中熟苹果杂种苗耐高温着色早期选择方法。
实施例中涉及的对各指标的检测均未常规方法，在此不再赘述，例如：
指标检测方法如下：
1叶片光合色素含量测定：
胡秉芬，黄华梨，季元祖，等.分光光度法测定叶绿素含量的提取液的适宜浓度.草业科学，2018，35(8)：1965-1974.
2过氧化氢酶CAT测定：
参照Nakano的方法测定过氧化氢酶(CAT)活性。
Nakano Y，Asada K.Hydrogen peroxide is scavenged by ascorbate-specificperoxidase in spinach chloroplasts.Plant Cell Physiol，1981：867-880.
3超氧化物歧化酶SOD测定：
用氮蓝四唑还原法测定超氧化物歧化酶(SOD)活性，参照李合生的方法。
李合生.现代植物生理学.3版.北京：高等教育出版社，2012.
4过氧化物酶POD测定：
用愈创木酚法测定过氧化物酶(POD)活性，参照李合生的方法。
李合生.现代植物生理学.3版.北京：高等教育出版社，2012.
5酚类物质总量测定：
用HPLC进行酚类化合物测定。
李小方，张志良.植物生理学实验指导.5版.北京：高等教育出版社，2016.
6脯氨酸测定：
茚三酮显色分光光度法测定脯氨酸含量，参照李合生的方法.
李合生.现代植物生理学.3版.北京：高等教育出版社，2012.
7可溶性糖测定：
可溶性糖含量采用蒽酮比色法进行测定。
刘海英，王华华，崔长海，等.可溶性糖含量测定(蒽酮法)实验的改进.实验室科学，2013，16(2)：19-20.
8可溶性蛋白测定：
考马斯亮蓝测定可溶性蛋白质含量，参照李合生的方法.
李合生.现代植物生理学.3版.北京：高等教育出版社，2012.
9褪黑素测定：
参考Zhao所述方法，通过高效液相色谱串联质谱(HPLC-MS/MS)进行褪黑素的测定。
Zhao Y,Tan DX,Lei Q,Chen H,Wang L,Li T,Gao A,Kong J.Melatonin and itspotential biological functions in the fruits of sweet cherry.Journal ofPineal Research,2013,55：79-88.
10吲哚乙酸测定：
采用高效液相色谱法测定吲哚乙酸含量。
11水杨酸测定：
采用高效液相色谱法测定水杨酸含量。
12SNAT、HIOMT、MYB、F3H、DFR基因测定：
基因表达采用荧光定量PCR(qRT-PCR)方法。
使用CTAB法从苹果叶片中提取总RNA，再使用Prime ScriptTMRT试剂盒进行反转录。荧光定量的染料来自于
试剂盒。
反应采用20μL体系，其中包括10μL SYBR、0.5μL正向引物、0.5μL反向引物、8.0μLdd H2O和1μL的cDNA模板。将苹果的MdActin作为内参基因，以2-ΔΔCt计算基因的差异倍数，每个样品设3次技术重复，试验所用到的引物如下。
SNAT：
F：TCACGCTGTGGTTGGAGGT；R：CTGCATGCTCCTGAACCAAC；
HIOMT：
F：GAATCACCGTCCACGCTCC；R：GAAATGCTTCCGATGTCCC；
MYB：
F：ATGGAGGGATATAACGAAAACCTG；R：CTATTCTTCTTTTGAATGATTCC；
F3H：
F：GGAAGGAGCTTTTGTGGTCAA；R：GGTTCTGGAATGTGGCTATGGA
DFR：
F：AGGAACTGTGAATGTGGAGGAGC；R：AGGAACTGTGAATGTGGAGGAGC；
MdActin：
F：TGACCGAATGAGCAAGGAAATTACT；R：TACTCAGCTTTGGCAATCCACATC。
以上仅为示意，并非是限定必须使用上述方法。
实施例1
一种早中熟苹果杂种苗耐高温着色早期选择方法，包括：
S1：杂种苗田间选择，对田间杂种苗进行生物学形态调查，主要剔除病害严重、畸形生长幼苗，选择生长健壮植株。
S2：实验材料选择，选取一年生枝上着生的功能叶作为试材。
S3：实验材料处理，对选取的功能叶片叶柄用无菌棉球包裹，喷水保湿，叶片置于光照培养箱中40℃、5000LX普通光下处理12小时。
S4：检测分析，对处理实验材料进行代谢物质、激素水平和着色相关基因表达水平检测分析。
S5：早期选择，检测分析样品中叶片光合色素含量、过氧化氢酶CAT、超氧化物歧化酶SOD、过氧化物酶POD、酚类物质总量、脯氨酸、可溶性糖、可溶性蛋白、褪黑素、吲哚乙酸、水杨酸上升，褪黑素合成相关基因SNAT和HIOMT，MYB转录因子，类黄酮合成途径中的F3H、DFR基因表达上调的植株作为选择对象。
实施例中，将上调定义为：处理组指标含量/对照组指标含量>2。
采用上述方法，从8000余株嘎拉与藤牧一号正反交的实生苗中，初选早中熟耐高温、着色好、优质丰产优系120份，决选36份，其中鲁丽、鲁艳等(参见图1)品种已获得国家农业农村部的植物品种权，并在在山东、陕西、河北、辽宁、山西、河南等苹果主产区示范推广30万余亩，取得了显著成效，有望成为我国中早熟苹果的主栽品种。
技术效果：
以‘鲁丽’为例：山东省果树研究所以‘藤木一号’和‘嘎啦’为亲本，选育出的新一代早中熟苹果优良品种。果实圆锥形，果面盖色鲜红，果面光滑，果点小，无锈，果肉淡黄色，肉质细、硬脆，汁液多，甜酸适度，香气浓，品质上等，果实发育期100天左右，早熟；适应性强，耐瘠薄土壤，早果、丰产性强。大苗定植后第二年有部分产量，第三年亩产达1000公斤左右，早果、丰产、稳产性很好。
选育的新品种解决了当前栽培的早中熟苹果品种中成熟期高温着色不良、品质差、不耐贮运、裂果炸果面果等问题，是新一代早中熟苹果优良品种，有望成为我国中早熟苹果的主栽品种。
定植第二年的鲁丽幼树：
鲁丽初选检测结果汇总参见表1
表1
需要说明的是：为达到严谨科学的筛选结果，在上述16项检测指标中，需至少满足10项以上。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (7)
1.一种早中熟苹果杂种苗耐高温着色早期选择方法，其特征在于，包括以下步骤：
(1)杂种苗田间选择；
(2)实验材料选择和处理；
(3)检测分析，筛选代谢物质、激素水平和着色相关基因表达水平中至少10项指标上调2倍以上的植株作为选择对象。
2.根据权利要求1所述的一种早中熟苹果杂种苗耐高温着色早期选择方法，其特征在于，步骤(1)所述杂种苗田间选择：对田间杂种苗进行生物学形态调查，剔除病害严重、畸形生长幼苗，选择生长健壮植株。
3.根据权利要求1所述的一种早中熟苹果杂种苗耐高温着色早期选择方法，其特征在于，步骤(2)所述实验材料选择：选取一年生枝上着生的功能叶作为试材。
4.根据权利要求1所述的一种早中熟苹果杂种苗耐高温着色早期选择方法，其特征在于，步骤(2)所述处理：选取的功能叶片叶柄用无菌棉球包裹，喷水保湿，叶片置于光照培养箱中40℃、5000LX普通光下处理8～12小时，以室温保存为对照。
5.根据权利要求1所述的一种早中熟苹果杂种苗耐高温着色早期选择方法，其特征在于，步骤(3)所述代谢物质：叶片光合色素含量、过氧化氢酶CAT，超氧化物歧化酶SOD，过氧化物酶POD、酚类物质总量、脯氨酸、可溶性糖和可溶性蛋白中的多个。
6.根据权利要求1所述的一种早中熟苹果杂种苗耐高温着色早期选择方法，其特征在于，步骤(3)所述激素：褪黑素、吲哚乙酸、水杨酸激素中的多个。
7.根据权利要求1所述的一种早中熟苹果杂种苗耐高温着色早期选择方法，其特征在于，步骤(3)所述着色相关基因：SNAT和HIOMT褪黑素合成基因、MYB转录因子、类黄酮合成途径中的F3H、DFR基因中的多个。
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