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毛白杨杂种三倍体的2n雌配子形成途径鉴定

旻昱 康宁 索玉静 田菊 康向阳

旻昱, 康宁, 索玉静, 田菊, 康向阳. 毛白杨杂种三倍体的2n雌配子形成途径鉴定[J]. 北京林业大学学报, 2017, 39(5): 17-24. doi: 10.13332/j.1000-1522.20170001
引用本文: 旻昱, 康宁, 索玉静, 田菊, 康向阳. 毛白杨杂种三倍体的2n雌配子形成途径鉴定[J]. 北京林业大学学报, 2017, 39(5): 17-24. doi: 10.13332/j.1000-1522.20170001
MIN Yu, KANG Ning, SUO Yu-jing, TIAN Ju, KANG Xiang-yang. Origin identification of 2n female gamete of Populus tomentosa triploid hybrids[J]. Journal of Beijing Forestry University, 2017, 39(5): 17-24. doi: 10.13332/j.1000-1522.20170001
Citation: MIN Yu, KANG Ning, SUO Yu-jing, TIAN Ju, KANG Xiang-yang. Origin identification of 2n female gamete of Populus tomentosa triploid hybrids[J]. Journal of Beijing Forestry University, 2017, 39(5): 17-24. doi: 10.13332/j.1000-1522.20170001

毛白杨杂种三倍体的2n雌配子形成途径鉴定

doi: 10.13332/j.1000-1522.20170001
基金项目: 

国家自然科学基金项目 31470667

内蒙古自治区科技重大专项 2014

北京市共建项目专项 2016BLREEE00

详细信息
    作者简介:

    旻昱。主要研究方向:林木倍性育种。Email : minyu421@126.com  地址:100083 北京市海淀区清华东路35号北京林业大学生物科学与技术学院

    责任作者:

    康向阳,教授,博士生导师。主要研究方向:林木倍性育种与细胞遗传学。Email: kangxy@bjfu.edu.cn   地址:同上

  • 中图分类号: S718.46;S792.117

Origin identification of 2n female gamete of Populus tomentosa triploid hybrids

  • 摘要: 由于受杨树大孢子母细胞减数分裂不同步性影响,通过施加理化处理诱导杨树大孢子染色体加倍授粉杂交获得的三倍体,可能既包括来源于第一次减数分裂核复原形成的2n雌配子(FDR),也有来源于第二次减数分裂核复原形成的2n雌配子(SDR),难以进行表型鉴别,而通过分子标记分析可以实现不同类型2n雌配子的区分。本研究以通过高温诱导毛白杨大孢子染色体加倍所获得的87株三倍体及其亲本为材料,从母本杂合且与父本有差异的SSR多态性引物中,筛选出5对适宜于2n配子诱导途径来源鉴定的重组率较低的SSR分子标记。在此基础上,利用这5对低重组率SSR分子标记,开展毛白杨杂种三倍体的2n雌配子诱导途径来源分析。结果表明,不同毛白杨杂种三倍体的2n雌配子相应简单重复序列区域的等位基因配置不同,利用筛选出的5对低重组率SSR标记可以实现毛白杨杂种三倍体的2n雌配子诱导途径鉴定,其中35株三倍体来源于FDR型2n配子,另52株来源于SDR型2n配子。此外,选用5个位于高重组率位点的SSR引物进行检测分析,结果验证了随机挑选的SSR引物用于鉴别结果是不完全可靠的,只有挑选低重组率的SSR位点才可能准确鉴定2n配子来源。开展毛白杨杂种三倍体的2n配子诱导途径鉴定研究,对于FDR、SDR不同形成途径的2n配子传递亲本杂合性分析,以及毛白杨三倍体育种策略制定等具有重要意义。

     

  • 图  1  毛白杨杂种三倍体SSR毛细管电泳分析

    亲本及三倍体子代植株在5对SSR位点处检测到的毛细管电泳结果。母本‘3119’及父本‘YX1’在所有位点多态且父、母本间没有相同等位基因,a、b、c、d代表父、母本的SSR位点所检测到的不同等位基因。异源三倍体植株的等位基因配置分为F型与S型两种类型,在F型中,母本所有杂合等位基因全部传递至三倍体中;在S型中,母本杂合等位基因中的1个以2倍剂量传递至三倍体中。GCPM_1577、GCPM_1120、GCPM_1153、GCPM_1390-1、GCPM_1175为研究中所应用的5对低重组率SSR引物。

    Figure  1.  Allelic configurations of the triploid hybrids

    Results of capillary electrophoresis for the five SSR markers were detected in two parents and triploid hybrids. Both female parent '3119' and male parent 'YX1' were heterozygous and two distinct alleles were detected at all loci. 'a', 'b', 'c', 'd' present different alleles detected at SSR loci of parents.The allelic configurations of the triploid hybrids were divided into two types, which were previously named F type and S type. In the F type allelic configuration, both female parent 3119 alleles were transmited to the triploid hybrid, whereas only one of two female parent 3119 alleles was transmited to the triploid hybrid in the S type allelic configuration. GCPM_1577、GCPM_1120、GCPM_1153、GCPM_1390-1、GCPM_1175 were the SSR primers applied in this study with low recombination rate.

    表  1  PCR扩增反应体系配置表

    Table  1.   Configuration of PCR reaction system

    成分
    Configuration
    体积
    Volume/μL
    无菌ddH2O Sterile double distilled water 7.20
    模板DNA DNA mould 2.00
    5′端接有M13序列的上游引物Forward primer with an universal primer M13 tail at the 5′end 0.08
    下游引物Reverse primer 0.32
    荧光引物(ROX,FAM,TAMRA,HEX) Fluorescently labeled primer(ROX,FAM,TAMRA,HEX) 0.40
    PCR Mix 10.00
    总体系Total system 20.00
    下载: 导出CSV

    表  2  适合毛白杨2n配子诱导途径鉴定的低重组率多态性SSR位点信息

    Table  2.   Information of SSR loci with low recombination frequencies

    标记名称
    Marker
    所在染色体
    Chromosome
    No.
    重复单元
    Repeat
    motif
    引物序列(5′→3′)
    Primer sequence
    (5′→3′)
    检测等位基因
    Detected allele/bp
    位点距着丝粒距离与
    所在臂长之比
    Ratio of distance from
    centromere of loci to
    chromosome arm length/%
    母本
    Female
    parent
    父本
    Male
    parent
    GC_1153 11 CTT 上游Forward TTCCTTTCACACAATGACAA 178/181 172/175 10.79
    下游Reverse TTTAAAAACTGGGTCCGTAA
    GC_1175 14 CTT 上游Forward TCATCAACCTGACCTCTACC 191/210 194/197 10.80
    下游Reverse CAAAGCAAAAACAAACACAA
    GC_1577 18 TA 上游Forward GAGAACATGTCAGCAGTTCA 238/242 234/240 16.44
    下游Reverse GCTTAAACATTGAGAAAGCG
    GC_1120 10 TAA 上游Forward TGCAAATCCAAACACAGATA 237/261 228/231 25.41
    下游Reverse ACGAATCATCTTACGCACTT
    GC_1390-1 13 CA 上游Forward CAAGAACGGTAAAATGCTTC 244/276 239/253 30.78
    下游Reverse TCCATTTTCCAAATCCTAAA
    下载: 导出CSV

    表  3  大孢子染色体加倍来源的毛白杨三倍体SSR分子标记鉴定

    Table  3.   Genetic composition of the 87 Populus tomentosa triploids analyzed by 5 SSR markers with low hDNA frequencies

    2n配子类型2n gamete type 基因配置类型Gene configuration 三倍体数Number of triploids 株数Total number
    FDR 5F0S 1 35
    4F1S 9
    3F2S 25
    SDR 2F3S 24 52
    1F4S 18
    0F5S 10
    下载: 导出CSV

    表  4  高重组率多态性SSR位点具体信息

    Table  4.   Information of SSR loci with high recombination frequencies

    标记名称
    Marker
    所在染色体
    Chromosome No.
    重复单元
    Repeat motif
    引物序列(5′→3′)
    Primer sequence
    (5′→3′)
    检测等位基因
    Detected allele/bp
    位点距着丝粒距离与
    所在臂长之比
    Ratio of distance from
    centromere of loci to
    chromosome arm length/%
    母本
    Female
    parent
    父本
    Male
    parent
    GCPM_2615 6 CTTT 上游Forward ATGTCAACGTCACTGACAAA 230/238 228/235 99.07
    下游Reverse ATTAGGCAATGCAGAACACT
    GCPM_3310-1 8 GAG 上游Forward GCCTCATCATGCTTCTAAAC 222/228 231/237 86.41
    下游Reverse CATGAACCTTCCACCATTAT
    GCPM_1418-1 16 CT 上游Forward GTACACTCAGCTGTGGTCCT 204/216 230/238 95.50
    下游Reverse TGGGAGTCATGAAATCTACC
    ORPM_422 1 TAA 上游Forward TGCAAATCCAAACACAGATA 222/234 218/232 81.36
    下游Reverse ACGAATCATCTTACGCACTT
    GCPM_719 15 AG 上游Forward AAAGTTATGCTTCAAGCCAG 211/239 207/227 80.57
    下游Reverse AAGGGTTGCAACTTTTCATA
    下载: 导出CSV

    表  5  SDR型(1F5S)三倍体在高重组率SSR位点的等位基因组成

    Table  5.   Allelic configurations of SDR triploids at the SSR loci with high recombination frequencies

    三倍体Triploid GCPM_2615 GCPM_3310 GCPM_1418-1 ORPM_422 GCPM_719
    SDR-1 Ho He He Ho Ho
    SDR-2 He He He Ho He
    SDR-3 He He He He He
    SDR-4 He He He He He
    SDR-5 He He He He He
    SDR-6 He He He He He
    SDR-7 He Ho Ho He Ho
    SDR-8 Ho He He He He
    SDR-9 He He Ho He He
    SDR-10 He He Ho He He
    注:He代表杂合型;Ho代表纯合型。Note: He, heterozygous; Ho, homozygous.
    下载: 导出CSV
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  • 收稿日期:  2016-12-28
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