寇春兰, 赵来宾, 刘梦, 郝明, 甯顺腙, 袁中伟, 刘登才, 张连全^*
四川农业大学小麦研究所 四川成都 611130
第一作者联系方式: E-mail: 510531588@qq.com
收稿日期:2015-11-04 接受日期:2016-03-14网络出版日期:2016-03-28基金:本研究由国家自然科学基金项目(31271723, 31201210), 四川省****基金项目(2011JQ0016)和四川省教育厅科研项目(14ZA0012)资助

摘要六倍体普通小麦( Triticum aestivum L., AABBDD, 2 n = 42)由四倍体小麦( T. turgidum, AABB, 2 n = 28)与节节麦( Aegilops tauschiiCosson, DD, 2 n=14)天然杂交, 然后通过染色体自动加倍形成。加倍过程主要受四倍体小麦未减数配子基因控制, 且不同四倍体小麦存在不同的遗传效应。本研究利用位于3B染色体上未减数配子基因 QTug.sau-3B的连锁SSR标记 Xgpw1146和高通量DArTseq分子标记, 筛选出可能转入四倍体小麦未减数配子基因的人工合成小麦改良后代。在105份改良材料中检测出17份具有四倍体小麦的 Xgpw1146等位位点, 表明四倍体小麦的未减数配子基因可能转入了这17份材料。利用DArTseq高通量标记技术分析人工合成小麦SHW-L1的88份改良后代, 发现含四倍体小麦 Xgpw1146等位位点的材料均具有来自SHW-L1、且可能包含 Xgpw1146的一个染色体区段, 表明未减数配子基因临近区域以一个区段传递到改良后代。这些人工合成小麦改良材料在加倍单倍体育种中有重要的应用潜力。

关键词:异源多倍体; 人工合成小麦; 未减数配子
Detection of the Molecular Marker and Chromosomal Segment linked to Unreduced Gamete Gene in Common Wheat
KOU Chun-Lan, ZHAO Lai-Bin, LIU Meng, HAO Ming, NING Shun-Zong, YUAN Zhong-Wei, LIU Deng-Cai, ZHANG Lian-Quan^*
Triticeae Research Institute, Sichuan Agricultural University, Chengdu 611130, China
Fund:This study was supported by the National Natural Science Foundation of China (31271723, 31201210), Sichuan Provincial Youth Fund (2011JQ0016), and the Scientific Research Foundation of the Education Department of Sichuan Province (14ZA0012)
AbstractHexaploid common wheat ( Triticum aestivumL., AABBDD, 2 n = 42) arose from spontaneous chromosome doubling of the hybrid between T. turgidumand Aegilops tauschiiCosson. The process of chromosomes doubling is mainly determined by unreduced gametes (UG) genes in T. turgidum. The genetic effects on the UG production may vary among T. turgidum lines. In this study, a SSR marker close to the UG gene QTug.sau-3B( Xgpw1146) and high throughput DArTseq genotyping technique were used to screen the UG gene in common wheat lines transferred from T. turgidum via synthetic hexaploid wheat (SHW) as a bridge. Out of the analyzed 105 SHW-derived elite lines, 17 had the Xgpw1146 allele from T. turgidum, indicating that the UG gene was probably transferred into these wheat lines. According to the DArTseq genotyping data on 88 lines derived from the synthetic hexaploid wheat SHW-L1, all these lines with the T. turgidum Xgpw1146 allele contained a chromosomal segment of SHW-L1, probably covering the Xgpw1146 locus. This indicates that the adjacent region of the UG gene as a chromosomal segment was transferred into wheat lines. These SHW-derived lines have important application potential on wheat doubled haploid breeding.

Keyword:Allohexaploid; Synthetic wheat; Unreduced gamete
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许多重要作物是异源多倍体。异源多倍体的产生通常包括两个关键过程, 一是不同物种的远缘杂交, 即异源过程; 二是远缘杂种的染色体自然加倍, 即多倍化过程。远缘杂种产生的未减数配子在染色体组自然加倍过程中发挥了重要作用^{[1, 2, 3]}。未减数的雌雄配子结合, 能够实现染色体自然加倍。未减数配子不仅在多倍体物种起源过程中发挥了重要作用, 而且在加倍单倍体育种和创制新双二倍体的遗传育种方面有重要应用价值^{[3, 4, 5, 6, 7]}。未减数配子能够让单倍体自动加倍, 从而克服了用秋水仙碱溶液等药品人工加倍存在的处理程序繁琐、成功率低、污染环境且导致加倍植株不正常等不利于大规模批量生产加倍单倍体的缺点^{[5, 6, 8]}。因而, 通过未减数配子实现染色体自动加倍, 可克服人工染色体加倍过程中的技术瓶颈, 从而大大加快小麦单倍体育种的进程。
普通小麦(Triticum aestivum L., AABBDD, 2n = 42)是最重要的粮食作物之一。该异源六倍体物种由四倍体小麦(T. turgidum, AABB, 2n = 28)与节节麦(Aegilops tauschii Cosson, DD, 2n = 14)天然杂交和染色体自然加倍形成的^[9]。研究表明, 一些四倍体小麦与节节麦的单倍体杂种F₁有好的育性, 并自交结实产生具有与正常普通小麦一样的AABBDD染色体组, 表明染色体发生了自动加倍^{[10, 11, 12, 13, 14, 15, 16, 17]}。染色体自然加倍来自于花粉母细胞第一次分裂核再组或单次减数分裂形成的未减数雌雄配子的结合, 因为花粉母细胞仅发生了单次分裂, 因此这样的减数分裂被称为“ 类似有丝分裂的减数分裂” (类有丝分裂)^[10]。事实上, 未减数配子的形成在许多小麦族物种的远缘杂种中是一个比较普遍的现象。因为减数分裂中期染色体不配对是未减数配子形成的一个重要特征^[18], 因此该过程也被称为“ univalent-dependent meiotic non-reduction” ^{[19, 20, 21]}。
四倍体小麦-节节麦杂种的未减数配子的形成主要受四倍体小麦主效基因控制^{[11, 12, 13, 14]}, 不同四倍体小麦存在不同的遗传效应^{[10, 17]}。控制未减数配子形成的基因可能通过延长第一次减数分裂的染色体分离过程促进了未减数配子的形成^[20]。四倍体小麦的未减数配子形成基因被导入六倍体小麦后, 仍然发挥作用。一些四倍体小麦-节节麦合成的六倍体小麦与黑麦^[22]和Ae. variabilis^[23]等物种的远缘杂种仍然能够形成未减数配子, 实现染色体自动加倍。最近, Hao等^[20]在四倍体小麦3B染色体上定位了一个未减数配子主效QTL, 即QTug.sau-3B。为了在小麦育种中利用QTug.sau-3B, 一项基础工作就是将该基因导入到普通小麦推广品种遗传背景中, 获得综合农艺性状优良的单倍体育种桥梁材料。
研究表明, 四倍体小麦AS2255、LDN和PI94666含有未减数配子基因^[17]。我们前期利用这些四倍体小麦与节节麦杂交创制的人工合成小麦, 与四川小麦新品种(系)杂交、顶交, 获得了一批综合农艺性状优良的小麦新品系。本试验利用与QTug.sau-3B紧密连锁的SSR标记和高通量DArTseq分型技术, 筛选出人工合成小麦衍生系中可能携带来自四倍体小麦AS2255、LDN、PI94666的QTug.sau-3B基因的材料, 为下一步的加倍单倍体育种研究提供亲本资源。
1 材料与方法1.1 植物材料105份人工合成小麦改良新品系来自11个组合(表1), 由人工合成小麦与普通小麦品种杂交而来, 其亲本包括18份普通小麦(SY95-71、700-011689、MY68942、川农16、Pm99915、04103、03-DH1959、南农02Y393、CN04-1、渝98767、ZL-21、川育18、郑麦9023、SW8188、川麦42、绵麦51、川麦47和川07001)和5份人工合成小麦。这105份人工合成小麦改良新品种(系)是从大量的新品系中挑选得到的综合农艺性状优良、丰产性状突出的优良品系。这5份人工合成小麦分别是SHW-L1 (系谱AS22 55/ AS60)、Syn-SAU-14 (系谱AS2255/AS2393)、Syn- SAU-7 (系谱LDN/AS77)、Syn-SAU-11 (系谱LDN/ AS2407)和Syn-SAU-39 (系谱PI94666/AS2407) ^[17]。上述所有改良品系、人工合成小麦及其四倍体小麦亲本AS2255 (T. turgidum ssp. turgidum)、LDN (T. turgidum ssp. durum)和PI94666 (T. turgidum ssp. dicoccon)均由四川农业大学小麦研究所提供。
表1
Table 1
表1(Table 1)

表1 合成小麦改良品系的分子标记检测结果 Table 1 Molecular marker assay in improved lines of synthetic hexaploid wheat (SHW) 系谱 Pedigree 品系数 No. of lines 有目标SSR标记的品系代号 Line code with the target SSR allele 检测标记 Marker used 有目标分子标记的组合 Hybrid combinations with the SHW allele SHW-L1/川农16//Pm99915-1///04103 F8-F9 16 L13-81, L13-316, L13-321, L13-326, L13-331, L13-341, L13-366 Xgpw1146+DArTseq SHW-L1/川农16//Pm99915-1///03-DH1959 F9 7 L13-461, L13-466, L13-471 Xgpw1146+DArTseq SHW-L1/SW8188//川育18///郑麦9023 F9 1 L08-1673 Xgpw1146 Syn-SAU-14/川麦42 F5 5 L35-8, L35-93, L35-16, L35-44, L35-87 Xgpw1146 LDN/PI94666//AS60///川麦47 F5 3 L14-6 Xgpw1146 无目标分子标记的组合 Hybrid combinations without the SHW allele SHW-L1/SY95-71//700-011689///MY68942 F7-F9 47 Xgpw1146+DArTseq SHW-L1/SY95-71//渝98767///ZL-21 F8-F9 13 Xgpw1146+DArTseq SHW-L1/SW8188//川育18///川麦42 F9 4 Xgpw1146+DArTseq SHW-L1/川农16//南农02Y393///CN04-1 F7 1 Xgpw1146+DArTseq Syn-SAU-7/绵麦51 F5 7 Xgpw1146 Syn-SAU-11/07001 F5 1 Xgpw1146 总数 Total 105

表1 合成小麦改良品系的分子标记检测结果 Table 1 Molecular marker assay in improved lines of synthetic hexaploid wheat (SHW)

1.2 SSR标记检测取新鲜叶片, 采用改良2× CTAB法提取基因组DNA^[24], 选用3个紧密连锁的SSR标记Xgwm285、Xcfp1012和Xgpw1146筛选未减数配子形成基因QTug.sau-3B^[20]。按Zhang等^[25]描述的反应体系和程序进行PCR扩增和产物电泳鉴定。
1.3 高通量DArTseq分型技术将DNA样品提供给澳大利亚Diversity Arrays Technology Pty Ltd. 公司(Canberra, Australia, http:// www.Triticarte.com.au)进行基于基因组简化和第二代测序技术的DArTseq分析, 人工合成小麦改良材料和亲本材料的基因型鉴定。从DArTseq获得的silicoDArTs标记为显性标记, 其结果分别赋值“ 1” (有)、“ 0” (无)和“ -” (不确定)。SilicoDArTs标记在3B染色体上的位置信息由Diversity Arrays Technology Pty Ltd. 公司提供。

2 结果与分析利用3个与QTug.sau-3B紧密连锁SSR标记检测表明, 只有Xgpw1146在四倍体小麦AS2255及合成小麦SHW-L1的扩增产物大小相同, 且扩增产物小于在普通小麦亲本04103、Pm99915、川农16中的扩增片段(图1-A)。以前的研究表明, Xgpw1146可作为跟踪QTug.sau-3B的理想标记^[26], 因此, Xgpw1146标记被进一步用于筛选人工合成小麦改良品系(图1-B)。
利用标记Xgpw1146检测105份人工合成小麦改良材料, 共检测到17份材料存在与人工合成小麦(或四倍体小麦)相同的等位位点(表1), 占所筛选后代的9.1%。其中, 源于四倍体小麦AS2255的改良新品系有16份, 占所筛选后代的17%, 涉及LDN的合成小麦改良后代仅有L14-6品系(图1-B)。由于该品系的人工合成小麦亲本涉及两个四倍体小麦(LDN和PI94666), 且这2个四倍体材料在Xgpw1146位点没有差异, 因此, 不能判断该材料导入的该位点来自哪个四倍体小麦。
选择人工合成小麦SHW-L1改良品系88个进行DArTseq分析, 其中10份材料含有SHW-L1的Xgpw1146标记等位位点(表1)。DArTseq分析获得815个位于3B染色体上且具有位置信息的SilicoDArTs标记。考虑到可能存在假阳性标记, 以1 cM为步长, 分别计算每个品系在这1 cM内与其对应亲本间的相同标记占标记总数的百分率, 并根据百分率的高低来判断该区段来源于哪个亲本。如果一个品系在该1 cM区段内大于90%的标记与其中一个亲本相同, 且和其他亲本相同的标记数小于90%, 则认为该区段来源于大于90%相同标记的亲本材料; 如果一个品系相邻的1 cM区段来源于同一个亲本, 则合并这些区段为一个更大的区段, 并重新计算该区段内分子标记和该品系各个亲本之间的相同率。根据已构建的遗传图谱, 与未减数配子基因QTug.sau-3B连锁的SSR标记Xgpw1146与DArT标记wPt-7152的遗传距离为4.66 cM ^[20](图2-A), 而DArTseq图谱上标记wPt-7152位于101.69 cM处(图2-B)。因此, 对包含wPt-7152标记的染色体区段101.67~107.37 cM和临近wPt-7152的93.78~98.97 cM区段重点分析, 因为QTug.sau-3B可能位于这2个区段之间。根据标记分布情况, 已检测到含有SHW-L1分子标记Xgpw1146等位位点的10个品系全部含有这2个染色体区段(图2-B), 表明这些品系可能具有源于四倍体小麦未减数配子基因QTug. sau-3B的染色体区段。另外, 虽然L13-336、L13-376和L13-71的93.78~98.97 cM区段可能也来源于SHW-L1, 但是包含wPt-7152标记的染色体区段101.67~107.37 cM及其Xgpw1146标记等位位点均不来自SHW-L1亲本。因此推测, 四倍体小麦的未减数配子基因可能没有导入这3个品系。
图1
Fig. 1

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图1 SSR分子标记检测 A: SSR标记Xgwm285、Xcfp1012和Xgpw1146(每份材料从左到右的3个泳道)在四倍体小麦、人工合成小麦及普通小麦的扩增结果。M: Marker; 1: 04103; 2: Pm99915; 3: 川农16; 4: AS2255; 5: SHW-L1。箭头示目标扩增片段。B: Xgpw1146在普通小麦、人工合成小麦及合成小麦改良品系的扩增结果。M: marker; 1: LDN; 2: Syn-SAU-39 (PI94666/AS2407); 3: Syn-SAU-7 (LDN/AS77); 4: Syn-SAU-11 (LDN/AS2407); 5: 绵麦51; 6: 川麦47; 7: 川07001; 8~13: 人工合成小麦改良品系, 其中12泳道为L14-6。箭头示目标片段。Fig. 1 PCR profiles of SSR markers A: Amplification profile of SSR markers Xgwm285, Xcfp1012, and Xgpw1146(from left to right in each line) in tetraploid wheat, synthetic hexaploid wheat (SHW), and common wheat. M: marker; 1: 04103; 2: Pm99915; 3: Chuannong 16; 4: AS2255; 5: SHW-L1. Arrows show the target band. B: Xgpw1146 amplification profile in common wheat, SHW, and improved lines of SHW, M: marker; 1: LDN; 2: Syn-SAU-39 (PI94666/AS2407); 3: Syn-SAU-7 (LDN/AS77); 4: Syn-SAU-11 (LDN/AS2407); 5: Mianmai 51; 6: Chuanmai 47; 7: Chuan 07001; 8-13: SHW lines, in which lane 12 is L14-6. The arrow shows the target band.

图2
Fig. 2

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图2 小麦基因QTug.sau-3B遗传连锁图(A)、DArTseq连锁标记wPt-7152的邻近区段 (B)及合成小麦改良品系中QTug.sau-3B邻近区段检测(C) A: QTug.sau-3B连锁图谱来自Hao等[20]; B: 可能含有与QTug.sau-3B相邻的2个染色体区段101.67-107.37 cM (红色矩形区域)和93.78-98.97 cM (绿色矩形区域)区段在DArTseq-3B图谱(由Diversity Arrays Technology Pty Ltd. 公司提供)上的位置。C: 2个染色体区段中任何一个和人工合成小麦亲本该区段相同标记数大于90%的株系, 其中, 蓝色横线表示检测含有SHW-L1的Xgpw1146等位位点的株系, 株系L13-81、L13-316、L13-326、L13-331、L13-341和L13-366中P1、P2、P3分别代表它们的小麦亲本川农16、Pm99915-1、04103; 株系L13-461、L13-466和L13-471中P1代表川农16, P2代表Pm99915-1, P3代表03-DH1959。Fig. 2 Linkage map of gene QTug.sau-3B (A), linked DArTseq marker wPt-7152 and its adjacent regions (B), and detection of adjacent regions harboring QTug.sau-3B in improved lines of synthetic hexaploid wheat A: Linkage map of QTug.sau-3Baccording to Hao et al.[20]; B: Location information of two chromosome segments nearby QTug.sau-3B on linking map of DArTseq-3B (provided by Diversity Arrays Technology Pty Ltd.), covering 101.67-107.37 cM (red rectangular region) and 93.78-98.97 cM (green rectangular region); C: The elite lines showed same markers more than 90%, compared to one of the two segments in synthetic hexaploid wheat. The blue lines represent the elite lines with the Xgpw1146allele from SHW-L1. P1, P2, and P3 represent Chuannong16, Pm99915-1, and 04103, respectively, which are their common wheat parents of lines L13-81, L13-316, L13-326, L13-331, L13-341, and L13-366. P1, P2, and P3 represent Chuannong16, Pm99915-1 and 03-DH1959, respectively, the parents of lines L13-461, L13-466, and L13-471.

3 讨论加倍单倍体在遗传和育种研究中有重要的应用价值。产生加倍单倍体包括两个关键步骤, 一是产生单倍体, 目前已经有产生小麦单倍体的比较成熟和可靠的方法, 如小麦-玉米杂交法; 二是对获得的单倍体进行染色体加倍, 目前常见的加倍方法是用秋水仙碱等药品, 处理, 但是该方法存在处理程序繁琐、工作量大、条件不易控制、成功率低、药品对植株有毒害作用、诱导遗传变异等缺点, 而且秋水仙碱等药品对人畜有毒, 容易造成环境污染。因此, 人工加倍方法不利于大规模的批量生产加倍单倍体, 从而限制了加倍单倍体在遗传育种中的实际应用效率。利用普通小麦单倍体自身具有的染色体自动加倍功能, 可以解决上述问题。本研究筛选出的可能具有未减数配子基因QTug.sau-3B、且综合农艺性状优良的新材料, 是单倍体育种潜在的育种亲本。下一个阶段的任务是将这些材料诱导成单倍体, 评价其实际加倍效果, 同时利用它们构建育种群体评估在加倍单倍体育种中的实际应用价值。
4 结论在人工合成小麦改良后代中筛选出17个具有未减数配子基因连锁标记的品系, 这些材料在小麦加倍单倍体育种中有非常重要的应用价值。
The authors have declared that no competing interests exist.


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