玉米优良自交系单片段代换系的构建

以优良自交系昌7-2为供体亲本、自交系9801为受体亲本,通过杂交、回交和供体染色体片段的SSR标记跟踪,构建以9801为遗传背景的昌7-2染色体单片段代换系群体,分析供体基因组成分在单片段代换系构建过程中的变化趋势。结果表明,共获得了74个以9801为背景的供体单片段代换系,片段长度为2.38～181.46 cM,平均长度为33.39 cM,导入片段总长为2 470.53 cM,染色体覆盖率为29%。     

控制水稻中胚轴伸长的QTL定位

水稻中胚轴能够为幼苗破土提供动力,培育长中胚轴水稻品种有助于水稻直播技术的推广,因此,研究水稻中胚轴伸长具有重要的理论和现实意义。为分析调控水稻中胚轴伸长的遗传基础,以Asominori和IR24重组自交系(RIL)群体为材料,结合其连锁图谱,对2017年杭州收获种子的中胚轴长度性状进行QTL定位。结果表明,在Asominori/IR24重组自交系群体中共检测到3个控制中胚轴伸长的QTL位点,分别位于第2、第3和第7条染色体上,LOD值在2.34～3.41之间,单个QTL对表型贡献率在7.25%～11.07%之间。同时用Asominori遗传背景的IR24染色体片段代换系进行验证,qML2对应的代换家系CSSL12的中胚轴与Asominori相比显著伸长,qML7对应的代换家系CSSL37的中胚轴与Asominori相比显著缩短,从而验证了qML2和qML7位点的存在。与先前研究比较,qML3和qML7在不同群体不同环境下稳定表达。同时利用重组自交系群体对2018年杭州大田环境株高性状进行QTL定位,共检测到2个QTL位点,均与中胚轴QTL位点不重合,说明控制中胚轴伸长与控制株高有着不同的遗传基础。     

普通野生稻苗期耐冷性QTL的鉴定与分子定位

 以两份普通野生稻核心种质资源DP15和DP30为供体、9311为受体构建染色体片段代换系鉴定苗期耐冷性QTL;利用苗期耐冷性最强的1个代换系构建QTL作图群体,用SSR标记对其主效QTL进行定位。研究结果表明,两个抗源DP15和DP30所含的苗期耐冷性QTL的数量、位点及耐冷性效应均存在明显的差异。在基本上覆盖两个亲本全基因组的230份BC4F2代换系中共发现19个苗期耐冷性QTL,分布在水稻12条染色体上,第3和第8染色体上有比较密集的苗期耐冷性QTL分布。这19个分布于全基因组的苗期耐冷性QTL被分别分离到不同的野生稻染色体片段代换系里,效应最小的微效QTL位点所在的代换系在苗期耐冷性鉴定中的活苗率仅为8%,而效应最大的主效QTL位点所在代换系的活苗率达到74%。这个主效QTL qSCT 3 1被定位在第3染色体着丝点附近长臂上的RM15031―RM3400区间,距离最近的标记RM15040、RM1164的遗传距离为1.8 cM。     

转录组测序技术在鉴定小麦染色体臂置换系染色体组成上的应用

为给染色体片段置换系的快速鉴定提供新的技术手段,用3个以普通小麦品种中国春(Chinese Spring,CS)为背景的野生二粒小麦(TTD140)染色体臂置换系(chromosome arm substitution line,CASL)进行转录组测序,并结合SNP分析,获得纯合的SNP,用于鉴定CASL材料中TTD140的置换区段。结果发现,CASL3AL的SNP主要分布在染色体3A的108～750 Mb区段;CASL7BS的SNP分布于染色体7B的0～536 Mb和5A的22～482 Mb区段;CASL4AL的SNP分布比较复杂,除集中在4A的40～745 Mb、7B的0～570 Mb以及5B的410～675 Mb外,还在1A、2A、3A、7A、2B、2D、5D、6D和7D染色体上成簇分布,表明该材料除在预期的染色体4A上保留TTD140的大片段外,还在其他染色体上残留许多TTD140小片段。通过97对多态性SSR标记验证CASL3AL的染色体组成,发现84对标记能检测到TTD带型,其分布范围与转录组数据鉴定结果基本一致。对SNP富集区域的320 bp PCR产物直接测序,发现CASL3AL与CS之间存在7个SNP位点,但与TTD140以及Zavitan参考序列一致。因此,本研究利用转录组测序技术能够有效鉴定小麦染色体片段置换系材料的染色体组成,且比传统的分子标记技术准确、方便、快捷。     

玉米株高qPH10的QTL定位及候选基因预测

利用掖478为轮回亲本、齐319为供体亲本构建的染色体片段代换系CL137为父本,与掖478杂交构建近等基因系F2分离群体,根据齐319、掖478重测序数据开发在双亲中具有多态性的Indel分子标记,在两个环境中对控制玉米株高的10号染色体QTL进行定位。结果表明,2017年的株高表型将QTL定位到标记mk8-bnlg1655之间,位于83.86~85.34 Mb(B73 RefGen_v3)的1.5 Mb区间,表型贡献率为7.57%;2018年株高表型将株高QTL定位到标记mk5-bnlg1655之间,位于82.76~85.34 Mb的2.5 Mb区间,表型贡献率为5.75%。同时检测发现,该QTL主要以加性效应为主,显性效应较小。通过对所定位的QTL重合区间内的基因进行功能注释,预测可能控制株高的候选基因,为后续精细定位第10号染色体株高QTL以及探索候选基因功能机制提供研究基础。     

玉米株高主效QTL qPH2.4的定位分析

课题组前期以玉米自交系郑58为轮回亲本,以昌7-2为供体亲本,通过分子标记辅助选择获得染色体单片段代换系Z12和W16。Z12和W16在bin2.07区域均含有1个来源于昌7-2的染色体片段,株高均显著高于轮回亲本郑58。利用郑58和Z12为材料构建F2分离群体,基于重测序和混合分离分析(BSA)策略,将株高主效QTL qPH2.4定位于第2染色体13.95 Mb(201 457 953~215 022 157 bp)的区域内。利用在目标区间内筛选出的20对多态性分子标记对包含743个单株的郑58×Z12 F2分离群体和包含1 720个单株的郑58×W16 F2分离群体进行基因型分析,结合田间株高数据进行QTL定位,将株高主效QTL qPH2.4定位在InDel分子标记ph-18和ph-19之间,区间的遗传距离为0.57 cM,物理距离为626 kb。参考B73基因组(RefGen_v4)注释信息,该区间内存在17个注释基因,其中,包含可以调控油菜素内酯信号的基因Zm00001d006677。     

基于玉米87-1综3单片段代换系的穗长QTL分析

以优良玉米自交系综3为供体、87-1为受体,利用回交和SSR标记辅助选择相结合的方法,获得了51份纯合的染色体单片段代换系(Single Segment Substitution Line,SSSL),每个SSSL内只含有一个来源于供体的染色体片段。51个代换片段不均匀分布在玉米10条染色体上,代换片段长度在1.75～172.45 cM之间,平均长度为24.44cM。覆盖玉米基因组的总长度为933.90 cM,覆盖率为40.20%。2008年在郑州利用17份SSSL材料及在三亚利用39份SSSL材料对玉米穗长进行了1年2点的表型鉴定,共检测到20个穗长QTL,加性效率百分率在-12.10%～19.18%之间。结果表明,这些单片段代换系存在较大的遗传变异,是用于玉米复杂性状的QTL鉴定、基因克隆及功能分析的理想材料。     

利用重测序的水稻染色体片段代换系定位控制稻米淀粉黏滞性谱QTL

 稻米RVA谱是评价稻米蒸煮与食用品质的重要指标之一,发掘新的控制稻米RVA谱QTL对稻米品质改良具有重要意义。利用以粳稻品种日本晴为受体、籼稻品种9311为供体并经高通量重测序的染色体片段代换系群体为材料,在两年两点的环境下对该群体中控制RVA谱特征值的QTL进行了定位分析。通过单因素方差分析和Dunnett多重比较, 分析染色体片段代换系与受体亲本之间相关RVA谱特征值的差异,以两年两点都能检测到的显著差异位点作为稳定表达的QTL。共检测到10个稳定表达的QTL,包括控制峰值黏度的4个QTL qPKV2.1、qPKV5.1、qPKV7.1和qPKV8.1,控制热浆黏度的2个QTL qHPV5.1和qHPV7.1,控制冷胶黏度的2个QTL qCPV5.1和qCPV7.1及控制消减值的2个QTL qSBV2.1和qSBV7.1。在两年两点的检测中10个稳定表达QTL的贡献率介于-31.8%～53.2%,这10个QTL分布在第2、5、7和8染色体上,其中位于第2、5和7染色体上的位点存在一因多效性。     

作物染色体片段代换系的研究进展

染色体片段代换系是用亲本杂交、回交和分子标记辅助选择技术建立的一系列近等基因系,可以提高对复杂农艺性状QTL或基因定位的精确性,尤其是具有遗传效应的较小的数量性状基因位点,并进行遗传效应分析。目前,已在多种作物中构建了染色体片段代换系。本文对染色体片段代换系的特性及其在主要作物中的应用进行了综述,以期对今后研究染色体片段代换系起到促进作用。     

基于昌7-2导入系发掘干旱胁迫下玉米产量相关QTL位点

以昌7-2为轮回亲本,自交系郑独青为供体亲本,采用回交和定向选择的方法构建高代导入系群体。通过玉米56K芯片对极端株系进行基因分型,以IciMapping逐步回归分析法进行穗重、穗粒重以及百粒重等QTL定位。结果表明,共获得分布于玉米第1、3、5、9、10共5条染色体上的10个QTL位点。其中,与穗重、穗粒重相关的各4个,与百粒重相关的2个。第1、5、10染色体上存在同时控制穗重和穗粒重的相同位点,加性效应均来源于郑独青,贡献率均在22%以上。此外,第10染色体相同位点还同时控制1个微效加性的百粒重QTL。在QTL定位的基础上,获得了多位点聚合的导入系,同时携带第1、5、10染色体上3个QTL位点的导入系,其产量性状表现优于轮回亲本昌7-2。     

玉米产量构成因子染色体代换系的构建及初步评价

以综3和87-1组配的重组自交系中株高和产量差异最大的RIL88和RIL279家系为材料,通过回交转育,结合分子标记辅助选择,建立一套玉米产量构成因子代换系。结果表明,平均代入片段2.88个,平均代入片段长度为26.5cM,全基因组覆盖率达到88.7%,平均背景回复率达到95.8%。同时,对这套代换系的表型效应进行初步评价,发现大多数调查性状具有广泛的变异。     

利用染色体片段置换系定位水稻芽期耐冷性QTL

以籼稻品种9311为受体、粳稻品种日本晴为供体构建的95个染色体片段置换系为材料,在5℃低温条件下进行芽期耐冷性鉴定。结果表明,6个置换系低温处理后的成苗率与受体亲本9311有一定差异,其耐冷性略强于9311。利用代换作图法共鉴定出4个与芽期耐冷性相关的QTL,分别位于水稻第5和第7染色体上。其中qCTB-5-1、qCTB-5-2和qCTB-5-3分别被定位在第5染色体RM267与RM1237、RM2422与RM6054及RM3321与RM1054之间遗传距离分别为21.3cM、27.4cM和12.7cM的置换片段上;qCTB-7被定位在第7染色体RM11-RM2752区间遗传距离为6.8cM的置换片段上。     

Detection of QTL for Cold Tolerance at Bud Bursting Stage Using Chromosome Segment Substitution Lines in Rice (Oryza sativa)

The cold tolerance at the bud bursting stage (CTB) was evaluated at 5°C by using a set of 95 chromosome segment substitution lines (CSSLs) derived from an indica rice 9311 and a japonica rice Nipponbare with a genetic background of 9311. The result showed that six CSSLs had slightly stronger effect on CTB than 9311. Total four quantitative trait loci (QTLs) for CTB were preliminary mapped on chromosomes 5 and 7 by substitution mapping. qCTB-5-1, qCTB-5-2 and qCTB-5-3 were mapped in the region of RM267-RM1237, RM2422-RM6054 and RM3321-RM1054, which were 21.3 cM, 27.4 cM and 12.7 cM in genetic distance on rice chromosome 5, respectively. qCTB-7 was mapped in a 6.8-cM region of RM11-RM2752 on rice chromosome 7.     

Functional roles of root plasticity and its contribution to water uptake and dry matter production of CSSLs with the genetic background of KDML105 under soil moisture fluctuation

Soil moisture fluctuation (SMF) stress due to erratic rainfall in rainfed lowland (RFL) rice ecosystems negatively affect production. Under such condition, root plasticity is one of the key traits that play important roles for plant adaptation. This study aimed to evaluate root plasticity expression and its functional roles in water uptake, dry matter production and yield under SMF using three chromosome segment substitution lines (CSSLs) with major genetic background of KDML105 and a common substituted segment in chromosome 8. The CSSLs showed greater shoot dry matter production than KDML105 under SMF, which was attributed to the maintenance of stomatal conductance resulting in higher grain yield. The root system development based on total root length of the CSSLs were significantly higher than that of KDML105 due to the promoted production of nodal and lateral roots. These results implied that the common substituted segments in chromosome 8 of the 3 CSSLs may be responsible for the expression of their root plasticity under SMF and contributed to the increase in water uptake and consequently dry matter production and yield. These CSSLs could be used as a good source of genetic material for drought resistance breeding programs targeting rainfed lowland condition with fluctuating soil moisture environments and for further genetic studies to elucidate mechanisms underlying root plasticity.     

利用染色体片段置换系定位水稻赖氨酸含量的QTL

 以籼稻品种9311为受体、粳稻品种日本晴为供体构建95个染色体片段置换系, 对水稻控制赖氨酸含量的QTL进行了定位。结果显示, 共有7个染色体片段置换系（chromosome segment substitution line, CSSL）的稻米赖氨酸含量与亲本9311差异显著。利用代换作图法共鉴定了4个与赖氨酸含量相关的QTL, 分别位于水稻第8、9和12染色体上。其中qHLY8和qHLY9.2来自高赖氨酸含量籼稻品种9311, 正向加性效应百分率分别为9.6%和8.5%;而qHLY9.1和qHLY12则来自低赖氨酸含量粳稻品种日本晴, 负向加性效应百分率分别为-16.0%和-21.3%。     

基于重测序的染色体片段代换系定位水稻抽穗期QTL

【目的】水稻的抽穗期是决定水稻产量及其适用性的重要农艺性状之一,是由多基因控制的数量性状。染色体片段代换系减少了个体间遗传背景的干扰,已经成为定位和克隆复杂性状QTL的重要材料。【方法】本研究以9311为受体,日本晴为供体构建的128个重测序的染色体片段代换系群体为试验材料,利用多元回归,结合Bin-map图谱,【结果】鉴定到6个在南京、扬州不同年份间稳定表达的抽穗期QTL,其中,qHD2.1被定位在第2染色体上的759 848 bp区间内;qHD2.2被定位在第2染色体上的45 286 bp区间内;qHD 3.1被定位在第3染色体上的147 931 bp区间内;qHD5.1被定位在第5染色体上的213 351 bp区间内;qHD5.2被定位在第5染色体上的442 305 bp区间内;qHD8.1被定位在第8染色体上的538 176 bp区间内。【结论】本研究为精细定位并克隆相应QTL,进而探明抽穗期QTL的分子调控机制奠定了基础。     

Identification of QTLs for Improvement of Plant Type in Rice (Oryza sativa L.) Using Koshihikari / Kasalath Chromosome Segment Substitution Lines and Backcross Progeny F2 Population

Abstract

Thirty-nine chromosome segment substitution lines (CSSLs) population derived from a Koshihikari / Kasalath cross was used for quantitative trait locus (QTL) analysis of plant type in rice (Oryza sativa L.). Putative rough QTLs (26.2～60.3cM of Kasalath chromosomal segments) for culm length, plant height, panicle number, chlorophyll content of flag leaf blade at heading and specific leaf weight, were mapped on the several chromosomal segments based on the comparison of CSSLs with Koshihikari in the field experiment for 3 years. In order to verify and narrow QTLs detected in CSSLs, we conducted QTL analyses using F₂ populations derived from a cross between Koshihikari and target CSSL holding a putative rough QTL. The qPN-2, QTL for panicle number was mapped on chromosome 2. In traits of flag leaf, the qCHL-4-1 and qCHL-4-2 for chlorophyll content was mapped on chromosome 4, and the qSLW-7 for specific leaf weight on chromosome 7. All QTLs were detected in narrow marker intervals, compared with rough QTLs in CSSLs. The qPN-2, qCHL-4-1 and qCHL-4-2 had only additive effect. On the other hand, the qSLW-7 showed over-dominance. It could be emphasized that QTL analysis in the present study with the combination of CSSLs and backcross progeny F₂ population can not only verify the rough QTLs detected in CSSLs but also estimate allelic effects on the QTL.     

The QTL Analysis of RuBisCO in Flag Leaves and Non-Structural Carbohydrates in Leaf Sheaths of Rice Using Chromosome Segment Substitution Lines and Backcross Progeny F2 Populations

Abstract

In rice (Oryza sativa L.), the maintenance of high photosynthetic rate of flag leaves and the carbon remobilization from leaf sheaths after heading is a critical physiological component affecting the yield. To clarify the genetic basis of RuBisCO content of the flag leaf, a major determinant of photosynthetic rate, and non-structural carbohydrate (NSC) concentration in the third leaf sheath at heading, we carried out quantitative trait loci (QTL) analysis with 39 Koshihikari/Kasalath chromosome segment substitution lines (CSSLs) and backcross progeny F₂ population derived from target CSSL holding the QTL/Koshihikari in the field. QTLs for RuBisCO content and NSC concentration at heading were detected between R2447-C1286 and R2447-R716 on chromosome 10, respectively, by comparing Koshihikari with four CSSLs for chromosome 10 (SL-229, -230, -231 and -232). The progeny QTL for RuBisCO content and for NSC concentration at heading qRCH-10 and qNSCLSH-10-1, respectively, were detected at similar marker intervals between RM8201 and RM5708. In addition, QTLs for RuBisCO content at 14 d after heading, qRCAH-10-1 and qRCAH-10-2, were detected in regions different from that of qRCH-10. No QTL for NSC concentration at 14 d after heading was detected between RM8201 and R716, the region analyzed in this study. The QTLs qRCH-10 and qRCAH-10-1 for RuBisCO content would have additive effects. These QTLs for RuBisCO content and NSC concentration newly found using CSSLs and their backcross progeny F₂ population should be useful for better understanding the genetic basis of source and temporary-sink functions in rice and for genetic improvement of Koshihikari in terms of their functions.     

QTL analysis of cold tolerance at seedling stage in rice (Oryza sativa L.) using recombination inbred lines

Cold tolerance at seedling stage of rice (Oryza sativa L.) is a favorable trait for the stable establishment in temperate and high-elevation areas. In the present study, 71 recombinant inbred lines (RIL) derived from the cross of Asominori (Japonica) and IR24 (Indica) were used to identify quantitative trait loci (QTL) affecting cold tolerance at seedling stage. The putative QTL was further confirmed using some chromosome segment substitution lines (CSSLs), in which IR24 was used as the donor parent and Asominori as the recurrent parent. The average seedling mortality was used as cold tolerance after cold treatment with 6 °C for 7 days and recovery culture with 25 °C for 4 days at three-leaf seedling stage. Three QTL affecting cold tolerance at seedling stage were detected on chromosomes 1, 5 and 6 with LOD scores ranging from 2.2 to 4.1 using composite interval mapping (CIM). Among them, qSCT-1 located in the region of XNpb87-2-C955 on chromosome 1 was a major QTL which explained 24.51% of total phenotypic variance and favorable allele came from japonica parent, Asominori. In addition, IR24 alleles at the other two loci (qSCT-5 and qSCT-6) increased cold tolerance. And these three QTL were confirmed by four lines from the IR24 CSSLs. Transferring favorable allele from japonica variety to indica background or pyramiding different QTL identified from indica is an effective way to improve cold tolerance of rice.