摘要
水稻(Oryza sativa L.)是世界上三大主要粮食作物之一。籼稻(O. sativa ssp. Indica)和粳稻(O. sativa ssp.Japonica)作为亚洲栽培稻的两个重要亚种,经历了漫长时间的进化,其基因组存在着高度的遗传分化,在产量性状和生态适应性等诸多方面表现出较大的差异。作为水稻两个典型的杂种优势群,籼稻和粳稻的杂交又能产生巨大的优势。因此,明晰籼(或粳)稻的等位基因在粳(或籼)稻遗传背景中的遗传特点,挖掘影响产量等相关性状的基因,对于籼粳亚种间优良基因的转移及杂种优势利用具有十分重要的理论和实践意义。水稻产量等性状一般属于多基因控制的数量性状,通常采用籼粳杂交衍生的分离群体,如F2、RIL以及DH等对控制这些性状的数量性状位点(quantitative traits locus, QTL)数目、染色体上的位置及遗传效应进行分析,也取得了一定进展。但这些传统的分析群体遗传背景复杂,估计的QTL往往不太准确,一些效应值较小的QTL很难检测到。染色体片段置换系群体(chromosome segment substitution lines, CSSLs)是指一套以相同亲本为遗传背景,置换了供体亲本一个或少数染色体片段的系列单株组成的群体,由于其遗传背景简单,群体变异小,便于研究者进行多年多点的重复试验等特点,备受研究者的重视,染色体片段置换系群体不仅大大提高了对复杂数量性状基因定位的精确性,而且通过构建次级分离群体可以对目标QTL进行精细定位和克隆分析。
本研究通过杂交、回交和自交,并在高世代构建DNA池,利用分子标记辅助选择的方法,将优良粳稻品种C418的染色体片段导入到了优良的、并已完成测序的籼稻品种9311的遗传背景中,培育出一套置换片段相互重叠、覆盖粳稻全基因组的水稻染色体片段置换系群体,旨在为水稻重要产量性状的基因(QTL)定位、图位克隆以及水稻杂种优势研究提供基础材料。研究结果如下：
1)从实验室公共的1000对SSR引物和自行设计的40对In/del标记中,筛选出158对在双亲间表现出多态性的标记,多态性比例为14.25%。多态性标记在不同染色体上的分布频率有一定差异,其中第3和6染色体最低,仅有11.11%；第9染色体相对较高,达24.39%。选择其中在12条染色体上分布相对均匀的136个标记,参考Temnykh, McCouch和the International Rice Genome Sequencing Project等发表的遗传图谱,构建了分子标记连锁图。该图谱覆盖全基因组1480.9 cM,标记间平均间距为10.89 cM,用于分析"C418/9311”BC3F4,BC3F6,BC3F8和SF2世代单株或DNA池的基因型,分子标记辅助选择目标单株。
2)利用上述的136对多态性标记分析了“C418/9311”BC3F4及其以后各世代群体的基因型,以每个单株含有尽可能少的供体染色体片段及所有家系包含的置换片段能最大程度覆盖整个供体亲本"C418”全基因组两个条件为标准,最终从BC3F8和SF2两个不同世代中筛选出108个株系,构成了一套新的籼、粳染色体片段置换系群体。
3)通过对置换系群体的遗传组成分析表明,置换系群体平均每个株系包含置换的分子标记数目占总标记数的比例为4.76%,置换的标记数变异范围在1-15个之间；其中5.96个为纯合供体基因型标记,0.51个为杂合基因型标记。
108个置换系家系置换的片段总长度为2586.3 cM,相当于水稻基因组总长度的1.7倍,代表了水稻的12条染色体。代表每条染色体的家系数目是有差别的,其中,第2染色体的代表家系数最多,有16个；其次是第4和7染色体,分别有11个；第5和10染色体的代表家系数较少,分别有6个和5个。每条染色体平均代表家系数目为9个。
4)以“C418/9311”染色体片段置换系为材料,采用改良的QTL IciMapping v2.2 Mapping软件,分析了2008年南京,2008年海南两个不同环境下籽粒性状的QTL(粒长、粒宽、粒厚、长宽比、长厚比和千粒重),共检测到70个籽粒性状QTL,分布在水稻的11条染色体上,其中11个QTL能在两个环境下重复检测到,即千粒重QTL-qTGW6.1和qTGW7,粒长QTL-qGL5、粒宽QTL-qGW5,qGW6.1和qGW7.2以及共同作用于长宽比的QTL-qLW5.1和谷粒体积QTL-qGV2,qGV7.4,qGV8.1和qGV9,其中,qTGW7位于第7染色体RM8261标记的染色体区段,是一个新的千粒重QTL。通过建立次级F2分离群体,将qTGW7定位在第7染色体的短臂上,与标记RM22034紧密连锁；后代近等基因系(NIL)的表型结果也证明了前期QTL定位的正确性,同时确定qTGW7与RM22034紧密连锁。利用C418/9311的置换系群体对籽粒性状研究的结果证实“C418/9311”染色体片段置换系应用于数量性状基因座分析是可行的。
5)选取了64个能覆盖C418 12条染色体全基因组的置换系家系分别与9311杂交形成了含64个家系的杂交组合群体(CSSLHs),采用QTL IciMapping v2.2软件分析了CSSLs和CSSLHs两群体中产量和产量相关性状具有显著遗传效应的位点(QTL)。2008和2009两年,分别检测到87和93个QTL,这些QTL大部分的贡献率都小于20%。在2008年,62.1%(54)QTL具有超显性效应,2009年61.3%(57)QTL具有超显性效应,表明在单位点水平上,超显性对产量及产量相关性状杂种优势的形成具有重要的作用,而含这些杂种优势位点的家系也成了杂交育种的理想材料。此外这些QTL中仅少数能在CSSLs和CSSLHs中同时检测到,表明水稻产量及产量相关性状和杂种优势可能由两套不同的遗传机理控制。
Rice is one of the most important crops in the world. Indica and Japonica are two subspecies of Asian cultivated rice and show high genetic differentiation. Great heterosis also exists in the inter-subspecific crosses between Indica and Japonica rice cultivars. The genetic effect of alleles from japonica (or indica) in a genetic background of indica (or japonica) and the efficiently utilization of inter-subspecific heterosis between Indica and Japonica has its important theory meaning and practical meaning. In the aforementioned studies, F2, F3 populations and the populations derived by backcrossing recombinant inbred lines (RILs) with the parents were usually used. Due to genetic background noise in these mapping populations, QTL location and its effect was not easily and precisely estimated. Especially, the identity of some minor QTL with a low LOD score could be ignored. Chromosome segment substitution lines (CSSLs), with each line carries a single or fewer defined chromosome segment of donor genome, have a pure genetic background from a recurrent genotype. Additionally, CSSLs are directly used in breeding programs when their genetic background is an elite cultivar. Moreover, secondary F2 population can be derived from a further backcross between a selected CSSL and the recurrent parent, and then be used in the fine mapping. For these reasons, the development of series of chromosome segment substitution lines (CSSLs) has been an effective way to isolate useful genes.
In order to breed a set of CSSL population for fine mapping of quantitative trait loci, map-based cloning and heterosis analysis, the present study used C418 and 9311, which are elite indica and japonica cultivars respectively, as the materials, to develop a set of chromosome segment substitution lines by using molecular marker aided selection. The main results are as follows:
1) A total 1000 pairs of SSR primers and 40 pairs of new developed in/del markers were used to detect the polymorphism between the parents, among them,158 pairs, which account for 14.25% of the total SSR markers, showed polymorphism. The frequency of the polymorphism markers varied from chromosomes, with the highest of 24.39% on chromosome 9 and the lowest of 11.1% on chromosome 3 and 6. The marker linkage map was built according to marker distance report by Temnykh et al., McCouch et al. and the International Rice Genome Sequencing Project, which is consisted of 136 molecualr loci, spanned a total of 1,480.9 cM on all 12 chromosomes with an average interval of 10.89 cM between adjacent markers. The candidate substitution lines were marker assisted selected from the progenies with these molecular markers.
2) The genotype of the BC3F4 lines and its consecutive progenies derived from "C418/9311" were analyzed with the 136 molecular markers. Eventually, the chromosome segment substitution lines, which were composed of 108 lines and chosen from BC3F8 and SF2 progenies, were developed with the following criterias:a) a high number of purely recurrent linkage groups;b) a wide coverage of the genome by the donor substituted segments.
3) The average percent of substitution markers in the 108 lines is 4.76%, with a range of 1 to 15, among them,5.96 and 0.59 markers exhibited homologous donor and heterozygous type, respectively.
The substitution genome length was 2586.3 cM, about as many as 1.7 times of the rice genome. The 12 chromosomes'genome was substituted by 108 lines, the most substitution lines were on chromosome 2, including 16 substitution lines;next were chromosomes 4 and 7, including 11 substitution lines;the least substitution lines were on chromosomes 5 and 10,6 and 5 respectively, the average substitution lines were 9.
4) The CSSLs were used to detect the quantitative trait locus (QTL) for kernel traits in two contrasting environments, and led to the identification of 70 quantitative trait loci (QTL), distributing on 11 chromosomes. Out of these QTL affecting kernel traits,11 QTL were simultaneously identified in both environments:1000-grain weight QTL-qTGW6.1, qTGW7; grain length QTL-qGL5;grain width QTL-qGW5, qGW6.1 and qGW7.2;grain length and width ratio QTL-qLW5.1 and grain volume QTL-qGV2, qGV7.4, qGV8.1 and qGV9. More detailed mapping of qTGW7 showed that it was co-segregated with RM22034 on the short arm of chromosome 7. These results indicated that the CSSLs were effectively to identify quantitative trait loci associated with important agronomy traits, and provided rich resource for rice molecular breeding synchronously.
5) 64 lines from these CSSLs were crossed one by one with the recipient parent to generate a set of corresponding CSSL hybrids (CSSLHs). These materials were field-tested over two years for yield and yield-related traits. A total of 87 (in 2008) and 93 (in 2009) QTL were detected. In 2008,62.1%(54) of the QTL were over-dominant, in 2009, this proportion was 61.3%(57), indicating that over dominance was a major contributor to heterosis. Some of the CSSLs harboured QTL associated with heterosis in both years;these should represent potential candidates as parents of F1 hybrids along with cv.9311 or other indica lines. Moreover, few of these yield-related QTL were detected in both CSSLs and CSSLHs, indicating that the main yield-related traits and heterosis were formed by two different genetic mechanisms.
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