【作者】 周斌；

【导师】 邢邯；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 大豆[Glycine max (L.) Merr.]是世界上最重要的经济作物之一,是人类蛋白质和脂肪的重要来源,在食品、饲料、工业上具有广泛的用途。消费需求的日益增加推动了大豆产量的不断提高。诸多的环境胁迫影响着大豆的产量和质量,其中干旱对大豆的生产危害最为严重。培育耐旱品种是解决干旱胁迫的根本途径。种质资源的耐旱性鉴定是耐旱育种的前提和基础,贯穿于育种过程的始终。耐旱QTL的挖掘和利用对阐明耐旱遗传机制、提高育种效率、加快育种进程有着重要意义。QTL定位的有效性与遗传连锁图谱的饱和度、精确度,以及实用性和通用性密切相关。为得到更高饱和度和精确度的大豆遗传连锁图谱,本文使用967对SSR引物对8个大豆重组自交系群体多态性进行检测,选择多态性高的群体分别构建遗传连锁图谱,然后利用各图谱共同标记整合成一张较高饱和度的遗传连锁图谱,并利用加密后的NJRIKY图谱对8个农艺性状进行了QTL重定位。利用隶属函数法以平均隶属函数值为指标鉴定了945份大豆种质以及大豆重组自交系群体NJRIKY的苗期耐旱性,采用两种QTL分析方法对苗期耐旱性进行了QTL分析,并对我国黄淮和南方育成品种抽样群体进行了耐旱性状与SSR的关联分析,对耐旱相关位点的优异等位变异进行了5个育成品种家族内的系谱追踪。全文主要结果如下：1、利用SSR标记对已有NJRIKY遗传图谱进行加密,获得一张含有553个遗传标记,25个连锁群,总长2071.6 cM,平均图距为3.70 cM的新遗传连锁图谱,其中SSR标记316个,RFLP标记197个,EST标记39个,形态标记1个。连锁群上大于20 cM的标记间隔由原来39个减少到2个。利用加密图谱将7个SMV抗性基因重定位于Dlb连锁群,与相邻分子标记距离均缩小,进一步验证了抗性基因成簇分布的现象。对本群体8个农艺性状进行QTL重定位,各QTL的标记区间明显缩短,与相邻标记的连锁更加紧密。2、利用筛选出的具明显农艺性状差异的4个大豆重组自交系群体分别构建了遗传连锁图谱,以图谱间共有SSR标记作为锚定标记,使用JoinMap 3.0进行图谱整合,得到一张包含20个连锁群,795个分子标记,总遗传距离2772.9 cM,平均图距3.49 cM的高密度整合图谱。与Song等的公共图谱比较,本图谱定位了5个在公共图谱上没有的SSR标记,另有6个SSR标记定位在不同的连锁群上,其余标记在连锁群上的分布和位置与公共图谱高度吻合。整合图谱可用于将标记分析或关联分析所获基因/QTL定位到连锁群区间,便于不同群体定位结果间的比较,并通过整合图谱寻找与之连锁更紧密的邻近标记。鉴于本图谱所用作图群体的亲本与国内育种常用材料的遗传来源相近,将更有利于国内育种性状QTL的相关研究。3、利用隶属函数法,以株高、叶龄、地上部分干重和地下部分干重四个指标的平均隶属函数值为标准评价了945份大豆种质的耐旱能力,以5级标准划分,鉴定出Ⅰ级耐旱材料3个(平均隶属函数值Fi≥0.8,强耐旱型),Ⅱ级材料48个(平均隶属函数值0.6≤Fi<0.8,较强耐旱型),Ⅲ级材料478个(平均隶属函数值0.4≤Fi<0.6,中间型),Ⅳ级材料401个(平均隶属函数值0.2≤Fi<0.4,干旱较敏感型),Ⅴ级材料15个(平均隶属函数值0.2<Fi,干旱极敏感型)。大部分品种的耐旱性属于中间型,两极端类型较少,品种间存在明显的遗传差异。对3个地理群体和3个品种群体分别进行了耐旱性评价。4、运用家系连锁分析方法在重组自交系群体NJRIKY中定位了3个耐旱QTL(LOD≥3.0):C1连锁群上qDr-C1-1的标记区间为Satt396-GMAC7L,表型变异解释率为14.1%;D1b连锁群上qDr-D1b-1的标记区间为BE475343-Satt271,表型变异解释率为7.8%;F2连锁群上qDr-F2-1的标记区间为Satt586-Satt343,表型变异解释率为7.9%。5、运用基于连锁不平衡的关联分析方法在173份黄淮及南方育成品种构成的群体中检测到5个与耐旱性状关联的SSR位点：位于C2连锁群上的Satt277、Satt307,位于D1b连锁群上的Satt005,位于F连锁群上的Satt334以及位于H连锁群上的Satt442。5个SSR位点的贡献率均大于6%,其中位于C1连锁群上的位点Satt277的贡献率达12.5%。耐旱材料矮脚青(南农编号：N03191,Fi=0.789)在5个耐旱关联位点中都存在增效等位变异,总效应达到了0.671,可作为优异种质运用于耐旱育种。6、对5个关联SSR标记的15个优异等位变异在5个大豆育成品种家族内进行系谱内优异等位变异追踪,发现不同的家族包含的优异等位变异数不同,各自的优势等位变异也不相同。Satt307-A183是5个家族共有的优异等位变异,但贡献率各异。徐豆1号、齐黄1号和南农493-1家族拥有各自的特异优异等位变异。家族内品种耐旱能力与所含有的优异等位变异数有一定的正相关。优异等位变异仅能解释部分耐旱效应,耐旱性不是优异等位变异效应的简单叠加。更多还原

【Abstract】 Soybean [Glycine max (L.) Merr] is now an essential and dominant source of protein and oil with numerous uses in feed, food, and industrial applications. It is the world’s primary source of vegetable oil for people and protein feed supplement for livestock. The steadily increasing consumption of soybean year by year requests more and more soybean seeds can be yielded from the field. Global soybean production and crop quality are severely affected by various environmental stresses and drought is the most devastating. The essential solution to drought stress is to develop soybean plants with enhanced drought tolerance. The identification of soybean germplasms with drought tolerance through the breeding process is the very basic work prior to all other efforts. So a set of 945 germplasms from over 15,000 of NJAU-NCSI resources collections is selected to identify their respective drought tolerance to find out the ones with high tolerance. Dought tolerance is a complex quantitative trait controlled by many minor effect genes called quantitative trait loci (QTL). The isolation and utilization of drought tolerance QTL can be very helpful to understand genetic mechanism of drought tolerance, speed up the breeding process and improve breeding efficiency. And these rely on a suitable genetic linkage map with high saturation.967 pairs primers of SSR were used to detect the polymorphism of 8 recombined inbred lines populations derived from Chinese domestic soybean varieties to find the ones with highest polymorphism to construct soybean genetic linkage maps and, an integrated map. Together with the trait phenotypic value, two QTL mapping strategies, family-based linkage mapping and linkage-disequilibrium-based association analysis, were applied to detect the drought tolerance QTL in the recombined inbred lines population NJRIKY and in a natural population that contains 173 released cultivars from Huanghuai and Southern China. and with that, tracing elite alleles of drought tolerance in the pedigree of major cultivar families in HuangHuai and Southern China was completed. The main results of present study are as follows: a) Total 401 polymorphic SSR markers were screened out from 967 ones for density-enhancement of the previous NJRIKY genetic linkage map. Along with other marker data, a new genetic linkage map was constructed by using Mapmaker/exp 3.0b, with 553 markers, including 316 SSR,197 RFLP,39 EST and one morphologic markers, spanning 25 linkage groups, covering total length 2071.6cM of the soybean genome, with an average marker interval distance of 3.70cM. In comparison with the old map, the number of gaps larger than 20 cM decreased from 39 to two on the enhanced map. Using this map to relocate the seven SMV resistant genes, Rsc-3, Rsc-7, Rsc-9, Rsc-13, Rsa, Rn1 and Rn3 were mapped on LG D1b again with distances to the flanking markers all less than 6cM, among them, Rsc-9, Rn1 and Rsa less than 1cM and Rsc-13 co-segregating with EST-SSR marker GMKF168a. After re-mapping the QTL of the eight agronomic traits,42 ones were detected on 12 linkage groups, with 20 of them accounted for more than 10% of the total variation, respectively, and their marker intervals obviously shortened, as well as some of the false QTL eliminated.b) By evaluating the polymorphism of the 8 RIL populations, the highest polymorphic three RIL populations, Nannong 87-23×NG94-156, Su 88-M21×Xinyixiaoheidou and Wan 82-178×Tongshanbopihuangdoujia derived from the crosses between distinct elite cultivars of Glycine max (L.) Merr. were used at first to construct individual genetic linkage maps with 560,223,195 and 133 markers, respectively, by using the software JoinMap 3.0. Then based on the common SSR markers across the four maps, the individual maps were integrated into a joint genetic linkage map by using the same software, which containing 795 markers spanning 2772.9 cM of the soybean genome, distributed on 20 linkage groups with the length of linkage groups varied from 77.1 cM to 224.7 cM, the marker number from 24 to 69, and an average marker distance of 3.49 cM. Among the linkage groups, C2, C1, N and F are obviously highly saturated. In comparison with Song et al.’s genetic linkage map, the present map shows a good coincidence except the six SSR markers located on other linkage groups and five new SSR markers added to the present map. The integrated map was used in QTL mapping and performed a reasonable result in comparison with the individual map itself. Therefore, the present map is potential in QTL mapping study, especially for domestic soybean breeding purposes since the parental materials of the four RIL populations are closely related to the breeding materials in Chinese breeding programs. c) A total of 945 soybean germplasms from the NJAU-NCSI soybean germplasm collection were evaluated for drought tolerance in rain-proof sheds in Nanjing and were ranked in one of the five drought tolerance degrees by their respective average membership function values(Fi). Among the 945 germplasms, three with the Fi over 0.8, the most tolerant, were ranked in Grade One (Ⅰ) according to the grading standard; 48 with Fi between 0.6 and 0.8, more tolerant, were ranked in Grade Two (Ⅱ); 478 and 401 with Fi lies in 0.4～0.6 and 0.2～0.4, the medium and more sensitive, were in Grade Three (Ⅲ) and Grade Four (Ⅳ), respectively; 14 germplasms, with the lowest Fi values, less than 0.2, were most sensitive to drought, belongs to Grade Five(Ⅴ). When sorting them by ecological regions and by genetic origins, a same tendency was seen on the distribution of germplasms of each status, ie. most germplasms are intermediate between tolerant and sensitive and only a few ones in the two terminals.d) The drought tolerance of 184 lines of RIL population NJRIKY were identified and then used in the QTL analysis by using the family-based likage analysis strategy with the CIM arithmetic of QTL software, WinQTLCart 2.5. Three QTL with LOD score greater than 3.0 were detected and mapped on linkage groups C1, Dlb and F2. The most significant QTL, qDr-C1-1, which located at the interval Satt396-GMAC7L on linkage group C1, accounted for 14.1% variation. The other two QTL, qDr-D1b-1 on LG D1b and qDr-F2-1 on LG F2, accounted of 7.8% and 7.9% variation, respectively.e) A set of 173 released cultivars from Huanghuai and Southern China were picked out from the 945 germplasm to detect the SSR loci which associated with drought tolerance by using the association analysis method. Five SSR loci were found to be associated with drought tolerance in the released cultivar population. These five loci distributed on 4 linkage groups and all of them acounted for over 6% variation. Among them, the locus Satt277 on LG C2 acounted for 12.5%. The cultivar Aijiaoqing (N03191), whose Fi was 0.789, had positive effect alleles in all the five SSR loci and the total effect value amazingly reached 0.671. Considering its highly pyramiding of elite alleles, maybe it can act as an excellent material and play a very important role in the drought tolerance breeding.f) 149 cultivars belonged to five mainly released cultivar families of Huanghuai and Southern in China,58-161, Xudou 1, Qihuang 1, Nannong 493-1 and Nannong 1138-2 were used to trace the drought tolerance elite alleles in each pedigree. The number of elite alleles differed in different families, also each family had its own elite alleles. Satt307-A183 was the only common allele among five families, but the variation is different. The families Xudou 1, Qihuang 1 and Nannong 493-1 had their own unique elite alleles. The general trend in five families was that the elite alleles number was positive correlated with the drought tolerance, the cultivars with more elite alleles often showed higher tolerance than those with fewer. Elite alleles can only acounted partial variation, tolerance was not a simple addition of them. Enhancing drought tolerance by pyramiding elite alleles was still far from realization.更多还原