Establishment And Integration Of Genetic Linkage Map And QTL Analysis Of Drought Tolerance At Seedling Stage Of Soybean

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.