| Soybean(Glycine max[L.]Merrill)not only serves as one of the most important edible oil and protein crops for human beings,but also provides a large amount of feeds for livestock and aquaculture in the world.Among the three main soybean production regions of China,i.e.Northeast China,Huang-Huai Valleys and Southern China,the latter,including the Middle and Lower Changjiang Valleys and their south,is characterized with complicated topography and multiple cropping systems.The soybean landraces in this region are especially diverse;more than half of the nation-wide soybean germplasm resources came from this region.Except the Middle and Lower Changjiang Valleys,the southeast part of this region is hilly areas(i.e.Hubei,Hunan,Jiangxi,Anhui,Zhejiang,Jiangsu Fujian and parts of Guangdong and Guangxi provinces),the souuthwest part is karst plateau region(i.e.Yunnan,Guizhou and mountainous region of Sichuan,Hunan and Hubei provinces),while it also includes Sichuan Basin and Jianghan Plain.Soybean is a short-day crop with sensitive response to changes of photo-thermal conditions,and differences in day-length and temperature caused by different geographical latitudes and elevations may lead to soybean diferentiation.There fore,the geographic factors play key roles in differentiation among soybeans in different areas along with its dissemination.The early years of agriculture was in a one-year full-season cropping system,as the old saying:ploughing in spring,weeding in summer,harvesting in autumn,and storing in winter.The ancient full-season soybean varieties have disappeared and the extant varieties are the products of the modern multiple cropping systems that can fit respective growing season.Soybeans were divided into spring-,summer-,autumn-sowing types(winter-sowing is in a seldom situation,such as winter nursery for adding generations),which is in fact a significant characteristic of soybeans in southern China.The day length changes with seasons in a year,therefore,sowing-season is another ecological factor determining the differentiation of soybeans in addition to the geographic factor.Since the genetic study on southern China soybean germplasm population was very few,while as the probable center of origin,this population is very important to the world soybean study.Therefore,it is worthwhile to make a thorough study on this germplasm population ecologically,genetically and evolutionarily.Southern China has a vast territory and is rich in illumination,water and temperature resources.Therefore,intercropping patterns of soybean is an important model in Southern China.Shade tolerance is an important factor that affects the yield of intercropped soybean,thus limiting the extension of intercropping patterns of soybean with high-altitude plants such as corn,sugar cane,cassava and fruit trees.It is necessary to establish an efficient,versatile and reliable shade tolerance identification system to be used in identifying the shade tolerant sources from germplasm populations,to clarify the shade tolerance of breeding materials and to study its genetic mechanism which can be used for shade tolerance breeding.Which promote the promotion of soybean intercropping mode and the development of the soybean industry.In this study,394 varieties were selected to form a representative sample of Southern China soybean germplasm population(SCSGP),covering four geo-regions in southern China,i.e.Eco-region ?,?,? and ?.Here we grouped all the spring-,summer-and autumn-sowing types into only SA and SP two types because their photo-thermal responses mainly can be distinguished as the two types,insensitive(spring-sowing)and sensitive(summer-and aurumn-sowing).According to the geographic and sowing-seasonal factors,we divided them into eight eco-populations,i.e.SA-?,SA-?,SA-?,SA-?,SP-?,SP-?,SP-?,and SP-?,where SA-? is the summer/autumn-sowing type in Eco-region ?;SP-? is the spring-sowing soybeans in Eco-region ?,the similar is for the others.The genome-wide 123,065 SNPLDB markers obtained by resequencing technology were used to analyze the genetic diversity,population differentiation and genetic relationship of different geo-seasonal populations,and 127 wild soybeans from the whole country were used as reference species to explore the origin of cultivated soybeans.At the same time,three shading degrees and three Identification period were set,and 26 traits were screened for shade tolerance based on heritability,error coefficient of variation,genetic variation coefficient and correlation with shade tolerance level Then shade tolerance of 915 soybean germplasm resources in the Southern China were identified under three environments.Finally using the newly developed"RTM-GWAS" association analysis method combined with SNPLDB to genetically analyze the shade tolerance and relevant ecological traits of soybean.The main results are as follows:1 Differentiation and evolution among geographic and seasonal eco-populations of soybean germplasm in southern ChinaThe geographic-seasonal differentiation of days to flowering(DTF)in the SCSGP was significant.DTF increased gradually from Eco-region ? to IV,VI,and V,in turn.The average,range and GCV in SA eco-population were higher than those in SP eco-population.Among the geo-seasonal eco-populations,the largest average DTF of SP geo-ecotypes(SP-V)was smaller than the smallest one of SA geo-ecotypes(SA-?).The largest differentiation between SA type and SP type in an eco-region was observed in Eco-region ?(15.9 d),followed by ?(13.0 d),?(12.2 d),and ?(7.0 d).The smallest genetic distance was between Eco-region ? and IV ecotypes,whereas Eco-region ? ecotypes was distant to that in other eco-regions.The ?2 test showed a significant correlation(P<0.0001)between ecotype grouping and Neighbor-Joining clustering,and the Mantel test showed that there was significant correlation(R=0.623,p<0.01)between the genetic distance matrix among ecotypes and the difference of ecological traits matrix,indicating that the ecological differentiation was closely related to genetic differentiation.Between the two sowing-seasonal ecotypes,the SA eco-population was characterized with a higher genetic diversity(? value 0.942 vs.0.938),a higher inherited allele(374,872 vs.361,357),less specific newly emerged alleles(2504 vs.2675),smaller Linkage disequilibrium(LD)(r2,0.304 vs 0.366),and smaller genetic distance to the wild soybean(0.4322 vs 0.4340),thus the SA eco-population might be the more primitive one.The wild accessions from Southern China have relatively small genetic distances to all cultivated ones from the four ecoregions,therefore,we inferred that the ancient G.sojapopulation from Eco-region ? might be the common ancestor of all the cultivated soybeans.SA-? was characterized with the closest genetic distance to the wild soybean(0.4155),the most inherited allele variation(319,995),the smallest LD level Therefore,the SA-?might be the most primitive type of cultivated soybean population.Among the four SA eco-populations,SA-? shared the largest number of multi-population specific present alleles(MPSPA,101675/0.63)with SA-? among the pairs of SA-? with other SA eco-populations,indicating that SA-IV might evolve gradually through adaptation from the most primitive SA-?.SA-? shared the largest number of MPSPAs(54206/0.39)with SA-? among pairs of SA-VI with other SA eco-populations,indicating that SA-? might evolve gradually from SA-?.SA-? shared the largest number of MPSPAs(87550/0.57)with SA-? among pairs of SA-? with other SA eco-populations,moreover,SA-? shared a large number of MPSPAs with SA-?,implying that SA-? might evolve gradually from SA-? and SA-?.The SP type was mainly evolved from its SA counterpart for shared the largest number of MPSPAs(except SP-?)in each eco-region.In addition,the Mantel test showed that there was significant negative correlation(R=-0.601,p<0.001)between the genetic distance matrix among ecotypes and the ratio matrix(the ratio of the MPSPAs shared between two groups to the total of MPSPAs in these two groups).From the above genetic relationship,the evolutionary route of cultivated soybeans in southern China was proposed:firstly,the original SA-? was domesticated from wild soybeans in the middle and lower reaches of the Yangtze River,and then SA-?,SA-? and SP-? were derived from SA-?,and then,SA-? and SP-? were derived from the original SA-?;SP-? were derived from SA-?;and SP-? mainly has three sources(SA-?,SA-?and SP-?).2 Establishment of an evaluation system of shade tolerance in soybean and its variation in southern china germplasm populationThe suitable shading treatment for shade tolerance evaluation was chosen among the 15%,30%and 60%three shading levels according to a reasonable lodging rate of the tested varieties.The 30%shading intension condition was better than other shading degrees.More specifically,there showed lower lodging rate of accessions(22%),higher phenotypic coefficient of variation(25%),and better distinction among accessions.The suitable shade tolerance indicators were chosen from the 26 traits,including plant height(PH,diameter,mean internode length(MINL),fifth intemode length,antepenult internode length,number of nodes in main stem(NN),fifth internode diameter,antepenult internode diameter,leaf length,leaf width,leaf shape index,petiole length,shoot fresh weight,shoot dry weight,root fresh weight,root dry weight,canopy reflectance spectrum,mumber of pods per plant,number of seed per plant,seed weight per plant,number of complete seed,weight of complete seed,100-seed weight,branch number,podding branch number and so forth.The shade tolerance indicator which composed of the plant height and the average internode length is better than other indicators after comprehensive tradeoff,with advantages as follows:?)It is more accurate due to its smaller error coefficient of variation(9.36%)and higher heritability(95.43%);?)It is more stable,as its correlation(0.92)between two environments is the largest;?)It has a better distinction degree,because its phenotypic coefficient of variation(31.25%)and genotypic coefficient of variation(30.52%)are larger;iv)It is more consistent to the field shade tolerance situation with the highest correlation(0.73)between the indicator and visual shade tolerance level.Compared to 40th and 60th day after sowing measuring times,the shade tolerance indicator on the 50th day after sowing is better,for example its correlation(0.87)between two environments is the highest and error coefficient of variation(7.75%)is the smallest.Therefore,the average of relative plant height and average length of internode was defined on the 50th day after sowing under 30%shading intension condition as the shade tolerance index(STI).The smaller STI,the more shade tolerance of the accession.The STI of 915 soybean varieties from southern China ranged from 1.14 to 2.60,with an average of 1.63 and there showed significant differences among the accessions.In addition,the heritability(90.07%)of these soybean varieties indicated that soybean shade tolerance had high accuracy from phenotypic selection.The STI in eco-region ?,?,? and ?showed abundant variations with the range of 1.15-2.56,1.14-2.60,1.17-2.49,and 1.22-2.46,respectively,which provide materials for the improvement of shade tolerance.Using the designed procedure,the shade tolerance of 453 soybean varieties from southern.China was evaluated,from which the tolerant sources were screened out.Under three environments identification,a number of excellent shade-tolerant germplasm resources were screened out,such as varieties Gongdou-7(Sichuan Province),Aijiaozao(Hubei Province),Hengxianheidou-2(Guangxi Autonomous Region),and QidongGuanqingj ia(Jiangsu Province).3 QTL-allele constitution its ecological differentiation of shade-tolerance and in southern China soybean germplasmThe genotype × environment was significant(P<0.01),which implies that the population performed quite inconsistently between the three environments.It suggested using G×E model in RTM-GWAS for mapping QTLs of STI.52 QTLs for STI were identified from the 394 materials,located on 19 chromosomes(except Gm 12)and up to 7 on chromosome 4.The main-effect accounted for 72.70%of the total phenotypic variation(PV);the G×E effect accounted for 9.46%PV.The locus 6LDB2137661121490108 had the largest phenotypic variation contribution(R2=15.25%).Among them,the 9 large contribution major QTLs(R2>2%)main effect explained 56.85%phenotypic variation,and the remaining small contribution major QTLs 43 QTLs explained 15.85%phenotypic variation.These results inferred that the genetic system of shade tolerance was the interaction of major loci and multiple genes.There are 320 alleles(150 negative effect alleles and 170 positive effect alleles),and The 52 detected shade tolerance loci with their 320 allele effects among the 394 varieties further designed into a 52 × 394(locus × accession)matrix.From the figure and the dataset,no accession included all positive or negative alleles for the 52 loci,and many positive alleles in non-shade-tolerant varieties and negative alleles in shade-tolerant varieties inferred that a high recombination potential for shade tolerance improvement existed in the SCSGP.SA and SP hold 11 and 6 specific-present allele(SPA),respectively,and all of SP are negative effect allele.There are 1 and 2 SPA in Eco-region IV and V,respectively.Of the 320 alleles,212 are MPSPAs(66.3%)and 2 are just present one population(SA-? and SA-?).Of the 17 SPAs between SA and SP,13 appeared at 6 large contribution major QTLs.Allele Shade.06.3-3(effect 0.036)involves 41 accessions,which is a SPA of SA;it is distributed throughout four SA geo-seasonal population with the highest frequency in SA-VI(0.60).It showed that allele not only specific present or absence in SP or SA,but also has a frequency diff-erence in the geo-seasonal population;Allele Shade.06.3-5(effect-0.060)involves 18 accessions,which is a SPA of SP;it just presented in SP-? and SP-? with higher frequency in former,which may be its origin population.Allele Shade.06.3-7(effect-0.325)involves 8 accessions,which is a SPA of SP;it just presented in SP-?,SP-? and SP-?.Among the 7 alleles of this QTL,5 are MPSPAs,3 are SPA of SP or SA,and the frequency distribution is extremely significant different(?2=49.44,p<0.001).In addition,the largest phenotypic contribution of this locus was the largest(15.25%),indicating that this locus is the main QTL leading to the differentiation of eco-population.QTL with more significant differentiation and higher large contribution are the focus of attention.Glyma.06g213100(6 SNP)was deemed the major gene of Shade.06.3,which is a transcriptional regulator of DELLA protein;moreover,DELLA protein is a key regulator of GA signal transduction pathway.In addition,the homologous gene(AT2G01570)in Arabidopsis thaliana is involved in the shade avoidance regulation network and regulates GA and phytochrome interaction factors(PIFs).Shade.05.3 contributed the second most to the phenotypic variation(R2=14.78%),and the function of major gene Glyma.05g231100(4 SNP)is indole-3-pyruvate mono-oxygenase,which confers the synthesis of auxin(conversion of IPA to IAA in auxin biosynthesis pathway);and its homologous gene(AT4G13260)is involved in shade avoidance response by coding YUC2 which confers the synthesis of auxin.The gene Glyma.16g050500(17 SNP)annotated on Shade.16.2(R2=7.75%)synthetizes F-box 3(the auxin signal).The gene Glyma.17g205300(6 SNP)annotated on Shade.17.2(R2=6.63%)is the gibberellin 3?-double oxygenase,which confers the synthesis of gibberellin;the homologous gene(AT1G15550)is involved in the signal transduction of gibberellin synthesis and the response to red and far-red changes.The gene Glyma.07g077100(37 SNP)annotated on Shade.07.1(R2=3.49%)confers the synthesis of COP9 which is involved in phytochrome signal transduction.4 Shade tolerance shared synergistic and unique characteristics with its composition and ecological traitsThe correlation between PH,NN,MINL,DTF,the sensitivity to seasonal photo-thermal changes(SPTC)and STI reached a significant level,respectively.69,55,50,63 and 73 QTLs were identified for the five traits,respectively;The main-effect accounted for 71.71%,75.53%72.58%,69.43%and 74.54%of the total PV,respectively;the G×E effect accounted for 15.11%,12.30%,10.66%,19.94%and 13.83%total PV,respectively.The number of their QTLs near or covered the QTLs for shade tolerance were 17,11,11,10 and 10 respectively,and the sum of the main effect R2 were 16.13%,11.26%,12.08%,8.68%and 11.79%respectively.A total of 35 shade-tolerant QTLs were involved,the total R2 was 41.42%,and 5 QTLs were completely coincident.For example,the annotation gene of 18LDB5564470055646203 is Dt2,indicating that the shade tolerance of soybean is closely related to the stem growth habit.In addition,there are 5,5,6,4 and 14 interactions.among the protein of the 21 candidate genes for shade tolerance with the protein of candidate genes for PH(5),NN(5),MINL(3),DTF(4)and SPTC(12).It is indicated that the shade-tolerant response and the stem morphogenesis process share some metabolic regulation pathways,which are induced or repressed by liht signals under shading conditions.The remaining 17 QTLs are unique to shade tolerance,such as Shade.16.2(R2=7.75%)appears only in the shade-tolerant response;some the shade-tolerant genes only interacts with the protein-protein of this trait.In summary,from the three levels of phenotype,QTLs and protein interaction,shade tolerance and its related ecological traits have a cooperative relationship with specific pathway and network.5 The genetic differentiation of geographic and seasonal ecological traits and its enlightenment on expanding the scope of soybean planting63 and 73 QTLs were detected in the DTF and SPTC with total 643 and 479 alleles,223 and 110 newly emerged alleles,420 and 367 inherited alleles,respectively.Among them,495 and 323 alleles only presented in one or several geo-seasonal populations.The difference in presence or absence is the main cause of differentiation,followed by allele with large differences in frequency distribution.These QTLs with qualitative or quantitative differences are the result of ecological adaptation and also contain a large number of evolutionary information.The proportion of newly emerged alleles in these two traits(36.8%and 23.0%)were much higher than the genome-wide level(8.2%).And there is significant differentiation among the geographic-seasonal differentiation,which is mainly due to the fact that there are more negative alleles in the newly emerged and inheritance of SP population(SP-? is the most),and more positive allele in the newly emerged and inheritance of SA population(SA-? is the most).It indicated that the negative allele variation of SP-? is subject to the strongest environmental pressure and artificial selection to adapt to the spring sowing environment in the Middle and Lower Changjiang Valleys,with a high degree of evolution,and the positive allele of SA-VI are the result of adapting to the tropical summer sowing environment in South China.Which supports that the SP is evolutionary type,the SA type is the primitive,and the photo-thermal insensitivity and the low-latitude photo-thermal sensitive type were derived from SA in the Middle and Lower Changjiang Valleys.Predicted DTF and SPTC of progenies in possible crosses in SCSGP were 4.9-92.4d and-0.06-0.67,respectively.The sensitive materials to the seasonal photo-thermal changes not only have sensitive alleles,but also excellent photo-thermal sensitive genetic structures with other sensitive materials;the not only have insensitive alleles,but also excellent photo-thermal insensitive genetic structures with other insensitive materials.At the same time,it was found that the excellent insensitive parents to the seasonal photo-thermal changes,Z-599 and Z-571,are also excellent shade-tolerant materials.Top 5 large positive effects just presented in SA ecotype may expand the scope of soybean planting to low latitude short day-length and high temperature areas.Top 4 large absolute value of negative effects mainly distributed in SP ecotype may expand the scope of soybean planting to long day-length and low temperature environments.6 Breeding potential prediction and optimal recombination design of the shade tolerance and ecological traits in the SCSGPZ-599 and Z-671 are both shade-tolerant materials,but their shade-tolerant alleles different,especially in the five LC QTLs.This indicates that the genetic structure of shade-tolerant materials is different,and the shade-tolerant QTL have abundant genetic diversity,which provides a genetic basis for parent selection and prediction of cross combinations.All possible crosses(77,421)among the 394 accessions were predicted the breeding potential,and 11,3,5,and 5 elite crosses were obtained within the Eco-region ?,?,?,and ?,respectively.Eco-region ? has obtained 3 adjacent elite crosses(?)and 1 non-adjacent lite cross(?);Eco-region ? obtained 10 adjacent elite crosses(4 with ?,6 with V).46 elite crosses were obtained among different eco-populations,and the best cross was between Z-597 and Z-671.Z-671(Hengxianheidou-2)is elite parent for recurring in 11 of the elite crosses.and match well with other materials in shade-tolerance genetic structure complement.Combining the obtained genetic information of shade tolerance and ecological traits,comprehensive prediction is made according to the actual production situation of intercropping.21 elite crosses match well with the conditions:stronger shade tolerance,more NN,shorter MINL,more photo-thermal insensitivity with the range of DTF from 35 to 40 d and the range of PH from 55 to 80 cm A total of 20 accessions were involved,among them,12 accessions are SP type,and 5,2,2 and 3 accessions come from Eco-region ?,?,?and ?,respectively;8 accessions are SP type,and 4 and 4 accessions come from the Eco-region ? and ?,respectively.More excellent parents are from the Eco-region ? and ?,indicating that the Eco-region ? and ? should introduce elite parents from the formers for the intercropping breeding. |
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