Development And Application Of Genomic SSR Markers From Wild Peanuts

Cultivated peanuts(Arachis hypogaea L.)are widely cultivated in China,India and the United States.It is an important oil crop and economic crop in the world.Besides being used for oil extraction,peanuts are also used for fresh food and processed into various kinds of food.Peanuts have great potential and important industrial status in increasing farmers’ income and adjusting agricultural structure in China.At present,the marker assisted selection(MAS)has become an important means of crop breeding.Compared with conventional breeding technique,MAS is more rapid,efficient,and can shorten the breeding period.However,the development of peanut MAS is still in its infancy.Small number of available molecular markers is an important limiting factor restricting peanut MAS.In this study,more than 110,000 simple sequence repeat(SSR)markers were developed according to the genome sequences of diploid progenitors of cultivated peanut,Arachis duranensis and Arachis ipaensis.These SSR markers were further applied in identification of true hybrid F1 and mapping reported QTLs for leaf spot disease.These results laid a foundation for the construction of high density genetic map of peanut,mapping of important genes,and the MAS.The main results are as follows:1.Development of genomic-SSR markers of peanutBased on the reference genome of the diploid progenitors of cultivated peanut,a total of 135,529 and 199,957 SSRs were identified from the A genome(Arachis duranensis)and the B genome(A.ipaensis),respectively.According to the flanking sequences of these identified SSRs,a total of 51,354 and 60,893 primers(SSR markers)were designed from A.duranensis and A.ipaensis,respectively.The densities of the SSR markers were one every 20.36 kb and one every 22.23 kb in A.duranensis and A.ipaensis genomes,respectively.To analyze the amplification efficiency of these SSR markers,in silico PCR was performed based on the genome sequences of cultivated peanut varieities Tifrunner.It was demonstrated that more than 77%(85,885)of these SSR markers could generate at least one PCR product in A.hypogaea,suggesting that these SSR markers might have high utilization value in peanut MAS.2.Application of peanut SSR markers in peanut hybrid breedingIn order to improve the efficiency of peanut hybrid breeding,it is necessary to identify the authenticity of F1 hybrids.To breed new peanut varieties that are resistant to disease or with special characters,crossing between ’Tifrunner’(susceptible to Aspergillus flavus)and ’GT-C20’(resistant to Aspergillus flavus),’J62’(susceptible to bacterial wilt)and ’J04’(resistant to bacterial wilt),’Weihua10’(high yielding variety in Shandong)and ’Yuhua29’(a peanut variety with black seed),’Fenghua1’(high yielding variety in Shandong)and ’SAAS2015ID’(a wild peanut variety)were conducted,respectively.In this experiment,the hybrid F1 was tested using selected SSR markers and reported MITE transposon markers.According to the results of molecular markers,a total of 96 real hybrids were identified from 254 seeds,and true hybrid rate is 37.79%.This study showed that molecular markers can be used to verify the authenticity of peanut hybrids.The identification of the true hybrids before sowing can save time and cost for the planting of subsequent materials,which laying the foundation for construction of genetic populations and breeding of new varieties.3.Constrution of the physical map using peanut genomic-SSR markers,and compared it with reported genetic map using functional comparative genomicsAll the developed SSR markers were anchored to the draft genome sequences of A.duranensis and A.ipaensis and constructed a genomic SSR-based physical map of peanut.The SSR-based physical map was further compared with the reported genetic linkage maps of peanut.In A01 linkage group,there were 34 out of 84 markers(40.5%)was matched with the SSR marker from this study.The alignment of physical map with known linkage group of peanut provided enough markers for increasing the density of genetic map for further fine mapping.In addition,through the comparion analysis of A04 linkage group with the SSR-based physical map,two reported disease resistance QTLs,q F2TSWV5 for Tomato spotted wilt virus(TSWV)and q F2LS6 for leaf spot(LS),were mapped in the region of 8.135 Mb of chromosome A04 of A.duranensis.From this genomic region,719 novel SSR markers were developed,which provide the possibility for fine mapping of these QTLs.Moreover,this region also harbors 652 genes and 49 of these are defense related genes,including two NB-ARC genes,three LRR receptor-like genes and three WRKY transcription factors.These disease resistance related genes could contribute to the resistance to TSWV and the LS in peanut.4.Analysis of the genetic diversity of peanut wild and cultivated germplasm resources using the peanut SSR markersWild peanut germplasm is an important resource for the genetic improvement of peanut.For a long time,the identification of wild peanut is mainly based on the observation of phenotype.To obtain a more accurate method for identification of wild peanuts,nine wild peanut germplasms and a cultivated peanut were used for SSR analysis.Through PAGE,14 out of 15 random selected SSR markers could generate clear and identifiable bands in at least seven germplasms,showing the SSR markers have high amplification efficiency in these wild peanuts.In addition,a total of 112 of cultivated peanut germplasm were also used for SSR analysis.The results could provide technical support for further utilization of these peanut materials.