| As an annual forage of the genus Sorghum(Poaceae),Sudangrass(Sorghum sudanense(Piper)Stapf.)has many advantages such as strong tillering ability,good regeneration,high nutrient content,and better palatability.Sudangrass can be mowed for many times as green forage,silage,or hay preparation,making it an important forage crop for animal husbandry development in arid and semi-arid regions of many countries,including southern China,where Sudangrass has become one of the best forage grasses for the "foodstuff to forage crop" planting pattern.However,drought,one of the main stress factors,seriously affects the seed germination and seedling growth of Sudangrass,causing the reduction in its yield and quality in the late growth stage.In this paper,drought tolerance was comprehensively evaluated in 10 Sudangrass seedling to study the physiological response to drought stress.Based on RNA-Seq of the screened Sudangrass(Wulate No.1)with strong drought resistant and a comparative analysis,several simple sequence repeats(SSR)primers were designed to reveal the abundant genetic diversity of Sudangrass and sorghum-Sudangrass.Through differential expressed gene analysis,five candidate genes related to drought tolerance were screened,and three positive transgenic plants were obtained by using the Sb BAM1 gene to transform Arabidopsis thaliana.Ultimately,the variety "Shucao 1," which has the characteristics of good drought tolerance,high grass yield,fast growth speed,and good quality,was obtained by crossbreeding and was registered by the Chinese Herbage Cultivar Registration committee,indicating that it is popularization for growth in southern China.The main objects and results of this paper are as follows:1.The physiological responses of seven Sudangrass and three sorghum-Sudangrass seedlings to drought stress were studied under a drought stress induced by polyethylene glycol 6000(PEG-6000).The drought tolerance were compared by using membership function model,which provids a reference for the breeding of new drought-resistant varieties of Sorghum and the cultivation of high-quality forage grass.The results showed that the germplasm with good drought tolerance had stronger antioxidant capacity,higher chlorophyll content,higher photosynthetic performance,and better integrity of cell membrane structure under drought stress.The relative water content(RWC),electrical conductance(EL),malondialdehyde content(MDA),chlorophyll content,chlorophyll fluorescence(Fv/Fm),net photosynthetic rate(Pn),water use efficiency(WUE),and antioxidant enzyme activities(SOD,POD,CAT,and APX)of 10 samples were determined to calculate their membership function value for drought tolerance.The results showed that two Sudangrass(No.5(CF019623)and No.7(CF019634))and ’Shucao1’ sorghum-Sudangrass(No.9)had stronger drought tolerance,followed by ’Wulate No.1’(No.4),’Shucao No.2’ sorghum-Sudangrass(No.8),and ’S1’ sorghum-Sudangrass(No.10).Sorghum-Sudangrass No.6(CF019626)was most sensitive to drought stress.The material of Sudangrass is sorted by drought resistance from strong to weak:CF019634 > CF019623 > ’Wulate No.1’ > ’S1’ > CF019621 > CF005161 > ’Yanchi’,and’Wulate No.1’ is the most drought-resistant variety available.2.RNA-Seq was used to identify the differentially expressed genes in ’Wulate No.1’Sudangrass(No.4)under drought stress and also to develop SSR markers.Based on RNA-Seq analysis,816,854,366 clean reads were remained after screening among852,543,826 raw reads,and 80,686 unigenes were obtained after sequence splicing,and45,065(55.9%)unigenes were identified as coding sequences(CDSs).31,444 unigenes were annotated by using gene Ontology analysis,and 11,778 unigenes were classified into 25 categories and 11,223 unigenes were classified into 280 KEGG pathways according to the cluster analysis of Eu Karyotic Orthologous Groups(KOG).RNA-Seq analysis revealed that five differentially expressed genes were significantly associated with drought stress,and these genes with unknown functions were up-regulated under drought stress.These results could be used for transgenic breeding,molecular marker development,and molecular marker assisted breeding.3.Based on the high-throughput sequencing results,EST-SSR markers for Sudangrass were developed in this study.The genetic diversity of 34 Sudangrass and two sorghum-Sudangrass were analyzed,and the developed EST-SSR primers were validated.Among the 80,686 sequences of Sudangrass transcriptome,the 17,548 SSR loci were distributed in 13,574 sequences with a frequency of 16.82%.Mononucleotide,dinucleotide,and trinucleotide repeats accounted for 38.59%,19.18%,and 39.09%,respectively,and the repeats of SSR bases ranged from 5 to 23 times.The amplification rate of 300 pairs of primers was 73.67%.Among them,54 pairs of polymorphic primers amplified 275 bands in 36 test samples,of which the number of polymorphic bands(NPB)was 205,the percentage of polymorphic bands(PPB)was 73.05%,the average polymorphic information content(PIC)was 0.41,and the genetic distance ranged from 0.3922 to0.9020.Thirty-six samples were classified into three categories by cluster analysis,and the same varieties were clustered into one category,indicating that the clustering between samples was related to geographical origin.These results indicated the feasibility of using Sudangrass transcriptome to develop SSR markers.The abundant genetic diversity among the test samples would provided a basis for the diversity and variation analysis of Sudangrass and its closely related species,as well as molecular marker-assisted breeding.4.Based on transcriptome sequencing and differential expressed gene screening,a BAM-like gene sequence named Sb BAM1 was cloned from Sudangrass.The gene has an open reading frame(ORF)of 1,641 bp encoding 547 amino acids,and its synthesized protein has a molecular weight of 59.58 k Da and a theoretical isoelectric point(p I)of7.96.The constructed plant over-expression vector p CAMBIA1300-Sb BAM1 was used to infect Arabidopsis inflorescences by Agrobacterium-mediated transformation.Eventually,three plants positive for p CAMBIA1300-Sb BAM1 were obtained after hygromycin screening and PCR detection,which indicated that the target gene had been successfully introduced into the Arabidopsis genome.This study laid a foundation for further exploring the role of the Sb BAM1 gene in the regulation of drought resistance in Sudangrass.5."S1" Sudangrass(No.10)with strong drought tolerance was crossed with different sorghum sterile lines to obtain the optimum hybrid combination.Finally,"72A*S1" sorghum-Sudangrass was screened to participate in the national regional trial.In the results of the trials,Sudangrass showed good adaptability in nine test sites,and "72A*S1" demonstrated good drought tolerance and high biomass yield.Compared with the control variety,the yield of "72A*S1" sorghum-Sudangrass increased by 14.36%～18.48% in 2016 and 12.27%～18.36% in 2017,as well as fast growth and good quality.Additionally,the crude proteins content in "72A*S1" sorghum-Sudangrass also increased by 15.6%–30.6%and the content of acid detergent lignin reduced by 23.08%～33.33%.Therefore,the China National Grass Variety Approval Committee approved the new variety "Shucao 1" sorghum-Sudangrass in 2018 as suitable for popularization and application in the Yangtze River Basin. |
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