Genetic Analysis And Heterosis Utilization Between Sorghum And Sudangrass

Sorghum[Sorghum bicolor(L) Moench]and sudangrass[S.sudanense(piper) stapf] are two members of the agriculturally-important genus Sorghum(Gramineal),but some disagreement exists as to whether they actually belong to the same species.Hybrids of these two species have exhibited favorable forage yields and overall improved quality and disease resistance.They are widely used in aquaculture,production of livestock food and environmental protection.It is urgent to study the morphological characteristics,heterosis, forage value and molecular genetic for hybrid.In this research,the genetics of sorghum(S.bicolor) and sudangrass(S.sudanense) was studied by numerical taxonomy,cytogenetics,physiology/biochemistry and molecular biology.Heterosis of hybrid between sorghum and sudangrass was utilized for the first time in China.DNA fingerprints for 42 sorghum and sudangrass varieties and 2 sorghum-sudangrass hybrids were constructed.The brown midrib gene(bmr-6) of sorghum-sudangrass hybrids was mapped.The results were listed as the following:1.Numerical Analysis of Genetic Variation in Sorghum[S.bicolor(L.) Moench]and Sudangrass[S.sudanense(Piper) Stapf]Sixty-four biological characteristics were evaluated for a comparative study between sorghum(S.bicolor) and sudangrass(S.sudanense) by means of variance,discrimination and cluster analyses.The significant differences in biological characteristics between sorghum and sudangrass were found.The F-test divergence values between the varieties of sorghum and sudangrass were highly significant.Discrimination analysis of all plants suggested that 100%of originally grouped cases were classified correctly.Cluster analysis showed that the 10 varieties of sorghum and sudangrass in the study fell into 2 groups:four sudangrass varieties making up one kind,and six sorghum varieties making up the other. Analysis of variance showed the characteristics chosen for cluster analysis were reasonable.The characters of significant positive correlation and significant negative correlation had been found.In terms of a combined line,64 characters were clustered to 5 groups.The taxonomic significance of these characters had also been analyzed.6 principal branch quantitative characters were determined using the principal component analysis.Principal component-â… was named for principal sudangrass factor and principal component-â…¡was named for sorghum principal factor.10 varieties could be divided into 2 main groups depending on the result of cluster analysis by principal component regression values. Sudangrass was clustered to 1 group,and sorghum was clustered to the other group.The results of quantity classification for 48 varieties,i.e.32 sorghum varieties and 10 sudangrass varieties and 4 relatives,supported basically above viewpoint.2.Cytogenetical Studies of Relationship on Sudangrass(S.sudanense) and Sorghum (S.bicolor)The Karyotypes of 4 sudangrass varieties and 6 sorghum varieties during mitosis were investigated using the method of wall degradation and hypotonic treatment of making chromosomal preparations.The results showed that the numbers of chromosome are 20 in sudangrass(S.sudanense) and sorghum(S.bicolor).The karyotypes of sudangrass varieties were 1A,and their karyotype formulas were shown as follows:No1 and No2 2n=18m+2sm(2SAT),No3 2n=18m+2sm(2SAT),No4 2n=20m(2SAT).Their karyotypes were not all same among sorghum varieties.Only No5 was 1A,and all the other varieties were 2A or 2B.The karyotype formulas of sorghum varieties were:No5 2n=20m,No6 2n=16m+4sm,No7 2n=18m+2sm,No8 2n=18m(2SAT)+2sm,No9 2n=16m+2sm(2SAT)+2st,No10 2n=16m+4sm.There were not different significantly between sudangrass and sorghum in short arm,long arm and absolute total length(P＞0.05).The Karyotypes of F₁ hybrids and their parents were analyzed,and the meiosis behavior of 3 F₁ hybrids and the chromosome numbers of 2 F₂ hybrids were observed The result showed that the karyotypic types of sorghum and sudangrass were 1A,and the karyotype formula of Sa was 2n=18m+2sm(sat);the karyotype formulas of 3042A and 3042A×Sa F₁ were 2n=20m,but the others were 2n=20m(sat).The difference among 3 groups(i.e.sudangrass,sorghum and sorghum-sudangrass hybrid F₁) weren't significant in absolute long arm,absolute short arm,absolute total length,arm ratio(l/s),relative total length from 1st to 10th chromosome pair(P＞0.05);This result supported above viewpoint. The paired chromosome configuration of sorghum-sudangrass hybrid F₁ in pollen mother cells at metaphase I was 2n=2x=20(10â…¡),and the chromosome pairing behavior was regular,but the frequencies of rod bivalent of Tx623A×S722 F₁,3042A×Sa F₁ and Tx623A×Sa F₁ were different,which were 4.887,5.710 and 5.126,respectively;that of ring bivalent were 5.113,4.290 and 4.874,respectively.At anaphase I,the paired chromosomes of sorghum-sudangrass hybrid F₁ could separate from each other.The chromosome numbers of F₂ hybrid were 20(2n=20).Therefore,the sudangrass/sorghum relationship is sufficiently close.3.Photosynthetic Characteristics,Peroxidase(POD) and Esterase(EST) Isozyme Analysis in Sudangrass and SorghumSix sorghums and four sudangrasses varieties was assayed by preoxidase(POD) and EST isozyme based on Polyacryamide gel electropheresis(PAGE),respectively.The results showed that preoxidase isozyme could determine different characteristics of different varieties with enzyme patterns difference,but could not distinguish sorghum with sudangrass.EST isozyme could determine characteristic of different varieties with enzyme pattern difference,and could distinguish sudangrass and sorghum.Four photosynthetic indices of sorghum and sudangrass were measured under strong and weak light conditions by CI-310 Portable Photosynthesis System.The results showed that the differences of net photosynthetic rate,transpiration rate,stomatal conductance of sorghum and sudangrass were significant between weak(PAR:353.07μmol·m^-2s^-1,in the overcast day) and strong(PAR:1331.86μmol·m^-2s^-1,in clear-cloudy day,) radiation,but internal CO₂ of sudangrass was not changed significantly.Under weak radiation,the difference of every index between sorghum and sudangrass was not significant.But transpiration rate and internal CO₂ were significant between sorghum and sudangrass under strong radiation.Under weak radiation,there were significantly positive correlated among net photosynthetic rate,transpiration rate and stomatal conductance in sorghum or sudangrass.Under strong radiation,there were not significant correlated among all indices of sudangrass;but there were significantly positive correlated among most indices in sorghum.4.Diversity Comparison and Phylogenetic Relationships of S.bicolor and S.sudanense as Revealed by RAPD and SSR markersThirty-two accession of S.bicolor,ten varieties of S.sudanense and two related species of S.bicolor were analyzed using random amplified polymorphic DNA(RAPD) markers.The results were as follows:(1)Atotal of 68 bands were amplified by 12 primers, among which 52(76.5%) bands were found to be polymorphic.(2)The similarity coefficient of S.bicolor and S.sudanense were 55%to 95%and 52%to 84%,respectively.The polymorphy is high in both.Restorer and maintainer lines of Sorghum bicolor can be differentiated by RAPD although the similarity of them was more than 89%.(3)Cluster analysis showed the 44 varieties fell into 10 groups by the threshold value of 0.66.The whole accession of the first group were S.sudanense and the whole accession of the 5,6,7 and 8 groups were S.bicolor.But there were S.sudanense and S.bicolor in the 2,3 and 4 groups.It suggested that S.bicolor and S.sudanense can not be distinguished by RAPD.Phylogenetic relationships among 48 accessions of sorghum(S.bicolor),sudangrass (S.sudanense) and their relatives(S.propinquum,S.halepense,Zea mexicana,Z.mays) were investigated using SSR markers.The 91 SSR primer pairs generated a total of 723 polymorphic alleles,with an average of 7.945 alleles per locus,and a range of 2 to 19 alleles.The average genetic diversity,as measured by the polymorphic information content (PIC),was 0.783.The average polymorphic rates were 84.530%and 78.483%within sorghum and sudangrass,respectively.The PIC values were 0.774 and 0.770,respectively, and there was no significant difference(P＞0.05) between sorghum and sudangrass. Additionally,the genetic distance(GD_sor-su) between sorghum and sudangrass was only 0.035,suggesting a high degree of genetic homogeneity.Genetic similarity(GS) values between all varieties ranged from 0.217 to 0.975 and were used to produce a dendrogram. The 48 accessions were clustered into five groups(GS=0.766),specifically,groupâ… (consisting of sorghum,sudangrass and sorghum-sudangrass hybrids),groupâ…¡(S. propinquum),groupâ…¢(S.halepense),groupâ…£(Z.mexicana) and group V(Z.mays). Results of our analyses suggest the sudangrass/sorghum relationship is sufficiently close to place them both within the same species-sorghum(S.bicolor).5.Heterosis Utilization of Hybrid between Sorghum[S.bicolor(L.) Moench]and Sudangrass[S.sudanense(Piper) Stapf]The morphological characteristics,heterosis and the forage value of hybrid between sorghum(S.bicolor) and sudangrass(S.sudanense) were studied.The results showed that the hybrid vigor was obvious.F₁ was higher than mid-parent,even higher than high-parent in leaf width,leaf length,plant height,tiller and fresh weight per plant.The forage yield of hybrid was higher significantly than that of sudangrass.Main stem diameter and leaf length had strong direct effects on fresh weight per plant,and the same was true for density,leaf length and weight per plant on yield per unit area.The path coefficient of density with yield per unit area(p=1.0299^**) was significant at 0.01 level.The best density suitable for the hybrid was 30×10⁴ plants/hm².The nutrient level of the hybrid was close to that of sudangrass.CN^- content in the hybrid was lower.Sugar content was 10.0%(Bx) at later stage of booting,and it was higher than other developmental stages.6.Establishment of DNA Fingerprinting and Mapping of the Brown Midrid Gene (bmr-6) for Sorghum/Sudangrass and HybridsNine RAPD primers with high polymorphism and repeatability were screened from 100 RAPD primers,and the polymorphism rate was 64.06%.Every accession could construct a special data fingerprint by 4 RAPD core primers in 42 sorghum and sudangrass accessions.One RAPD fingerprint was constructed by primers F-01,and could differentiate two sorghum-sudangrass hybrids,but didn't distinguish Wancao No 3 and Sa.73 SSR primer pairs were screened from 95 SSR primer pairs,and the polymorphism rate was 86.06%.Each of 42 sorghum and sudangrass accessions could construct a special data fingerprint by 3 SSR core primer pairs.Primer txp18 showed obvious complementary band between the two sorghum-sudangrass hybrids.One SSR fingerprint was constructed by primer txp18,which could distinguish Wancao No 2,Wancao No 3 and their parents.The midrib and stem marrow were brown in the brown midrib sorghum/sudangrass varieties.The scarfskin was dark brown.The content of lignin reduced 40%to 60%than normal varieties.So the digestibility increased effectively.F₁ and F₂ population were constructed by the hybrid of Tx623A(brown midrib,bmr-6) and Sa-line(white midrib).F₁ generation showed white midrib.The ration of white and brown midrib was suitable to 3:1 in F₂ generation.The results showed that the brown midrib gene(bmr-6) was a single recessive gene.Using microsatellite(SSR) markers and F₂ segregation population,one SSR marker was linkage with the brown midrib gene.The linkage map distance was 4.2 cM.The SSR marker was in LG-07.Therefore,the brown midrib gene(bmr-6) was mapped in LG-07.