| Saccharum spontaneum L.is a crucial wild origin parent for sugarcane genetic breeding,which can improve the resistance to abiotic or biotic stress,adaptation and cane yield of sugarcane cultivars.China is one of origin and diversity center of S.spontaneum,lots of clones collected from different regions in China performed rich diversity in phenotype and chromosome types.The successful utilization of different ploidy clones in breeding plans had improved the resistance to environmental stress,ratoon capability and cane yield of sugarcane cultivars,these achievements had promoted the rapid development of sugar industry.At present,lots of different ploidy clones were conserved in China National Nursery of Sugarcane Germplasm Resources,which were collected from different regions throughout the efforts of several generations.To date,only few ploidy clones were explored in sugarcane breeding plans,their genetic background and excellent traits or genes remained unknown.In view of this,the genetic diversity of the different ploidy clones of S.spontaneum were evaluated by using phenotypic traits,genomic-SSR marker and rDNA-ITS sequence data;and the distributing of genetic variations from genome and the genes related to tress tolerance were estimated by using genomic-SSR and gene markers data with commonly used parents as controls.Finally,the gene diversity and haplotype distribution of GluAl resistant to smut and HTD2 related to tillering were also screened by using their genomic sequences with other sugarcane origin species parents,backbone parents and main varieties as controls.These results obtained above can provide direction for the utilization of different ploidy clones of S.spontaneum.The main findings were as follows:1.According to the phenotypic data of 16 morphological traits and 4 quality-related traits,the phenotypic diversity of 62 different ploidy clones of S.spontaneum were evalated,the results showed that there were no high diversity in qualitative traits from morphological traits because of the low Shannon-Wiener index with a range from 0.0000 to 1.0680,of which the 57 hair group exhibited the richest diversity with a Shannon-Wiener index of 1.0680,followed by leaf color,but stalk shape and bud placement exhibited no diversity.Different ploidy clones performed high diversity variations in five yield-related traits from morphological traits with the mean coefficient of variation of 35.94%,of which these clones from decaploid and octaploid possesses more rich genetic variations.For the five yield-related traits,the stalk weight performed the highest variations with the coefficient of variation of 51.16%,followed by lamina width and stalk length.To four quality-related traits,their genetic variation was lower than that of five yield-related traits for different ploidy clones,and the decaploid and octaploid clones possessed more abundant genetic variations than the nonuploid and dodecaploid clones.Among four quality-related traits,the biggest coefficient of variation(32.23%)was obtained in sucrose in cane,followed by apparent purity.The significant positive correlations were found among yield-related traits or quality-related traits,but there were no correlations between yield-related traits and quality-related traits except negative correlations occuring between stalk diameter andquality-related traits,the stalk diameter can be used to determine the performance of quality-related traits of different ploidy clones of S.spontaneum.The significant correlations were also found betweenelevation or latitude and quantitative traits,the results indicated that theseclones with big stalk and low sugar can be collected in low elevation or latitude regions,and these clones with small stalk and high sugar might appear in high elevation or latitude regions.All clones of different ploidy were divided into three obvious clusters according to the Nei's 1972 genetic distances calculated by using phenotypic trait data.Finally,ten clones such as Yunnan 82-110 and Yunnan 84-268,which performed very well in yield-related traits and quality-related traits,should be considered in improving cane yield of sugarcane varieties.2.The molecular diversity and genetic relationships among different ploidy populations of S.spontaneum were screened by using 30 pairs of genomic-S SR primer and rDNA-ITS sequences,the results showed that the decaploid and octaploid populations had abundant genetic diversity with high polymorphic content value(0.9281 and 0.9248)more than 0.92 at genomic-SSR loci.For rDNA-ITS sequences,more variation sites(58)and parsimony informative sites(20)were found in the decaploid population,followed by the octaploid population with 43 variation sites and 17 parsimony informative sites.Among four ploidy population,there were no apparent genetic divergent because of small genetic divergent coefficient(genomic-SSR:Gst = 0.0880 and rDNA-ITS:Gst = 0.0700)and high gene flow(genomic-SSR:Nm = 5.1840 and rDNA-ITS:Nm =3.3200).Based on the Nei's unbiased(1978)genetic distance and cluster analysis among four ploidy populations,there is a close genetic relationship between decaploid and octaploid populations which were divided into same group,and dodecaploid popualtion have further relationship with decaploid and octaploid populations,but exhibiting a close relationship with nonaploid population.By combinating marker data and the collecting recording information,we presumed that nonaploid clones might be cross offspring from between decaploid clones and octaploid clones;dodecaploid clones might be from the combination of hexaploid gametes from nonaploid clones.According to the molecular UPGMA dendrogram,the clustering relationships of different ploidy clones have corelations with their collecting locations,these clones from same or adjacent regions were clustered into same groups.3.The distribution of marker bands from genomic-SSR and resistant gene markers of different ploidy populations of S.spontaneum were screened with 63 sugarcane commonly used parents as controls.The results showed that about 515 genomic-SSR and 116 resistant gene marker bands were obtained from different ploidy populations,but only 363 genomic-SSR and 120 resistant gene marker bands got from sugarcane commonly used parent population,these implied that there area large number of genetic variations in genomic level and resistant gene loci for different ploidy populations especially for decaploid and octaploid populations.Low proportion of common bands forgenomic-SSR marker(64.60%)between different ploidy populations and sugarcane commonly used population showed that lots of variations were not introduced into sugarcane parents in genomic level,but high proportion of common bands for resistant gene marker(82.35%)between them indicated that many variations have been introduced into sugarcane parents in resistant gene loci,that is reason that the artificial selection of resistance were executed in the utiliaztion of S.spontaneum.For four resistant gene marker,more variations from AQP and DREB genes had been introduced into sugarcane parents than from HSP70 and WRKY1 genes.Comparing the number of amplification marker bands from per clone,four clones from ploidy S.spontaneum such as Fujian 89-1-17,Guangdong 16,Sichuan 79-2-16 and Fujianxianyou possess lots of genomic-SSR marker bands more than 170 bands,and 7 clones from sugarcane commonly used parents which included ROC 16,ROC 25,ROC 22,F 172,CP 72-1312,FR 93-435 and ROC 20 have more than 150 genomic-SSR bands,these indicated that they possessed rich genetic variations in genomic level.To resistant gene marker,Guizhou 78-1-11 and Yunnan 83-213 belong to S.spontaneum got many bands more than 60 bands;some elite parent in resistant breeding such as Yacheng 82-90,CP 72-1210 and F 160 got more bands,:indicated that these clones aboved can be utilizated in sugarcane breeding program for improving varieties.4.The genomic sequences of GluAl resitance to Smut and HTD2 related to tillering were cloned from these ploidy clones of S.spontaneum with high resistance to sugarcane smut and strong tillering capability,and other origin species parents(Saccharum officinarum,Saccharum robustum,Saccharum barberi,Saccharum sinense),sugarcane backbone parents and sugarcane main varieties as controls.Total 1040 sequences were obtained according to 20 sequences per clone because of their complex multiploidy.The gene component structure analysis indicated that two genes were comprised of two exons and one intron.There are more variations in HTD2 gene sequences than in GluAl gene sequences.For GluA1 gene,exon2 region possessed more nucleotide diversity,follow by intron region,the exonl region perform more conservative with low variation sites.For HTD2 genes,the intron region exhibited richer variations than two exon regions,the exon2 region was conservation.there are more InDel sites in HTD2 gene sequences than in GluA1gene sequences.The origin species parents have richer variations and more haplotypes of code region than in sugarcane backbone parents and sugarcane main varieties.In origin species parents,HATUNI(S.barberi)and Uba(S.sinense)exhibited high gene diversity in GluAl genomic sequences,follow by two ploidy clones,and the Black cheribon(S.officinarum)and 51NG63(S.robustum)performed low gene diversity.For HTD2 genomic sequences of origin species parents,two ploidy clones possessed high genetic diversity,same low gene diversity was also found in Black cheribon and 51NG63.The low haplotype distribution of two ploidy clones in other origin species parents,sugarcane backbone parents and sugarcane main varieties indicated there were big genetic differences in two gene loci between two ploidy clones and other origin species parents,it was pity that only few of haplotypes were introduced into sugarcane backbone parents and sugarcane main varieties,that implied that the different clones have big potential in improving the resistance to smut and tillering capability of sugarcane varieties.By comparison amino acid sequence of haplotype protein between high disease-resistant clones and low disease-resistant clones or between high tillering capability clones and low tillering capality clones,1 amino acid variable sites in GluAl and 1 amino acid variable sites in HTD2 may have relationships with disease-resistance and tillering capability of thesed sugarcane materials respectively. |
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