SNPs and Indels identified by re-sequencing
Genome wide re-sequencing (WGRS) was performed on 206 samples. These included 89 Chinese core collections, 4 local varieties and 113 other collections from different countries and regions, representing the genetic, geographical and morphological diversity of forage sorghum.
After comparing the reference genomesand the SNP calling, we identified 14,570,430 SNPs with MAF ≥ 0.05 and 1,967,033 indels In principal component analysis, based on all collected SNPs, the first and second principal coordinates (PC) explained 13.16% and 4.78% of SNPs molecular variance, respectively (Fig. 1a). Genetic relationship of 206Sorghum reflected the diversity of Sorghum distribution, however the genetic diversity of European varieties is relatively scarce compared with other regions (Fig. 1b).
All materials can be divided into two categories by NJ tree analysis (Fig. 1c). GroupⅠmainly includes most American accessions and groupⅡ includes sorghum breeding lines from all other regions. Accessions from all breeding lines were mainly but not completely divided into several subgroups or minor groups, which indicated that the genetic mixture of breeding lines and local varieties was widespread in sorghum breeding.
Linkage disequilibrium analysis
SNP data were used to analyze the genome-wide linkage disequilibrium patterns of Asian and American, Asian × American, Asian × Australian and European germplasms, respectively. In the same linkage group, the speed of LD decay can indicate whether the group is selected or not to some extent. Generally speaking, LD of wild population decreased faster than that of domesticated population, and LD of cross pollinated plants decreased faster than that of self pollinated plants. For example, maize: local variety 1KB, inbred line 2KB, commercial inbred line 100kb. In Asia, America, Asia × America, Asia × Australia and Europe subgroups, the decay rates are very different, which may lead to different resolution of association mapping in different genome regions. The LD decay rate of European varieties was the lowest, which may be due to self-fertilization(Fig. 1d).
Association analysis of Agronomic Traits
ADF content of 206 forage sorghum materials showed continuous distribution (Fig. 2a),.A total of 8 candidate regions (− log(P) > 6) spanning 100 kb were significantly associated with ADF content on chromosomes 2, 5, 6, 7, 8 and 9supported by the strong LD among associated SNPs (Fig. 2band c).
ADF content variation analysis between haplotype
In order to understand the origin and development of haplotypes, the geographical distribution of SNPs of 8 QTLs in 206 accessions was analyzed (Fig. 3). Allelic distribution for C2_S6746476 show that the 5 accessions contain all haplotypes from AA to TT, ADF content of America×Asia was significantly lower than other accessions, except in genotype of AA,CC,GG,TT. Europe accessions was the opposite (Fig. 3a). For C5_S5473787 genotype of AA, CC, GG, TT, were still high. But among the four genotypes, European sorghum had the lowest ADF content. However, among the other genotypes, European sorghum had the highest ADF content. As a whole, the ADF content of different genotypes was similar (Fig. 3b). For C6_S60364789 the content of ADF in Asia × Australia sorghum is relatively wide. There were varieties with high and low ADF content. Even some genotypes were higher than Europe sorghum. America and America×Asia sorghum had the lowest ADF content( Fig. 3c). For C7_S60623290 the ADF content of all the haplotypes was similar, In AA, GG, CC and TT haplotypes, the ADF content of European sorghum was still the lowest, but in other haplotypes, the ADF content of Asia×Australia sorghum breeding lines were higher than that of European sorghum and became the highest among the five sorghum varieties (Fig. 3d). For C8_S56789460 All haplotypes were classified into two groups: AA, CC, GG, TT and others, The ADF content of these haplotypes were also relatively consistent (Fig. 3e). For C9_S40246128 genotype of AA, CC, GG and TT, ADF content of European sorghum was lower and concentration was relatively concentrated. Among the other genotypes, the ADF content of Asia×Australian and European genotypes was the highest, and the difference of ADF content in Australian varieties was large (Fig. 3f).
Genetic collinearity analysis
The eight candidate regions of ADF content showed about 200000 SNPs on chromosomes 2, 5, 6, 7, 8 and 9 (Table 1), including 91 genes. The results of collinearity analysis showed that 44 of 91 ADF related loci were highly collinearity with previously reported gene families (Table 2). Sorbi_3006G272600 is highly collinear with Osaae3 (Os04g0683700) (Kikuchi s et al. 2003) in rice, which regulates rice blast resistance, floret development and ADF biosynthesis.
Table 1
Candidate QTL regions significantly associated with ADF
Chr
|
QTL
|
Start
|
End
|
2
|
C2_S6746476
|
6,746,476
|
6,946,476
|
5
|
C5_S5473787
|
5,473,787
|
5,673,787
|
6
|
C6_S60364789
|
60,364,789
|
60,564,789
|
7
|
C7_S60623290
|
60,623,290
|
60,823,291
|
8
|
C8_S56789460
|
56,789,460
|
56,989,479
|
9
|
C9_S40246128
|
40,246,128
|
40,446,128
|
Table 2
Gene collinearity of ADF content between sorghum and crops (rice and corn)
Gene ID
|
Ortholog name
|
Description
|
SORBI_3002G068100
|
Os07g0193600
|
Similar to Aminoacyl-tRNA synthetase
|
SORBI_3002G068300
|
Os07g0194000
|
putative vesicle-associated membrane protein 725
|
SORBI_3002G068600
|
Os07g0194500
|
probable prolyl 4-hydroxylase 6
|
SORBI_3002G068800
|
Os07g0194800
|
hydroxyproline O-galactosyltransferase GALT6
|
SORBI_3002G068900
|
Os07g0195100
|
ras-related protein RABE1c
|
SORBI_3002G069000
|
Os12g0265400
|
Glycosyl transferase, family 31 domain containing protein
|
SORBI_3002G069000
|
Os07g0195200
|
hydroxyproline O-galactosyltransferase GALT6
|
SORBI_3005G054800
|
Os11g0169700
|
aldehyde oxidase GLOX
|
SORBI_3005G055000
|
Os11g0169900
|
V-type proton ATPase 16 kDa proteolipid subunit-like
|
SORBI_3005G055300
|
Os03g0269100
|
tropinone reductase homolog At5g06060
|
SORBI_3005G055400
|
Os11g0170000
|
Similar to Amidase family protein, expressed
|
SORBI_3005G055600
|
Os11g0170200
|
Protein of unknown function DUF869, plant family protein
|
SORBI_3006G272200
|
Os04g0683100
|
pre-mRNA cleavage factor Im 25 kDa subunit 2
|
SORBI_3006G272400
|
Os04g0683400
|
nuclear transcription factor Y subunit C-2
|
SORBI_3006G272600
|
Os04g0683700
|
oxalate–CoA ligase
|
SORBI_3006G272700
|
Os10g0133166
|
putative disease resistance protein RGA3
|
SORBI_3006G272800
|
Os04g0683800
|
FT-interacting protein 1
|
SORBI_3006G273000
|
Os04g0683900
|
AT-hook motif nuclear-localized protein 9
|
SORBI_3006G273400
|
Os04g0684500
|
white stripe leaf5
|
SORBI_3006G273700
|
Os04g0684800
|
ubiquitin-conjugating enzyme E2 variant 1C
|
SORBI_3006G273800
|
Os04g0684900
|
probable CCR4-associated factor 1 homolog 11
|
SORBI_3006G274000
|
Os04g0685000
|
transcription repressor OFP13
|
SORBI_3006G274200
|
Os04g0685200
|
hypothetical protein
|
SORBI_3006G274300
|
Os04g0685300
|
NDR1/HIN1-like protein 2
|
SORBI_3007G171000
|
Os08g0557600
|
monodehydroascorbate reductase 4, cytosolic-like
|
SORBI_3007G171400
|
Os08g0557400
|
putative low molecular weight protein-tyrosine-phosphatase slr0328
|
SORBI_3007G171500
|
Os05g0301700
|
cytochrome c1-2, heme protein, mitochondrial
|
SORBI_3007G171600
|
Os08g0557200
|
manganese-dependent ADP-ribose/CDP-alcohol diphosphatase-like
|
SORBI_3007G171800
|
Os08g0557100
|
28 kDa ribonucleoprotein, chloroplastic
|
SORBI_3007G172100
|
Os08g0556900
|
KDEL-tailed cysteine endopeptidase CEP1
|
SORBI_3007G172300
|
Os08g0556600
|
NADH dehydrogenase [ubiquinone] iron-sulfur protein 5-B
|
SORBI_3007G172400
|
Os08g0556550
|
Hypothetical conserved gene
|
SORBI_3007G172500
|
Os08g0556400
|
probable protein S-acyltransferase 19
|
SORBI_3007G172600
|
Os08g0556200
|
Similar to Dihydroneopterin aldolase
|
SORBI_3007G172700
|
Os08g0556000
|
Similar to YTH domain protein 2
|
SORBI_3007G172800
|
Os08g0555700
|
zinc finger protein 2
|
SORBI_3007G172900
|
Os08g0555800
|
protein MULTIPOLAR SPINDLE 1
|
SORBI_3007G173000
|
Os08g0555200
|
Nonaspanin (TM9SF) family protein
|
SORBI_3007G173000
|
Os08g0554900
|
transmembrane 9 superfamily member 8
|
SORBI_3007G173000
|
Os08g0555000
|
Transmembrane 9 superfamily protein member 2 precursor (p76)
|
SORBI_3002G068100
|
Zm00001d018852
|
DDT domain-containing protein DDR4
|
SORBI_3002G068100
|
Zm00001d039545
|
DDT domain-containing protein DDR4
|
SORBI_3002G068300
|
Zm00001d007767
|
vesicle-associated membrane protein 722
|
SORBI_3002G068300
|
Zm00001d019091
|
vesicle-associated membrane protein 722
|
SORBI_3002G068800
|
Zm00001d007764
|
Hydroxyproline O-galactosyltransferase GALT4
|
SORBI_3002G068900
|
Zm00001d019093
|
Small GTP-binding protein
|
SORBI_3002G069000
|
Zm00001d007762
|
hydroxyproline O-galactosyltransferase GALT6
|
SORBI_3002G069000
|
Zm00001d019094
|
hydroxyproline O-galactosyltransferase GALT6
|
SORBI_3002G069000
|
Zm00001d033748
|
Hydroxyproline O-galactosyltransferase GALT4
|
SORBI_3005G054800
|
Zm00001d053008
|
glyoxal oxidase
|
SORBI_3005G055000
|
Zm00001d004821
|
vacuolar ATP synthase 16 kDa proteolipid subunit
|
SORBI_3005G055000
|
Zm00001d053010
|
V-type proton ATPase 16 kDa proteolipid subunit
|
SORBI_3005G055400
|
Zm00001d004817
|
Fatty acid amide hydrolase
|
SORBI_3005G055501
|
Zm00001d004796
|
E3 ubiquitin-protein ligase RZF1
|
SORBI_3005G055600
|
Zm00001d053011
|
filament-like plant protein 3
|
SORBI_3005G056000
|
Zm00001d004818
|
CBS domain-containing protein CBSCBSPB3
|
SORBI_3005G056000
|
Zm00001d053014
|
CBS domain-containing protein CBSCBSPB2
|
SORBI_3006G272200
|
Zm00001d001787
|
Pre-mRNA cleavage factor Im 25 kDa subunit 2
|
SORBI_3006G272400
|
Zm00001d026648
|
Nuclear transcription factor Y subunit C-4
|
SORBI_3006G272600
|
Zm00001d026649
|
Oxalate–CoA ligase
|
SORBI_3006G272800
|
Zm00001d001785
|
C2 calcium/lipid-binding plant phosphoribosyl transferase family protein
|
SORBI_3006G273000
|
Zm00001d001784
|
AT-hook motif nuclear-localized protein 5
|
SORBI_3006G273000
|
Zm00001d026652
|
AT-hook motif nuclear-localized protein 5
|
SORBI_3006G273200
|
Zm00001d001780
|
probable serine/threonine-protein kinase BSK3
|
SORBI_3006G273200
|
Zm00001d026653
|
protein kinase APK1B
|
SORBI_3006G273300
|
Zm00001d001779
|
plant integral membrane protein TIGR01569 containing protein
|
SORBI_3006G273400
|
Zm00001d026654
|
Pentatricopeptide repeat-containing protein chloroplastic
|
SORBI_3006G273800
|
Zm00001d001774
|
hypothetical protein
|
SORBI_3006G273900
|
Zm00001d001773
|
pentatricopeptide repeat-containing protein At3g51320
|
SORBI_3006G274000
|
Zm00001d026661
|
Transcription repressor OFP13
|
SORBI_3006G274000
|
Zm00001d026663
|
hypothetical protein
|
SORBI_3006G274200
|
Zm00001d001771
|
aspartic proteinase nepenthesin-2
|
SORBI_3006G274300
|
Zm00001d001772
|
harpin inducing protein
|
SORBI_3006G274300
|
Zm00001d026662
|
hypothetical protein
|
SORBI_3007G170900
|
Zm00001d005344
|
histidine-containing phosphotransfer protein 2
|
SORBI_3007G170900
|
Zm00001d049952
|
his-containing phosphotransfer protein
|
SORBI_3007G171000
|
Zm00001d049954
|
hypothetical protein
|
SORBI_3007G171100
|
Zm00001d049958
|
Putative peptidyl-prolyl cis-trans isomerase and WD40 repeat domain family protein
|
SORBI_3007G171400
|
Zm00001d031963
|
Low molecular weight protein-tyrosine-phosphatase slr0328
|
SORBI_3007G171400
|
Zm00001d046049
|
protein tyrosine phosphatases;protein tyrosine phosphatases
|
SORBI_3007G171500
|
Zm00001d031967
|
cytochrome c1, heme protein
|
SORBI_3007G171600
|
Zm00001d049960
|
manganese-dependent ADP-ribose/CDP-alcohol diphosphatase
|
SORBI_3007G171800
|
Zm00001d031969
|
RNA-binding protein CP31B chloroplastic
|
SORBI_3007G172100
|
Zm00001d031971
|
Vignain
|
SORBI_3007G172300
|
Zm00001d031975
|
Fb14
|
SORBI_3007G172400
|
Zm00001d049974
|
peroxisome biogenesis protein 1
|
SORBI_3007G172500
|
Zm00001d031977
|
putative protein S-acyltransferase 19
|
SORBI_3007G172600
|
Zm00001d031979
|
dihydroneopterin aldolase 1
|
SORBI_3007G172600
|
Zm00001d031995
|
Dihydroneopterin aldolase 2
|
SORBI_3007G172700
|
Zm00001d031981
|
evolutionarily conserved C-terminal region 5
|
SORBI_3007G172800
|
Zm00001d031985
|
protein LATE FLOWERING
|
SORBI_3007G172900
|
Zm00001d050498
|
multipolar spindle 1
|
SORBI_3007G173000
|
Zm00001d031986
|
transmembrane 9 superfamily member 9
|