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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (6): 829-838.doi: 10.3724/SP.J.1006.2019.84111

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Mapping of genes confessing same height of tiller and main stem in sorghum

Rui WANG1,*,Liang LING2,*,Peng-Jie ZHAN1,Ji-Zhen YU1,Jian-Qiang CHU1,Jun-Ai PING1,*(),Fu-Yao ZHANG1,*()   

  1. 1 Sorghum Institute, Shanxi Academy of Agricultural Sciences / Key Laboratory of Genetic and Germplasm Innovation in Sorghum for Shanxi Province, Yuci 030600, Shanxi, China
    2 Institute of Edible Fungi, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, China
  • Received:2018-08-21 Accepted:2019-01-19 Online:2019-06-12 Published:2019-06-12
  • Contact: Rui WANG,Liang LING,Jun-Ai PING,Fu-Yao ZHANG E-mail:pingja1029@163.com;zfy5607@163.com
  • Supported by:
    This study was supported by the Doctoral Research Fund of Shanxi Academy of Agricultural Sciences(YBSJJ1606);the China Agriculture Research System(CARS-06);the Advantage Research Group of Shanxi Academy of Agricultural Sciences (YCX2018D2YS11).

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Abstract:

In this study, an F2 population derived from a cross between two sorghum lines with same height and different heights of tiller and main stem respectively was used to construct pools. In order to map genes related to same height of tiller and main stem, BSA and SLAF-seq technique were developed. Genetic analysis showed that the trait of same height of tiller and main stem was controlled by a single recessive nuclear gene. Reference genome of sorghum was used to design markers by simulating the number of markers produced by different enzymes. The SLAF library was conducted and sequenced by paired-end sequencing. The restriction enzyme was Rsa I + Hae III. The fragment length was 364-414 bp. The quality of Q30 was up to 91.70% and the GC content (45.79%) was low enough to perform sequencing. Compared with the sequencing data of rice, the construction of SLAF library fitted well to the standard, with its paired-end mapped reads reaching to 93.35% and normal digestion ratio reaching to 90.60% in sorghum. In total of 30.80 M reads and 133,246 SLAF labels were obtained and 319,428 SNPs were found. The associated region was located by SNP-index, Euclidean distance, and their combination. The candidate regions had a size of 1.95 Mb at nucleotides 54,788,026-56,740,873 on Chr.9. The SNPs locating at the associated region were analyzed between the two parents. Four non-synonymous-coding SNPs were found in this region. By verification, these SNPs were considered to be related to same height of tiller and main stem. Corresponding to three candidate genes (Sobic.009G197901.1, Sobic.009G213300.1, and Sobic.009G221200.1), these genes may be functional genes directly related to the traits.

Key words: sorghum, same height of tiller and main stem, SLAF, SNP

Table 1

Number of SLAF labels on each chromosome"

染色体
Chromosome		 染色体长度
Chromosome length		 预测SLAF标签数
Expected SLAF number		 平均SLAF间距
Average SLAF distance (bp)
Chr.1 84007898 11859 7083.89
Chr.2 88663305 12527 7077.78
Chr.3 84691115 11983 7067.61
Chr.4 77402132 10684 7244.68
Chr.5 70937750 9529 7444.41
Chr.6 70774438 9694 7300.88
Chr.7 73201405 10007 7315.02
Chr.8 63096686 8854 7126.35
Chr.9 67846938 9319 7280.50
Chr.10 69378333 9446 7344.73
合计Total 750000000 103902 7218.34

Fig. 1

Distribution of SLAF on reference genome Abscissa represents the length of the chromosome; the deep color area is SLAF label concentrated distribution area."

Fig. 2

Distribution of observed control insert size"

Table 2

Alignment analysis of obtained reads in Oryza sativa"

样品
Sample		 双端比对
Paired-end
mapped reads (%)		 单端比对
Single-end
mapped reads (%)		 未比对成功
Unmap reads
(%)		 正常酶切比例
Digestion normally
(%)		 部分酶切
Digestion partly
(%)
水稻 Oryza sativa 93.35 2.79 3.86 90.60 9.40

Table 3

Statistics of sequencing for each sample"

样品编号
Sample ID		 总reads数
Total reads		 Q30 (%) GC (%)
父本 Male parent 4040595 92.05 45.96
母本 Female parent 3591789 92.49 45.96
混池aa Pool aa 10734806 92.02 45.58
混池ab Pool ab 12433150 90.24 45.67
水稻(对照) Rice (control) 192955 92.22 43.57

Table 4

Statistics of SLAF"

样品编号
Sample ID		 SLAF标签数
SLAF number		 测序总深度
Total depth		 平均测序深度
Average depth
父本 Male parent 117244 2922769 24.93×
母本 Female parent 119317 2438178 20.43×
混池aa Pool aa 131993 8387924 63.55×
混池ab Pool ab 132818 8011244 60.32×

Table 5

Statistics of SNP information"

样品编号
Sample ID		 SNP总数
Total SNP		 SNP个数
SNP number		 杂合率
Heterozygote ratio (%)
父本 Male parent 319428 221552 3.42
母本 Female parent 319428 233073 5.54
混池aa Pool aa 319428 265665 48.54
混池ab Pool ab 319428 312515 68.51

Table 6

Distribution statistics of SLAF and SNP on genome"

染色体
Chromosome		 SLAF标签数
SLAF number		 SNP数
SNP number
Chr.1 12565 31481
Chr.2 14026 40153
Chr.3 14064 35216
Chr.4 11159 24654
Chr.5 10947 29394
Chr.6 11411 31105
Chr.7 11939 24799
Chr.8 10144 25585
Chr.9 10634 27338
Chr.10 10933 25189
其他Other 15424 24514
合计Total 133246 319428

Fig. 3

Distribution of SLAF and SNP on genome Abscissa represents the length of the chromosome; the darker area is SLAF label concentrated distribution area."

Fig. 4

Distribution of SNP-index associated values on genome The red line represents the threshold line of 0.99. The blue line represents the threshold line of 0.95, and the green line represents the threshold line of 0.90."

Table 7

Statistics of associated region information"

染色体
Chromosome		 关联区域起始位置
Start		 关联区域终止位置
End		 关联区域大小
Size (Mb)		 关联区域内的基因数量
Gene number
Chr.9 54788026 56740873 1.95 265
Total — — 1.95 265

Fig. 5

Distribution of ED associated values on genome"

Table 8

Statistics of associated region information"

染色体
Chromosome		 关联区域起始位置
Start		 关联区域终止位置
End		 关联区域大小
Size (Mb)		 关联区域内的基因数量
Gene number
Chr.9 30964929 38410608 7.45 44
Chr.9 43824552 59633348 15.81 1538
合计Total — — 23.26 1582

Table 9

Information of SNP"

染色体
Chromosome		 位置
Position		 碱基替换
Transform		 基因
Gene
Chr.9 55037659 C→A Sobic.009G197901.1
Chr.9 55037906 T→G Sobic.009G197901.1
Chr.9 56069487 T→G Sobic.009G213300.1
Chr.9 56613839 A→G Sobic.009G221200.1

Table 1

0 Sequence and information of the primers"

引物名称
Primer ID		 引物序列
Forward primer (5'-3')		 引物序列
Reverse primer (5'-3')		 产物大小
Size (bp)
SNP1 AAAATCACGTGGATAGGCCA AGATCCGGCAATGATAGTGG 269
SNP2 CCACTATCATTGCCGGATCT ATCTGAGGAAGCAAGCGAGA 233
SNP3 GGAGGAAATGCTGTGCTTCT AAGCGATGGTCCATTGAATC 245
SNP4 TGATTCCTTCGTTGGAGGAG TCACTAACATTGGGCAACCA 199

Fig. 6

Sequencing results of the parents with primer SNP1(A), SNP2(B), SNP3(C), and SNP4(D)"

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