Ruiyun Wng*,Higng Wng,Xiohun Liu,Xu Ji,Ling Chen,Ping Lu,Minxun Liu,Bin Teng,Zhijun Qio,*

aCollege of Agriculture,Shanxi Agricultural University,Taigu 030801,Shanxi,China

bInstitute of Crop Germplasm Resources of Shanxi Academy of Agricultural Sciences,Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau,Ministry of Agriculture,Shanxi Key Laboratory of Genetic Resources and Genetic Improvement of Minor Crops,Taiyuan 030001,Shanxi,China

cInstitute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081,China

dAnhui Provincial Key Lab of Rice Genetics and Breeding,Institute of Rice Research,Anhui Academy of Agricultural Sciences,Hefei 230001,Anhui,China

Keywords:

A B S T R A C T A set of 132 accessions of common millet,Panicum miliaceum L.,from 12 provinces of China were assessed for endosperm starch type(waxy or non-waxy)using I2-KI staining,amylose and amylopectin contents using the dual-wavelength colorimetric method,and genotype of Waxy genes conditioning amylose content by gene sequencing. Endosperm starch content varied from 57.69%to 74.70%,while the amylose and amylopectin contents of the starch ranged from 0 to 23.29%and from 41.99%to 70.24%,respectively.Sequencing two Wx genes,including Wx-L(intron 5-exon 7 and intron 8—9)and Wx-S(exon 9—intron 10)revealed several polymorphisms(S0,S?15,LC,LF,LY).Marker M5-R11 linking to the Wx-S gene may be used to discriminate waxy common millet accessions from non-waxy ones.Among the 132 accessions,68 with the S?15 allele had waxy endosperm starch with the amylose content range 0—2.58%and 64 accessions with the S0allele had non-waxy endosperm starch with amylose content range 3.94%—23.29%.Five genotypes including S?15/LF(45%of the accessions),S0/LF(25%),S0/LY(12%),S0/LC(11%),and S?15/LY(7%)were identified.Six new SNPs were detected at the Wx-L locus.These results will facilitate common millet breeding,especially of cultivars free of amylose.

1.Introduction

Starch is an important component of the endosperm in cereal grain,and is composed of amylose and amylopectin.Many cereals are rich in amylopectin[1—11].High-amylopectin kernels of waxy sorghum[Sorghum bicolor(L.)Moench]have high economic value in the food and bioenergy industries because of their increased starch digestibility and higher ethanol conversion rate compared with those of wild-type sorghum[1].High-amylopectin grains of waxy common millet show high adhesiveness after cooking,properties that are preferred by people of East Asia[2].Amylose content affects the edible quality of the grain:waxy starch has a lower amylose content,resulting in a glutinous nature.For this reason,waxy types are preferred,and non-waxy types contain a higher amylose content.Waxy and non-waxy endosperm occur in many plants including rice(Oryza sativa),wheat(Triticum aestivum),maize(Zea mays),barley(Hordeum vulgare),foxtail millet[Setaria italica(L.)P.Beauv]and Job's tears(Coix lacryma-jobi L.)[3—11].In rice,a G—T mutation at the fifth splicing site of the first intron of the Waxy(Wx)gene was associated with low amylose content and glutinous phenotype[5,12].In wheat,an insertion of a 173-bp transposable element in Wx-A1b was detected[4,13].In maize,DNA insertions of 1—131 bp and five spontaneous deletions were determined and insertion of transposable elements resulted in the expression of the Wx gene[3].In barley,a C(nucleotide 2453)to T mutation in the fifth exon,as well as 191-bp and 15-bp insertions in the first intron and second exon,respectively,together with the deletion of a 403-bp sequence of Wx gene,were detected in waxy endosperm accessions These alterations gave rise to waxy protein expression and produced an amylose-free phenotype[7,14—16].Four classes of Wx alleles,Wx(a)—Wx(d),have been generated in sorghum[1,6].Among them,a Glu/His polymorphism was detected at the Wx locus in the waxy line BTxARG1.The Wx(c)allele has a G deletion at the 5′splicing site of the ninth intron,causing a shift of the 5′cleavage position,and the resulting reading frame shift led to premature translation termination.The Wx(d)allele contains a mutation at the splice site of the 10th intron,leading to a splicing site shift and the deletion of five amino acids(GTGKK).All of the above mutations in the Wx gene contributed to the waxy phenotype.In foxtail millet,seven kinds of transposons inserted in the Wx gene were detected in several waxy endosperm phenotypes[9,10].Types IV,IVa,and IVb harbored TSI-2,TSI-4&TSI-2,TSI-5&TSI-2,respectively,in the first intron[9,10].Type VII harbored TSI-9 in the 10th exon[9,10].Type VIII was characterized by TSI-11&TSI-10 in the 12th intron[10].Types V and X were characterized by TSI-7 and TSI-8,respectively,in the third exon[9,10].In Job's tears,a 275-bp deletion in exons 10—11 of the Wx gene was detected,corresponded to the amylose-free phenotype,and caused a lack of the Wx protein[11].

Common millet originated in China and is widely grown in the northern part of the country.It is drought-resistant and plays an important role in local people's daily life and diet.Many studies of this crop have focused on genetic diversity,common millet-specific SSR marker development,and drought-induced transcription[17—21],but research into the starch properties of this minor grain crop has lagged behind.Previous studies have revealed starch contents ranging from 67.6%to 75.1%in common millet[22].Common millet is tetraploid with 2n=4x=36 and carries two Wx loci,as revealed by crossing experiments and DNA sequencing of lines[2,23].The waxy trait is controlled by the Wx gene,which encodes the GBSSI enzyme regulating amylose synthesis.Graybosch and Baltensperger[2]found that the amylose content of waxy endosperm starch was 3.5%and that the waxy trait was controlled by the recessive alleles wx-1/wx-2;the amylose content of non-waxy endosperm starch was 25.3%,and the non-waxy trait was controlled by the dominant alleles Wx-1 and Wx-2.Hunt et al.[23]named the two Wx loci Wx-S and Wx-L and identified five alleles:S0,S?15,L0,LF,and LY.Starch composition and protein function assays showed that these two loci were responsible for amylose content and reduced synthesis capacity of GBSSI protein[24].However,only 17 accessions from China were investigated in that study.

A total of 9885 germplasm resources are maintained in the National Gene Bank of the Institute of Crop Science,Chinese Academy of Agricultural Sciences(Beijing,China).There may be new alleles at the Wx locus.To further elucidate the genetic regulation of starch production,we assessed the genotypes and phenotypes of 132 common millet accessions from different provinces of China.New single-nucleotide polymorphisms(SNPs)identified in the process could enhance our understanding of the genomic regulation of starch properties in this old species.

2.Materials and methods

2.1.Plant materials and endosperm type evaluation

Table 1 describes the 132 common millet accessions originating in 12 provinces of China (Table S1). Seeds of each accession were sown in plastic pots (diameter, 10 cm), and grown under greenhouse conditions at the experimental station of the Agronomy College, Shanxi Agricultural University (37°25′N, 112°35′E), Taigu, Shanxi, China. The endosperm type (waxy or non-waxy) was determined by an iodine color reaction using milled grain. Five seeds obtained from self pollinated plants were cracked and soaked in a 3% KI and 1% I2solution, and the non-waxy and waxy phenotypes in the endosperm starch were classified by their blue-black and reddish-brown colors, respectively.

2.2.Measurement of amylose and amylopectin contents

The amylose and amylopectin contents of starch were detected using a dual-wavelength colorimetric method[22].The absorbance of the solution was measured with a UV1601 spectrophotometer(Ruili Corp.,Beijing,China),detection wavelengths of amylose and amylopectin were evaluated at606 and 538 nm(Fig.1),respectively.The amylose and amylopectin contents were calculated from a standard curve(Fig.2)generated with amylose and amylopectin blends(Sigma-Aldrich,St.Louis,MO,USA).Starch content(%)=amylose content(%)+amylopectin content(%).

Table 1–The types of starch in the endosperm of common millet.

Fig.1 –Scanning spectrum of absorption of amylose and amylopectin(400–850 nm).

2.3.Genomic DNA extraction,PCR amplification and Waxy gene genotyping

Total genomic DNA was extracted from 15-day old seedling leaves using the modified CTAB method[25].Every sample consisted of 2 g of leaves pooled from 3 to 4 seedlings of an accession.DNA quality was checked by 1%agarose gel electrophoresis and quantified at 260 nm using a spectrophotometer.

PCR amplification was performed with three sets of primers(Table S2)synthesized by BGI Tech Solutions Co.,Ltd.(Beijing,China).Primer sets int5Lf-R3 and M12-R12 were used to amplify the intron 5—exon 7 and intron 8—9 sequences,respectively,of the Wx-L gene[23,26].Primer set M5-R11 was used to amplify the exon 9—intron 10 sequence of the Wx-S gene.PCR amplification was performed in a solution of 25 μL total volume containing 2.5 μL 10× PCR buffer(ZOMANBIO,Beijing,China),0.5 μL of each primer(0.5 μmol L?1),2 μL dNTP(200 μmol L?1),0.5 μL DNA polymerase(1.3 U),16 μL ddH2O,2.5 μL 10× Taq buffer,and 3 μL DNA template.M5-R11 and M12-R12 were amplified using the following temperature cycling parameters:94°C for 2 min,followed by 30 cycles of 94 °C for 30 s,54 °C for 30 s,and 72 °C for 1 min 30 s,with a final extension at 72°C for 7 min.The Int5Lf-R3 region was amplified at 94 °C for 2 min,followed by 35 cycles of 94 °C for 45 s,54 °C for 30 s,and 72 °C for 2 min,followed by a final extension at 72°C for 7 min.PCR amplicons with single discrete bands from agarose gel electrophoresis were purified.

PCR amplicons of all 132 accessions were sequenced by BGI Tech Solutions.The sequencing results were analyzed using Chromas 2.3(Technelysium Pty Ltd.,Brisbane,QLD,Australia).Alignment of the sequences with GU199253(Wx-L)and GU199261(Wx-S)for common millet in NCBI,was performed with DNAMAN 6.0(Lynnon Biosoft Inc.,San Ramon,CA,USA).

Fig.2–Standard absorbance curve of amylose and amylopectin blends at 606 and 538 nm.

Fig.3–Starch granules of common millet stained with 3%KI and 1%I2solution.(A)Waxy type,stained reddish-brown(accession 00000714,plant 3);(B)Non-waxy type,stained blue-black(accession 00005716,plant 1).

3.Results and discussion

3.1.Determination of starch phenotypes

The I2-KI reaction revealed that 68 of the 132 common millet accessions had a waxy endosperm starch phenotype(reddish-brown stain), whereas the other 64 were non-waxy,showing a blue-black color after staining (Fig. 3, Table S3).

3.2.Starch content of common millet endosperms

The common millet starch content varied from 57.69% to 74.70% among the 132 accessions (Table S3). This range is similar to a previous finding of 59%—80% [27]. The contents of amylose and amylopectin varied from 0 to 23.29% and 41.99%to 70.24%, respectively. Among the waxy accessions, the range of amylose content was between 0 and 2.58%, in agreement with a previous report of 0—3.7% [28]. The range of amylose contents calculated from the 64 non-waxy accessions (3.94%—23.29%) was broader than that of 4.5%—12.7% reported previously [29]. The discrepancy may result from the high diversity of these traits in the more diverse germplasm resources used in our study. As for accessions from Shanxi,about 70% (43 of 63) were waxy-type, probably as a partial result of selection against non-glutinous cultivars during domestication [30].

Bred in Shanxi province,Jinshu 1 and Jinshu 8(accessions 65 and 70)are two amylose-free cultivars of common millet.Their starch contents were(68.20±3.64)%and(69.41±1.84)%,respectively,in broad agreement of previous results(72.72%and 71.53%,respectively)[21].

With respect to the amylose content in endosperm starch,common millet accessions were classified into several types(Table 1).Among them,three cultivars were elite waxy lines,including Jinshu 1,Jinshu 8,and Nianshu(accession 83)with 0.45%amylose content,and 11 were elite non-waxy lines.All 14 elite cultivars could be used as parent materials in breeding to facilitate the development of the common millet industry.

3.3.Determination of Waxy gene alleles

Aligning the sequences of 132 accessions with GU199253 and GU199261 revealed five known Waxy allele haplotypes(LC,LF,LY,S0,and S?15),with representative results shown in Fig.4.Comparison of the sequences amplified by int5Lf-R3 and M12-R12 with that of GU199253 revealed three alleles.The LCallele is the wild-type haplotype,LYindicates a G to A substitution at the 967th nucleotide position in the 7th exon(Fig.4-A),and LFhas an A insertion at the 2113rd nucleotide position in the 9th exon(Fig.4-B).Comparison of the sequences amplified by M5-R11 with that of GU199261 revealed two alleles.The S0allele is the wild-type haplotype,whereas S?15contains a 15-bp deletion(5′-AACAAGGAGGCGCTG-3′)in the 10th exon(Fig.4-C).Among the 132 accessions,68 harbored the S?15and 64 the S0allele at the Wx-S locus.With respect to the relative capacities of the Wx-S and Wx-L loci for amylose synthesis,Wx-S made a major contribution to amylose content,whereas Wx-L contributed little[24]:68 accessions with S?15showed the waxy phenotype and 64 with S0showed the non-waxy one irrespective of the mutation at the Wx-L locus.

We identified a marker(M5-R11)near the 5′end of exon 10 for the Waxy gene polymorphic site(Wx-S).M5-R11 is an InDel polymorphism closely linked to the Waxy gene.S0is harbored by the wild type with non-waxy phenotype and S?15by the mutant with a 15-bp deletion.The PCR amplification product of M5-R11 in all 68 phenotypically waxy accessions carried the 15-bp deletion at the Wx-S locus,whereas none of the 64 nonwaxy accessions carried this deletion.There was thus 100%correspondence between the deletion and the waxy phenotype.A similar observation was reported by Hunt et al.[24],who found 48 waxy individuals with S?15and 130 non-waxy individuals with S0.Thus,primer pair M5-R11(with forward primer 5′-GGACGTCAGCGAGTGGGACC-3′and reverse primer 5′-CAGGCACACTGCTCCCAATG-3′)appear to amplify a useful marker closely linked to the Waxy gene for discriminating waxy from non-waxy common millet lines.

Six novel SNPs were detected in seven accessions(Fig.5).An A(nucleotide 2170)to G substitution was detected in accessions 00005159(Fig.5-A)and 00007156.A G(nucleotide 2300)to C substitution and a C(nucleotide 2274)to G mutation were identified in accessions 00000060 and 00000069,respectively(Fig.5-B).A T(nucleotide 1888)to C polymorphism was detected in accession No.80(Fig.5-C),whereas a T insertion at nucleotide 2337 and a G(nucleotide 2350)to C change were observed in accessions 81 and 15,respectively.

3.4.Waxy genotyping of 132 accessions

We identified 11 genotypes in the 132 accessions (Table S3,Tables 2, 3). Among them, five known genotypes were identified in 125 accessions (Table 2) and six new ones in seven accessions (00007156 and 00005159 showed the same genotype, Table 3).

For the five known genotypes,the percentage of each is displayed in Table 2.Genotype S?15/LFaccounted for the most(45%),followed by S0/LF(25%),whereas S0/LY,S0/LC,and S?15/LYaccounted for 12%,11%,and 7%,respectively.Thus,among the waxy accession genotypes,S?15/LFshowed the highest frequency,suggesting that this mutation has been preferentially maintained by strong positive selection for glutinous texture by the human population.This finding corroborates the conclusion of Hunt et al.[24]that waxy genotypes were associated in China mainly with S?15/LF.

Accessions with genotype S?15/LYor S?15/LFwere characterized by waxy/glutinous texture,whereas those with genotypes S0/LY,S0/LC,or S0/LFhad a non-waxy/non-glutinous texture(Table 2).This finding was in agreement with previous reports[23,24]in which 24 accessions including 37 individual plants from China were investigated,with all 10 waxy individuals carrying genotype S?15/LFand the 27 non-waxy individuals carrying three genotypes(S0/LY,S0/LC,S0/LF)[24].That we detected genotype S?15/LYnot found in previous studies may be due to our use of the larger germplasm panel of 132 accessions,harboring more Wx alleles.To date,many waxy cultivars have been developed,including Plateau,the first American waxy cultivar with Chinese accession Lungshu 18(PI 436626)in its pedigree[31—33],and seven landraces from Yulin,Shaanxi,China[34].A previous study of Korean andJapanese common millet revealed that plants with genotype S?15/LYproduce starch with free amylose[35],and a similar finding was observed for Jinshu 1 in our study.Jinshu 1 is a cultivar released by the Institute of Alpine Region Crops of Shanxi Academy of Agricultural Sciences in 1989,which had been selected by line breeding from the landrace Mawu shuzi.It has been reported[24,33]that the glutinous/non-glutinous trait was associated with a genetic cluster based on PCR and SSR markers.To date,almost ten thousand common millet accessions in China,among them over 1000 are in Shanxi province.Because the glutinous nature of food made from waxy grains of common millet suits to consumer's taste,waxy cultivars were selected during the cultivation history[30,36].

Table 2–Accession numbers and percentages of 125 common millet accessions in each genotype,together with their observed phenotypes.

We could not find the S?15/LCgenotype in 132 accessions,in agreement with previous observations[24].It is noteworthy that partially waxy accessions(with the S?15/LCgenotype)are either extremely rare in or absent from the common millet gene pool.In contrast to common millet,partially waxy lines were present in wheat[37].There are two possible explanations for the lack of partially waxy common millet lines.One is that the corresponding alleles at the two paralogous loci are limited to distinct geographic or evolutionary groups,leaving few chances for them to combine.The other is that this phenotype has been lost owing to positive selection for the sticky phenotype.However,SSR and GBSSI data showed clearly[24]that gene flow has occurred between differentiated populations.Thus,it is impossible that the absence of lines with S?15/LC,conferring a partially waxy phenotype,can be fully explained by genetic or geographic isolation of groups.Artificial selection against partially waxy lines was responsible for the absence of this phenotype[24].

Six new genotypes were detected(Table S3,Table 3)including three homozygous(S0/L1,S0/L2,S0/L3)and three heterozygous(S?15/LF/L4,S?15/LF/L5,S?15/LF/L6)genotypes.All three homozygotes showed the non-glutinous phenotype with one allele was S0.All three heterozygotes showed the glutinous phenotype with two alleles were S?15/LF.Previous reports[23,24]also mentioned one heterozygous genotype(S0/LC/LF);however,our finding of three heterozygotes revealed several new alleles at the Wx-L locus.Future research into these mutations' functions may shed light on the population structure of common millet.

Table 3–Genotypes of seven common millet accessions with novel SNPs at the Wx-L locus.

4.Conclusions

Waxy allelic diversity is abundant in common millets of China.Non-waxy phenotypes with genotype(S0/LC,S0/LYor S0/LF)contained 3.94%—23.29%amylose.Waxy phenotypes with genotype(S?15/LYor S?15/LF)contained 0—2.58%amylose.M5-R11 was a useful InDel polymorphic marker for discriminating the waxy from the non-waxy phenotype of common millet accessions.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2018.02.004.

Acknowledgments

This study was supported by the National Natural Science Foundation of China(31271791),Research Project supported by Shanxi Scholarship Council of China(2016-066),the Subject Construction Project of Anhui Academy of Agricultural Sciences (16A0102),China Agriculture Research System(CARS-06-13.5-A16),the Key Research and Development Program(General Project)(Agriculture)of Shanxi Province of China(201603D221003-5),the Special Program of Crop Germplasm Resources Protection and Utilization of Ministry of Agriculture of the People's Republic of China(2017NWB036-21),and the Subplat form Project of National Proso Millet Germplasm Resources of China(NICGR2017-027).

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