A dCAPS marker developed from a stress associated protein gene TaSAP7-B governing grain size and plant height in wheat

WANG Yi-xue, XU Qiao-fang, CHANG Xiao-ping, HAO Chen-yang, LI Run-zhi, JING Rui-lian

1 College of Agronomy, Shanxi Agricultural University, Taigu 030801, P.R.China

2 National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

1. Introduction

Stress-associated proteins (SAPs) are a class of zinc-finger proteins composed of A20/AN1 domains. These proteins have recently been identified as novel stress regulatory proteins in plants (Giriet al.2013). SAP genes are widely distributed in plant species. At present, 14 SAP genes inArabidopsis, 18 in rice and 11 in maize have been reported(Vij and Tyagi 2006; Jinet al.2007; Liuet al.2011). Majority of SAP genes have been found to be stress inducible and their over-expression in plants conferred tolerance to abiotic stresses (Huanget al.2008; Ben Saadet al.2010; Dixit and Dhankher 2011; Charrieret al.2013).OsSAP1was the first reported SAP gene. It affected the expression of 43 endogenous genes involved in stress response and enhanced stress tolerance in rice and tobacco(Mukhopadhyayet al.2004; Dansanaet al.2014). In the same way,OsSAP8andOsSAP11enhanced tolerance to high salt, drought, and cold stresses in transgenic plants(Kanneganti and Gupta 2008; Giriet al.2011).

Wheat is a staple food commodity for more than one third global population. Its growth and development are often impacted by abiotic stress which leads to yield reduction. Exploitation and utilization of superior genes and allelic variations can be helpful approaches for improving wheat production. Enormous amount of allelic variations are present in wheat germplasms. Developing functional markers using these allelic variations will provide the basis for marker-assisted selection breeding. Association analysis possesses the advantages of shorter research time and higher mapping resolution, therefore it is considered as a powerful approach for identifying superior allelic variations(Myleset al.2009).

Previous studies on SAPs focused on the domains composed of A20 and AN1 domains, but SAPs composed of two AN1 domains are still unknown. In this study, the polymorphism of theTaSAP7-B, a member of SAP gene family from B genome of wheat including two AN1 domains was detected by sequencing 32 wheat accessions. A dCAPS marker was developed based on the polymorphic site in the promoter region. Furthermore, association analysis between the marker and agronomic traits using a natural population consisted of 262 accessions was implemented. Consequently, the marker conferred to plant height (PH) and 1 000-grain weight (TGW) was developed for the molecular marker-assisted selection in the breeding program.

2. Materials and methods

2.1. Plant materials and measurement of agronomic traits

Hanxuan 10 is a drought-tolerant cultivar and used as the plant material for cloning geneTaSAP7-B. The nullitetrasomic lines of Chinese Spring wheat were used for chromosomal location. A doubled haploid (DH) population was used for genetic mapping. Thirty-two highly diverse accessions were used to identify nucleotide polymorphism ofTaSAP7-B. Population 1 (262 winter wheat accessions)was initially used for association analysis. These accessions were mainly from the Northern Winter Wheat Zone and Yellow and Huai River Valleys Facultative Wheat Zone. This population was sown at Shunyi (40°23´N, 116°56´E) and Changping (40°13´N, 116°13´E), Beijing, over three growth cycles (2010–2013). Two water treatments, i.e., well-watered(WW) and rain-fed (drought stress, DS), were supplied at each site. The WW plots were irrigated with 750 m3ha–1(75 mm) at the pre-overwintering, booting, flowering, and grainfilling phases, though, the DS plots were rain-fed. The rainfall in three growing seasons was recorded as 131, 180, and 158 mm, respectively. Another two populations were also used to verify the results of initial association analysis,geographic distribution, and frequencies analysis of allelic variation. Population 2 (348 modern cultivars) and population 3 (157 landraces) were taken from Chinese wheat core collection and Chinese wheat mini-core collection,respectively (Haoet al.2011). Population 2 was sown at Luoyang (36°41´N; 112°45´E) in Henan Province in 2002 and 2005, and at Shunyi, Beijing in 2010. All plant materials were sown in the first week of October and harvested in the following mid-June. Each experimental unit consists of four 2 m rows having 40 plants in each row. Row-to-row distance was maintained at 0.3 m. Agronomic traits of populations 1 and 2 were measured by random selection of five plants to calculate the mean in each accession, including PH, peduncle length (PL), length of penultimate internode (LPI), number of spike per plant (NSP), and TGW.

2.2. Cloning TaSAP7-B gene in wheat

Based on conserved sequence of the AN1 domain,reference sequence ofTaSAP7-Bwas obtained from URGI (Unité de Recherche Génomique Info) website(https://urgi.versailles.inra.fr/blast/). Specific primers for B genome (Sap7BF, 5´-TATAGGAGAAACTCCGCGAG-3´and Sap7BR, 5´-TGACACGTTGTAGATGAGTTC-3´) were designed to amplifyTaSAP7-Bgene from Hanxuan 10,including the 5 and 3 flanking regions.

2.3. dCAPS marker development

A pair of primer was developed based on the SNP site at –260 (C/T) in the promoter region ofTaSAP7-B. The primers were named as MF1 (5´-TCCGGAGCTGACCGG ATCGATCCAGGAGC-3´) and MR1 (5´-CTTGCGTTCGGG TGCGAAG-3´). MF1 was designed by one base mismatching at –264 bp, then a restriction enzymeSacI recognition site was produced. By two rounds of PCR, the first round was to amplify fragments ofTaSAP7-Bwith the genomicspecific primer pair of Sap7BF/Sap7BR; the second round was performed as follows: first round PCR products were diluted 100 times, followed by taking 1 μL as template for the second round PCR using the primer pair MF1/MR1(annealing at 57°C for 30 s, and extension at 72°C for 30 s).The PCR products were digested bySacI and separated in 4% agarose gels.

2.4. Genetic mapping

A DH population (150 lines) was established from the cross between Chinese wheat cultivars Hanxuan 10 and Lumai 14. A genetic linkage map including 395 marker loci was established from data on 150 lines (Haoet al.2003).Allelic variation (C/T) was found at the site –260 bp in both parents. The genotypes of the 150 lines were identified.According to the genotypic results,TaSAP7-Bwas mapped on the genetic linkage map of the DH population using the MAPMAKER/EXP 3.0.

2.5. Association analysis between TaSAP7-B allelic variation and agronomic traits

Correlation betweenTaSAP7-Ballelic variation and agronomic traits were identified by TASSEL 2.1 Software following mixed linear model (MLM). Associations were considered significant difference atP＜0.05. One way analysis of variance (ANOVA) was performed by using SPSS v16.0.

3. Results

3.1. Clone and nucleotide diversity of TaSAP7-B

The full-length of theTaSAP7-Bgene (2.0 kb) including promoter, coding region and 3´-UTR was cloned with primers Sap7BF and Sap7BR. The length ofTaSAP7-Bcoding region was 543 bp and coded 180 amino acid residues.Structure prediction showed that TaSAP7-B including 2AN1 zinc-finger domains which were the specific domains from SAPs. Using a highly diverse population consisted of 32 wheat accessions, no evidence for intron and nucleotide variation was found in the coding region, but two nucleotide variation sites (one insertion-deletion (InDel) and one single nucleotide polymorphism (SNP)) were detected in the promoter region ofTaSAP7-Bgene (Fig. 1).

3.2. Marker development of TaSAP7-B

Although the InDel was identified by sequencing, it was not used for subsequent research, because very low proportion(3.05%) of accessions possessed deletion among 262 accessions. On the basis of SNP site (–260 bp), one dCAPS was developed, named as SNP-260. The primers MF1/MR1 were designed to obtain the PCR product of 202 bp containing the variation site. Below mentioned sequence paved a way for restriction enzymeSacI recognition between C and T genotype accessions. The T genotype accessions digested bySacI exhibited two bands (175 and 27 bp) while the C genotype accessions showed only one band (202 bp)when separated the digested PCR products on 4% agarose gel (Fig. 2).

3.3. Chromosomal location and genetic mapping of TaSAP7-B

TaSAP7-Bwas located on chromosome 5B using a set of nulli-tetrasomic lines of Chinese Spring. The dCAPS marker SNP-260 ofTaSAP7-Bwas used to scan the DH lines derived from Hanxuan 10×Lumai 14.TaSAP7-Bwas mapped on chromosome 5B flanked byAX-99395950(2.86 cM) andXwmc380(5.26 cM) (Fig. 3).

3.4. Association analysis between marker SNP-260 and agronomic traits

Population 1 was divided into two subpopulations,comprising 126 and 136 accessions according to the analysis of population structure by 209 whole-genome SSR markers with STRUCTURE v2.3.2. Zhanget al.(2014) reported the highest deltakatk=2. Significant association was noted between the marker SNP-260 and agronomic traits in the natural population, including PH in 10 environments. The marker SNP-260 was also significantly associated with PL, LPI, NSP, and TGW in environments 3, 3, 5, and 4, respectively (Table 1).

Based on the SNP (C/T) at –260 bp, population 1 was divided into two groups. The proportion of two genotypes possessing C and T alleles was 84 and 16%, respectively.The genotypes possessing T allele had significantly higher PH, PL, and LPI in all 10 environments as compared to C allele genotypes. Similar results were obtained for NSP in seven environments. The genotypes possessing C allele had significantly higher TGW in eight environments.Therefore, the base C was considered as the superior allele (Fig. 4).

Fig. 1 Clone and sequence polymorphism analysis of TaSAP7-B. A, TaSAP7-B isolated from Hanxuan 10. M is marker III; lane 1 is the PCR product of TaSAP7-B. B, schematic diagram of TaSAP7-B structure. ATG and TGA are the initiation and termination codon, respectively.

3.5. Verification of function with the marker SNP-260

Population 2 was used to validate the association analysis results obtained from population 1. The results showed that the genotypes possessing C allele have shorter PH and higher TGW than the T allele genotypes. TGW of C allele genotypes were significantly higher in all three environments,whereas, PH of C allele genotypes were significantly lower in two environments (Fig. 5). The functions of the marker SNP-260 are consistent in both populations under multienvironmental conditions.

3.6. Geographic distribution of TaSAP7-B allelic variation in Chinese wheat production zones

Fig. 2 Marker development of TaSAP7-B. A, scheme of dCAPS primer MF1 design. The sequence in the box is the primer MF1. The elliptic boxes indicate mutated bases: A→G. ▼ is the enzyme cutting site of SacI. B, PCR products of dCAPS marker SNP-260 was restrictively digested by SacI. M is 100 bp DNA ladder; C, thymine; T, cytosine.

Fig. 3 Location and genetic mapping of TaSAP7-B. A, TaSAP7-B was located on chromosome 5B using nulli-tetrasomic lines of Chinese Spring by the primer pair Sap7BF and Sap7BR. M is marker III. B, TaSAP7-B was mapped on chromosome 5B flanked by AX-99395950 and Xwmc380 using dCAPS marker SNP-260.

Table 1 Association analysis between marker SNP-260 and agronomic traits in wheat1)

On the basis of agro-ecological regions, the China has 10 major zones for wheat production. The geographic distribution ofTaSAP7-Ballelic variation was evaluated using the accessions from populations 2 and 3 covering all 10 major zones. Wheat genotypes with C allele were less than half of proportions in Chinese landraces in Zones I–III and V. However, the base C was the dominant allele in Chinese modern cultivars in all 10 major zones (Fig. 6).

Fig. 4 Phenotypic comparisons of TaSAP7-B allelic variation in 10 environments. PH, plant height; PL, peduncle length;LPI, length of penultimate internode; NSP, number of spike per plant; TGW, 1 000-grain weight. E1, 2010-CP (Changping)-WW (well-watered); E2, 2010-CP-DS (drought-stressed); E3,2010-SY (Shunyi)-WW; E4, 2010-SY-DS; E5, 2011-SY-WW;E6, 2011-SY-DS; E7, 2012-CP-WW; E8, 2012-CP-DS; E9,2012-SY-WW; E10, 2012-SY-DS. T, cytosine; C, thymine.＊, P＜0.05; ＊＊, P＜0.01. Error bars indicate standard error.

3.7. Frequency of TaSAP7-B allelic variation in Chinese wheat breeding programs

To evaluate fluctuations in the allelic variation frequency over a period of time, 335 accessions from 348 modern cultivars with known release dates were splitted into five groups according to released decade (pre-1950s, 1960s, 1970s,1980s, and post-1990s). A continuous reduction (from 21.6 to 2.9%) in the proportion of genotypes with T allele was noted since the pre-1950s until the post-1990s; meanwhile,the proportion of genotypes with C allele continuously increased from 78.4 to 97.1%. The proportion of superior C allele had been maintained in a quite high level (Fig. 7).It is suggested that the superior C allele were apparently selected in Chinese wheat breeding programs.

4. Discussion

Single nucleotide polymorphism (SNP) is a solo nucleotide variation that occurs at a specific position in the genomes,and is recently developed as genetic markers (Garces-Claveret al.2007; Zhanget al.2013). As the 3rd generation molecular marker, SNPs were used as a tool for a series of research program, such as, marker-assisted selection,genome mapping and association mapping (Rounsley and Last 2010; Yueet al.2015). Recent developments in bioinformatics and sequencing technology are rapidly promoting SNP development in wheat progressively achievable (Winfieldet al.2012). For example, 14 078 putative SNPs have been identified in 6 255 diverse reference sequences with Illumina GAIIx data from different wheat genotypes (Allenet al.2011). Database for wheat SNP (http://wheat.pw.usda.gov/SNP/new/index.shtml)contains more than 8 000 primer pairs, 2 200 polymorphic sequence tag sites (STS) and 11 200 polymorphic loci have been developed from diploid progenitors of wheat.

Fig. 5 Phenotypic comparisons of TaSAP7-B allelic variation in population 2. PH, plant height; TGW, 1 000-grain weight. The three environments were at Luoyang (LY) in 2002, 2005 and Shunyi (SY) in 2010. T, cytosine; C, thymine. ＊＊, P＜0.01. Error bars indicate standard error.

Fig. 6 Geographic distribution of TaSAP7-B allelic variation in 10 major wheat zones of China. A, distribution of TaSAP7-B allelic variation in 157 Chinese landraces. B, distribution of TaSAP7-B allelic variation in 348 Chinese modern cultivars. I, northern winter wheat zone; II, Yellow and Huai River Valleys facultative wheat zone; III, middle and low Yangtze Valleys autumn-sown spring wheat zone; IV, southwestern autumn-sown spring wheat zone; V, southern autumn-sown spring wheat zone; VI, northeastern spring wheat zone; VII, northern spring wheat zone; VIII, northwestern spring wheat zone; IX, Qinghai-Tibet Plateau spring-winter wheat zone; X, Xinjiang winter-spring wheat zone. T, cytosine; C, thymine.

Stress associated proteins (SAPs) family plays pivotal roles in stress responses in plants. Proteins containing A20/AN1 zinc-finger domains have been reported in different organisms. GeneTaSAP7-B, containing two AN1 domains, was obtained by the conserved sequence of the AN1 domain. SNPs are abundantly present in non-coding region as compared with coding region (Nasuet al.2002).In the current study, polymorphism analysis ofTaSAP7-Bshowed that the nucleotide diversity was found in the promoter region but not in coding region. Aforementioned result was in consistent with anotherSAPmember in wheat,TaSAP1(Changet al.2013).TaSAP7-Bwas mapped on chromosome 5B flanked byAX-99395950(2.86 cM) andXwmc380(5.26 cM). Previous research showed that QTLs for PH and TGW were in the close vicinity ofTaSAP7-Bregion (Wuet al.2010, 2012; Liet al.2012).

Association analysis is an efficient tool for genetic dissection of complex and superior traits as compared to linkage analysis, such as higher mapping resolution, shorter research time and wide range of genetic variation (Guptaet al.2005; Zhuet al.2008). Population structure is a critical factor causing false association (Rafalski 2010). A MLM approach was applied in association analysis betweenTaSAP7-Ballelic variation and agronomic traits to reduce false association effect caused by population structure.Previous studies indicated thatTaSAP1allelic variation significantly correlated with agronomic traits, such as TGW,spike length and number of spikelet per spike (Changet al.2013, 2014). In this study, elite allele inTaSAP7-Bgene was identified by the dCAPS marker and association analysis.The allelic variation ofTaSAP7-Bassociated with PH and TGW. The results implied that the two SAP proteins may have some common functions in wheat. PH is a trait easy to measure and remains constant after flowering. Breeders prefer wheat PH ranged between 85 and 100 cm (Cuiet al.2011; Zhanget al.2011). The genotypes possessing C allele had ideal PH. Lengths of peduncle and penultimate internode positively contribute to PH, and the genotypes possessing C allele also had proper PL and LPI. Being a staple food crop, breeding for high yielding wheat varieties is the utmost objective of the breeders. TGW is a vital trait that consistently attracts the attention of breeders (Barreroet al.2011). The genotypes with C allele had a higher TGW than that of T allele under all environments indicating the superiority of C allele genotypes over T allele genotypes.

Wheat modern breeding is a process of accumulating superior allelic variation (Wanget al.2012). Geographic distributions ofTaSAP7-Ballelic variation showed that the proportion of the genotypes with C allele was increased from landraces to modern cultivars in all 10 major wheat zones of China. According to the frequencies ofTaSAP7-Ballelic variation in different decades, the genotypes with C allele were apparently selected in Chinese wheat breeding programs. All in all, the dCAPS marker SNP-260 ofTaSAP7-Bcould be used in target trait selection to improve the efficiency of wheat breeding program.

Fig. 7 Frequencies of TaSAP7-B allelic variation in Chinese wheat breeding programs in different decades. T, cytosine;C, thymine.

5. Conclusion

One InDel and one SNP were identified in the promoter region ofTaSAP7-Bgene. The dCAPS marker SNP-260 developed from the SNP was significantly correlated with plant height (PH), peduncle length (PL), length of penultimate internode (LPI), number of spike per plant(NSP), and 1 000-grain weight (TGW). Accessions in the natural population were divided into two genotypes by the marker SNP-260. The genotypes possessing T allele had higher PH, PL, LPI, and NSP than that of C allele genotypes in all environments. The base C at the site–260 bp was an allele for higher TGW, therefore the base C was considered as the superior allele to increase TGW.The superior C allele was apparently selected in Chinese wheat breeding programs. The dCAPS marker SNP-260 ofTaSAP7-Bcould be helpful in marker-assisted selection in wheat improvement.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2016YFD0100605) and the National Natural Science Foundation of China (31271720).

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