Wheat is the largest cultivated and the most consumed cereal crops in the world to feed more than 35% of the global population as well as to provide the necessary nutrients of human beings in 20% of energy and protein (Sun et al., 2009; Yang et al., 2004). As the development of the world’s economy and changes in the structures of foods in developing countries, particular in the rapid growth of the world's population, wheat is considered to be the crop with the world's largest growth in demand (Rajaram, 2002). It is estimated that global demand for wheat in 2020 will reach 840 million tons to 1050 million tons, which means that wheat production needs to increase by 2.0% annually on the basis of the existing production of 560 million tons (Gill et al., 2004; Sun et al., 2009). Clearly, a substantial increase of wheat production would be the major solution to solve this problem. Therefore, breaking yield barriers and enhancing yields per unit would be the great challenges for wheat breeders. However, breeders applied a small number of well-known backbones of parents in long-term of wheat breeding and improvement, leading to narrow the genetic base of cultivated wheat varieties narrow. There are serious genetic erosion resulting in pandemics of pest and disease as well as great loss of yield in wheat production (Porceddu et al., 1988; Yang et al., 2004), which seriously constrains the further improvements in wheat breeding program. In wheat germplasm resources, the multiple-spikelet wheat (the numbers of spikelet is 30 or more) is specific germplasm with the traits of multiple spikelet and grains having the potential to increase wheat yield. Therefore, Utilization of this specific resource, a kind of multi-spikelet wheat, to change the panicle morphological characters as well as to enhance the number of spikelet and grains have become one of the ideal ways in high-yield breeding of wheat, of which has made encouraging progress so far (Rajaram, 2002; Yen et al., 1995; Swaminathan et al., 1966; Koric, 1973; 1980).

Multiple spikelet traits can be divided into three types, including branch type multiple spikelet, complex spike type multiple spikelet and straight-spike type multiple spikelet (no branch) (Millet, 1987; Li and Zhao, 2000). Tibetan triple spikelet wheat (Triticum aestivum L. Concv. Tripletum, 2n=42, AABBDD), belonging to the straight-spike type multiple spikelet, was a unique landrace collected in 1990’s in the Tibet of China, with the traits of three spikes bearing on each rachis of ear (Figure 1), while common wheat (Triticum aestivum L., 2n=42, AABBDD) has only one spike on each rachis of panicle (Figure 1) (Yan and Yang, 1999; Yang et al., 2004). The characteristics of triple spikelet in Tibetan triple spikelet wheat have genetic stability, similar to the triple spikelet of the six-rowed barley. Although the ear length of Tibetan triple spikelet wheat is quite similar with the ordinary ear length, the plant can bear more than 60 spikes each ear with more than 170 spikelets as well as has the normal tillering and full filling seed, of which the numbers of spikes and spikelets reach 2 or 3 times than that of common wheat (Yang et al., 2004). So far Tibetan triple spikelet wheat is the only one of triple spikelet wheat found in the world, which will provide a new valuable gene resource to mine new favorable genes as well as to develop high yield variety of wheat. Currently, most of studies showed that the trait if multiple spikelet (supernumerary spikelet) would be a recessive trait, governed by one or two genes (Yang et al., 2005; Martinek and Bednar, 2001; Peng et al., 1998a; Millet, 1986; Pennell and Halloran, 1983; Sharman, 1967). Some of studies found that the multiple spikelet might be dominant or partially dominant trait inheritance controlled by one or two genes (Sun et al., 2009; Sun et al., 2000; Chapman and McNeal, 1971; Dencic, 1988). Also some studies found that multiple spikelet traits could be quantitative inheritance, usually jointly controlled by a major gene and several minor genes (Zheng, 1994a; 1994b; Klindworth et al., 1990a; 1990b; Millet, 1987). Previous studies on chromosome analysis by using monomer and nullisome of Chinese Spring had some multiple spikelet gene mapped on the chromosomes of 2A, 4A, 5A, 7A, 2B, 4B, 2D, 3D and 6D (Peng et al., 1998a; 1998b; Zheng et al., 1994b; Yen et al., 1995), of which there was a strong genetic effect on the chromosome 2D (Peng et al., 1998a). It has reported that the second homology group of wheat chromosome might carry the genes controlling trait of multiple spikelet by studies on multiple spikelet characteristics of the tetraploid and hexaploid wheat (Peng et al., 1998a; Laykova et al., 2005; Klindworth et al., 1997;). Sears (1954) found that the deletions in the chromosome 2A or 2D of hexaploid wheat might generate LSS trait of which the gene inhibiting this trait located on chromosome 2DS and 2AL.

Previous studies of our group have shown that (Yang et al., 2005; Yang et al., 2004) the triple spikelet trait was no any association with the undesirable traits of Tibetan triple spikelet having low kilo-grain weight and low seed setting rate and so on. We obtained excellent triple spikelet individual from the progenies derived the triple spikelet germplasm, which possessed multiple spikelet with more flowers and more grains as well as high weight of 1000 grain weight and single panicle. It has confirmed that it would be feasible to improve the yield of wheat by the use of triple spikelet trait, as well as found that Tibetan triple spikelet trait was controlled by two independent recessive genes based on genetic analysis (Yang et al., 2005; Yang et al., 2004). However, there is no any study on mapping on the the triple spikelet trait so far. In this study, we employed the advanced line TTSW-5 with triple spikelet trait derived from the progenies of the cross between Tibetan triple spikelet wheat and Sichuan common wheat to mate with cultivated common wheat Jian 3 and Chuanmai 55 building F₂ populations in order to analyze the genetic characteristics of triple spikelet trait and detect the QTL associated with the triple spikelet trait through SSR markers.

1 Results and Analysis
1.1 Genetic analysis of triple spikelet trait
TTSW-5 was mated with Jian 3 and Chuanmai 55 to generate hybrid F₁ plants of 47 and 11, respectively. All of F₁ plants self-crossing were to generate F₂ plants of 1107 and 176, respectively. The panicle phenotypes of F₁ and F₂ were identified in ear maturity. The results showed that all of F₁ from the crosses of TTSW-5/Jian 3 and TTSW-5/Chuanmai 55 exhibit the ear phenotypes of common wheat as the same as the parents of Jian 3 and Chuanmai 55, indicating that trait of triple-spikelet of TTSW-5 would be controlled by a recessive gene.

F₂ individuals derived from TTSW-5/Jian 3 and TTSW-5/Chuanmai 55 were phenotyped in mature ear, in which 1040 of 1107 individuals of F₂ population from TTSW-5/Jian 3 exhibited the same ear phenotype as the parental Jian 3, while 67 individuals exhibited the triple-spikelet-like trait in different levels from 30%~100% of triple–spikelet panicles accounting for the total spikelets. Likewise, 161 of 176 individuals of F₂ population from TTSW-5/Chuangmai 55 exhibited the same ear phenotype as the parental Chuanmai 55, while 15 individuals exhibited the triple-spikelet-like trait in different levels from 35% to 100% of triple–spikelet panicles accounting for the total spikelets. The chi-square test showed that the ratios of two F₂ populations were χ²<χ²_0.05 (χ²_0.05=3.841), being consistent to 15 to 1 (Table 1), which indicated that triple spikelet traits of TTSW-5 should be governed by two independent genetic recessive genes, this result was identical to our previous results in genetic analysis of population derived from Tibet triple spikelet wheat itself.
 

1.2 Polymorphic analysis among parents and triple spikelet and common spikelet
944 SSR markers were chosen from the genomes of A, B and D in common wheat to screen the polymorphisms between the triple spikelet wheat TTSW-5 and Jian 3 of cultivated common wheat, of which 189 markers were detected to be differences accounting for 20% of the total markers with well electrophoresis. 69 polymorphic markers were detected in A genome with 7.3% of polymorphic ratio, 66 polymorphic markers detected in B genome, with 7.0% of polymorphic ratio and 54 polymorphic markers detected in D genome, 5.7% of polymorphic ratio. 14 of 189 SSR markers exhibiting polymorphisms between the parents, including Xgwm377, Xgwm296, Xcfd160, Xcfa2263, Xwmc144, Xgwm95, Xbarc1064, Xbarc11, Xbarc15, Xgwm122, Xgwm425, Xgwm275, Xgwm296 and Xwmc453, showed polymorphisms between the pools in the triple spikelet and the common spikelet wheat.

1.3 QTL detection
The markers having ploymorphisms in the pools and parents of the triple spikelet and common spikelet wheat were applied to scan the F₂ population derived from TTSW-5/Jian 3 for the separation of the triple spikelet trait. Genetic linkage map of chromosome 2A were built by 10 SSR markers covering the 112.8 cM in length of total chromosome (Figure 2). A QTL was detected in 2A on chromosome 1 by composite interval mapping analysis with LOD value of 6.19, tentatively named qTS2A-1, explaining 33.1% of the phenotypic variation. The detected QTL was located between SSR markers, Xgwm275 and Xgwm122, in span of 6.6 cM in the genetic distance (Figure 1). The results indicated that qTS2A-1 might be the major QTL associated with the triple spikelet traits. However, previous genetic analysis showed that the triple spikelet trait of TTSW-5 might be controlled by two independent genetic recessive genes, but there was only a major QTL for triple spikelet trait detected in this study, indicating that another one should not detected yet. It might be possible due to limited SSR markers or too far distance among SSR markers. So regarding another recessive locus, we managed to us new genetic population to identify the location and its effect of the tentative locus.

2 Discussions
A lot of analysis on genetic models of multiple spike traits has been made already. Patil (1958) considered that the branching traits of durum wheat might be controlled by single recessive gene. Pennell and Halloran (1983) studied that unstable branching trait of durum wheat lines AU S4531 controlled by two recessive genes. Aybeniz et al (2011) found that the novel branching panicle traits derived from the hybrid of synthesized wheat line 171ACS and hardy wheat were also controlled by the single recessive gene. Sun et al (2009) studies have shown that multiple spikelet wheat line 51885 was controlled by two pairs of dominant genes. Koric et al (1996) study suggested that branching panicle traits of common wheat were controlled by two pairs of dominant complementary and pyramiding genes. Our research group has used the Tibetan triple spikelet wheat to mate with Chuanmai 38 and SW8488, respectively to generate F₂ and backcross populations for genetic analysis, indicating triple spikelet trait of Tibetan triple spikelet was controlled by two independent recessive genes (Yang et al., 2005; Yang et al., 2004). Although the genetic model of multiple spikelet traits might be difference due to using different materials that may carry different genes, our results in this study had confirmed the above mentioned conclusions previous done by our research group (Yang et al., 2005; Yang et al., 2004).

Klindworth et al (1990b) found a recessive major gene associated with branching trait in the chromosome 2AS, but its linkage molecular marker was unavailable. Qxana et al (2009) had multiple spikelet gene Mrs1 mapped on chromosome 2DS, co-separating with SSR locus Xwmc453, meanwhile rye multiple spikelet gene Mol gene mapped on chromosome 2R. Although in this study the maim effective QTL closely related to triple spikelet traits was detected on chromosome 2A, a multiple spikelet locus detected by Qxana, Xwmc453, using to scan the parents and populations did not exhibit any correlation with the triple spikelet trait of Tibetan triple spikelet wheat, indicating that the QTL detected in this study should be difference from the Mrs1 locus mapped by Qxana et al (2009).

3 Materials and Methods
3.1 Materials used in this study
Tibetan triple spikelet wheat is a unique landrace in Tibet of China provided by the Sichuan Agricultural University, possessing similar triple spikelet trait of six-rowed barley with more than 60 spiklets and more than 170 florets, which should be 2 or 3 times than that of common wheat. TTSW-5 is the triple spikelet line derived from the progenies of the cross between the Tibetan triple spikelet wheat and Sichuan wheat Chuanmai 38. Jian 3 is the breeding line of common wheat developed by Crops Research Institute of Sichuan Academy of Agricultural Sciences. Chuanmai 55 is new variety of common wheat developed by Sichuan Academy of Agricultural Sciences. F₂ populations from the cross between triple spikelet wheat TTSW-5 and Jian 3 was employed for triple spikelet trait mapping and QTL detection. F₂ population from the cross of TTSW-5 and Chuanmai 55 were used for further genetic validation on phenotype.

3.2 Phenotypic identification
TTSW-5 was mated with Jian 3 and Chuanmai 55 to generate hybrid F₁ plants of 47 and 11, respectively. All of F₁ plants self-crossing were to generate F₂ plants of 1107 and 176, respectively. The panicle phenotypes of F₁ and F₂ were identified in ear maturity. The phenotypic methods were as following: harvesting the mature individual, measuring the number of triple spikelet per ear and total number of spikelet of whole ear to calculate the percentage by the number of triple spikelet to total of spikelet, the average percentage of each plant was assigned to be a phenotypic data of each ear.

3.3 DNA extraction and SSR analysis
Genomic DNA was extracted from the parents and F₂ individuals derived from TTSW-5/Jian 3 by using CTAB method. 944 SSR markers encoded with Barc, Xgwm, Xgdm, Xwmc, Xcfd etc. (Röder et al., 1998; Pestsova et al., 2000; Somers et al., 2004; Song et al., 2005) applied to scan the polymorphism between TTSW-5 and Jian 3. DNA pool of triple spikelet and DNA pool of common spikelet were established by using eight triple spikelet individuals and the common according to the results of phenotypic identification, respectively, find the polymorphism locus between the traits of triple spikelet and common spikelet by using the bulked segregant analysis (BSA) method.

PCR reaction was performed in the total volume of 20 μL containing 1×buffer (100 mmol/L Tris-HCl, pH 8.3, 1.5 mmol/L MgCl₂), 0.2 mmol/L dNTPs, 50 ng random primers, 1 U Taq DNA polymer enzyme, 50~100 ng template DNA. PCR amplification procedures were following as 94℃ pre-denaturing for 5 min and then following 35 cycles as 94℃ denaturing for 1 min, 50℃/55℃/60℃ annealing for 1 min, 72℃ extending for 1 min and finally 72℃ extending for 10 min. PCR amplification was carried out in a PTC-200, SSR primers were synthesized by the TaKaRa Company. PCR products was scored by 6% polyacrylamide gel electrophoresis with 1×TBE of electrophoresis buffer and 400 V voltages for 40 min, stained with nitrate silver and digitalized by digital camera.

3.4 Genetic linkage map and QTL analysis
Linkage map was constructed by the Software of Map Manager QTXb20, recombination rate was converted to genetic distance (centiMorgan, cM) with Kosambi function. QTL were detected by using composite interval mapping of QTL Cartographer 2.5 software.

Author’s contribution
JL completed the experimental design, result analysis and manuscript preparation and revision; JW, HTW and XRH are the main executor of the experimental work, completed data analysis and involved in writing. WYY conceived the project and designed the experiments as well as wrote and revised manuscript. All authors had read and agreed the final text.

Acknowledgement
This study was jointly supported by the National Foundation of Natural Sciences (30871532), National Key Basic Research Projects (2011CB100100), Specific Funds of Modern Agricultural Technology System (CARS-3-2-41), Sichuan Provincial Breeding Project, Sichuan International Cooperation Project (2010HH0052, 2011HH0026) and Youth Fund and Excellent Breeding Articles of Sichuan Province Finance.

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