The thinner pericarp of the kernel are associated with the tenderness of fresh waxy corn, we evaluated 60 waxy maize inbred lines in generation S₆-S₈. To determine thickness pericarp, we used micrometer and identified 38 out of 60 lines have favorable thickness pericarp ranged from 35 to 60 µm according to fresh quality. Using 5 SSR markers umc2189 – ZCT131; bmc1396-mmc0143; umc2118-bmc1325; umc1757-umc1550; umc2038-dupssr28 to detect QTL control thinner pericarp of waxy maize kernel. result shown that interelation between phenotype of 21 lines containing QTL. There were nine lines origined from Vietnamese traditional maize varieties (D14, 161, 21, 22, 42, 45, 60, 61, and 90), one line origined from Laos (D18) and eleven lines origined from China (D29, 30, 31, 52, 601, 70, 71, 72, 74, 82, and 85). Based on the evaluation of phenotype and molecular marker, we selected seven elite lines that suitable agronomical characters and thninner pericarp to develop waxy maize hybrid for market fresh quality those were D29, 161, 71, 601, 61, 86, and 74. These lines were crossed in diallel according to Griffing 4 to produce 21 possible F₁ hybrids. Twenty-one F₁ and their seven parents planted side by side in replicated trials to evaluate agronomical characteristics on phenotype and molecular markers. We selected 10 hybrids with favorable characteristics and thinner pericarp those were THL1, TH2, TH3, TH4, TH5, TH6, TH8,TH9, TH16 and TH20. They will be further evaluated to develop waxy maize hybrid have high quality in Northern of Vietnam.

Waxy maize (Zea mays L. var. certain Kulesh) is a special cultivated type of maize, and was first discovered in China in 1908 and then in other Asian countries (Collins GN ,1909; Fan LJ, 2009). Waxy maize with many excellent characters in terms of starch composition and economic value has grown in China for a long history and its production has increased dramatically in recent decades (Zheng H, 2013). There is a wide genetic diversity of waxy maize in agronomic traits such as plant height, maturity, resistance to insects and diseases and yield components. According to plant morphology, geographical distribution and biological characters, combined with historical data and folklore, waxy maize was considered to be derived from one single gene mutant from flint corn (Tian ML., et al. 2009). Fergason (2001) indicated that waxy maize varieties are now equal in yield to ‘dent’ field corn, and average variations in yield do not exceed 5%. But a real breakthrough in the process of selection of new varieties over the last 20 years has been observed in China (Agnieszka Klimek-Kopyra., et al. 2012). Introduction waxy corn as a fresh vegetable has been cultivated throughout Southeast and East Asia for more than a century for local and export markets continue to expand (Danupol Ketthaisong., et al. 2014).

Although normal corn is widely cultivated and used in food, forage, industrial and bioenergy, waxy corn or sweet corn, which is a special type of cultivated maize, is only used in food production. Waxy corn has become a popular and valuable crop in East Asian countries. Both demand and consumption of waxy corn have soared in recent years (Sa., et al. 2010). Along with increased consumption of waxy corn, consumers are also demanding more delicious new cultivars of waxy corn (Ki Jin Park., et al. 2013). Such as improvement of waxy corn quality is one of the most important aims in current breeding programs. To facilitate the development of new varieties with high eating quality, it is necessary to understand the genetic basis of such traits. Eating quality of waxy maize has been extensively studied consisted tenderness; flavor, taste, sweetness and kernel color depend on the market (Danupol Ketthaisong., et al. 2013). Kernel pericarp thickness and ear architectural traits are important selection criteria in fresh waxy corn breeding programs as they are associated with consumer sensory and visual preferences (Eunsoo Choe, 2010). Waxy maize breeding with high quality, special tenderness is priority in Vietnam therefore we conducted this study with the aim was identifying waxy maize inbred lines and crosses have high yield and quality for fresh waxy maize production in Northern of Vietnam

Plant materials
The waxy maize inbred lines consisted of 60 lines in generation S₆ to S₈ those were developed from germplasm have deference origin by self-polination, among them 30 lines from open-polination populations of Vietnam, 7 lines from Laos, and 23 lines from China with HQ6 and HN88 are check varieties as follows:

Methods
The field experiment was implemented at Crop Resaerch and Development Institute (CRDI),Vietnam National University of Agriculture following the method of Gomez, K. A., & Gomez, A. A. (1984).

Waxy inbred lines were evaluated in spring 2014, treatments were designed in randomized complete block design(RCBD) with two replications, plot area was 10m², spacing 60cm x 25cm, plant density was maintained at approximately 67,000 plants per hectare. A seven inbred lines namely: D29, D61, D71, D601, D161, D86 and D74 were selected based on the performance that shown in inbred lines experiment in spring 2014, aiming to identify the ncombinations between parental lines with high and low perfomance in traits related to eating quality and thickness pericarp traits. Parental lines were crossed in Griffing 4 diallel cross to produce 21 possible F₁ hybrids introduced in trail with designed in randomized complete block design (RCBD) with three replications, plot area is 14m² with a row-to-row distance of 70 cm and plant-to-plant distance of 25 cm. Plant density was maintained at approximately 57,000 plants per hectare. Standard agronomic practices were applied to all trials by Vietnamese testing regulation QCVN01-56:2011/BNNPTNT.

Measuring thickness of mature corn pericarp by micrometer method Kernels were steeped in water for 3 to 4 hours at room temperature, crown cap and tip cap were cut and removed. Pericarps were peeled, excised pericarp strips were placed in a 1:3 water:glycerol solution (by volume) and evacuated. Strips were left in the evacuated solution for 24 hours at room temperature. Pericarp strips were removed from the liquid, blotted dry, and placed in an equilibration environment of 25oC at 50% RH for 24 hours. Pericarp thickness was measured with a micrometer (Model 105-01-0), plunger diameter 1.66 mm using a 1-g weight) at the levels. Five regions of pericarp thickness traits consisted are upper germinal (UG), lower germinal (LG), upper abgerminal (UA), lower abgerminal (LA), and Crown (CWN). In addition, in this study, taste, flovor, sweetness and kernel color evaluated by tasting method, this are parameters associated with quality of the fresh waxy maize

SSR markers were used to detect QTL for pericarp thickness traits those were five specific primers umc2189 – ZCT131, bmc1369-mmc0143, umc2118-bmc1325, umc1757-umc1550, umc2038-dupssr28. Eunsoo Choe, (2010) can detect thikness pericarp in 5 regions of the kernel pericarps UG, LG, UA, LA and CWN of the 60 waxy inbred lines and 21 hybrids.

DNA extraction and SSR genotyping conducted according to CIMMYT, (2005): Genomic DNA from parental and F1 analysis to detect QTL related to thickness pericarp was isolated from young leaves. SSR amplifications were performed in a total volume of 30 μl and consisted of 20 ng genomic DNA, 1× PCR buffer, 0.3 M of forward and reverse primers, 0.2 mM deoxyribonucleotide triphosphate (dNTP) and 1 unit Taq Polymerase. The PCR profile consisted of a 5-minute initial denaturation period at 94°C followed by two cycles each of 1-minute denaturation at 94°C, 1-minute annealing at 65°C and 2-minute extension at 72°C. The annealing temperature was gradually decreased after the second cycle by 1°C following every second cycle until a final temperature of 55°C was reached. The last cycle was then repeated 20 times. Upon completion of the cycles, the reaction was extended for 10 minutes at 72°C. Five microliters of the final reaction product was mixed with 10 μl electrophoresis loading buffer (98% formamide, 0.02% BPH, 0.02% Xylene C and 5 mM NaOH). Remove combs only when ready to load samples. Pour enough 1X TBE buffer into the gel rig to cover the gel by at least 0.5 cm. Run samples into gel at 100 volts, constant voltage, for about 2-3 h, until the bromophenol blue dye has migrated to just above the next set of wells. Remove tray from rig and stain in 1 μg/ml ethidium bromide (100 μl of 10 mg/ml ethidium bromide in 1000 ml dH2O) for 20 min with gentle shaking. Rinse gel in dH₂O for 20 min, slide gel onto a UV transilluminator, and photographed.

Statistical procedures and data analysis: The experimental data were analyzed using analysis of variance (ANOVA) for individual environments. Homogeneity of error variance was tested before combined analysis across environments (Gomez and Gomez, 1984). The least significant difference (LSD) analysis, analysis of the diallel for general combining ability(GCA) and specific combining ability (SCA) for yield were based on the Model 4, The statistical model for the mean value of cross (i × j) in Griffing’s analysis is:
Yij= m + gi+ gj+ sij+ rij+1/b∑ eijk,
Software used for ANOVA, CV% and LSD analysis was IRRISTAT ver 5.0 and GCA, SCA used DTSL software Nguyen Dinh Hien (1995).

Evaluation of the 60 waxy inbred lines in spring seasons 2014
Choe, Eunsou (2010) reported the thickness pericarp of the waxy ranged from 35 μm to 60 μm was suitable to fresh waxy maize. In this study, there were 35 inbred lines have thickness pericarps ranged from 35-60 μm and grain yield was higher 1.5 ton per hectare, among them 14 inbred lines origined from Viet Nam (D2, D14, D161, D21, D22, D33, D36, D42, D45, D60, D61, D68, D89, and D90), four inbred lines origin from Laos (D18, D26, D27, and D88), 18 inbred lines origin from China (D15, D28, D29, D30, D31, D52, D601, D70, D71, D72, D73, D74, D75,D76, D77, D78, D80, and D86. There were three inbred lines have thickness pericarp siminar check line HQ6 from Korea.

Indentification of the waxy inbred lines that contained QTLs control for pericarp thickness traits referance from Choe Eunsoo., (2010) with 5 primers flanked QTLs, which located at the chromosome 1, 2, 3 and 4. Detection of the QTLs in a set of waxy maize inbred lines include 60 inbred lines and check inbred line HQ6. Result was showed in table 3

Screening methods by marker SSR for identifying QTL control thickness pericarp traits, the marker umc2189 detected QTL relate to thickness pericarp traits in three regions are UA, LA and CWN was identified 51 bands with size ranged 100 to 200bp, such as 51 lines among the 60 lines contained QTL for thickness pericarp trait in three regions of the kernel pericarp. Marker ztc131 detected 59 bands with size from 100 to 200bp, correspond to 59 lines have contained QTL control thickness pericarp traits in three regions are UA, LA and CWN among 60 waxy inbred lines. Marker umc2118 identifed 59 bands with size ranged from 100 to 200bp. There were 59 inbred lines contained QTL control thickness pericarp traits of the five regions of the kernel pericarp (UG, LG, UA, LA and CWN). Marker bmc1325 identified 58 bands with size ranged 100 to 200bp, this marker identified 58 lines have contained QTLs control thickness pericarp in five regions of kernel pericarp. Another markers bmc1369 identified 59 lines contained QTL in fieve regions of kernel pericarp. Marker mmc 0143 identified 41 lines, Marker dupsr28 indentifed 59 lines (figure 2).

Marker umc2038 identified 55 bands in 55 lines those contained QTL control thickness pericarp trait in two regions (UA, LA). Among 60 waxy inbred lines, marker bmc 1550 identifed 50 bands such as correspondence to 55 lines have contained QTL control thickness pericarp trait in region (LA). Among 60 waxy inbred lines, marker umc1757 identifed 51 lines have contained QTL control thichkness pericarp in region LA of kernel (Figure 3).

Correlation between phenotypic and molecular markers was ditermined in 24 out of 60 lines those have thinner pericarp trait. Combination of the thinner pericarp trait and yield was selected 21 lines within 24 lines those were considered promising lines, 9 lines origined from Viet Nam (D14, D161,D 21, D22, D42, D45, D60, D61 and D90), one line from Laos (D18) and 11 lines from China (D29, D30, D31, D52, D601, D70, D71, D72, D74, D82 and D85).

In addition, selected based on the other quality traits as taste, flovor, tenderness were picked out 7 lines put into combining ability evaluation were D29 (1,71); D61 (3,74); D71 (4,27); D601 (4,28); D161 (4,62); D86 (4,46); D74 (4,48). These lines have pericarp thickness trait values and selected best lines based on pericarp thickness phenotype and the selected best line based on pericarp thickness favorable QTL alleles on each line measured in spring season 2014.

Combining ability analysis of the waxy maize inbred lines and detected QTL related thickness pericarp in F1 progenies in autunm-winter season 2014

The seven waxy inbred lines were crossed in diallels designe by Griffing 4 method to produced 21 hybrids. The experiments were evaluated 21 hybrids in a complete randomized block design with three replications, where each plot consisted of two 10-m long rows. Rows were spaced 0.7 m and plants 0.25 apart, with a total area of 10m2. Recorded data on the fresh yield, average thickness pericarp and some quality charecteristics showed that the fresh yield of the hybrid ranged from 8.16 t/ha (THL4) to 11.58t/ha (THL8), there were 16 THLs have fresh yield equal check variety HN88. The thickness pericarp was thinly ranged from 45.1 µm (THL15) to 60.0 µm, equal check variety HN88 and all hybrids belong to thickness pericarp appropriated to fresh waxy maize market (Eunsoo Choe, 2010) and accepted consumers in Viet Nam. Another quality traits flavor, tenderness, taste in similar to check variety

Diallel analysis of combining ability was carried out using Method 4 of Griffing (1956). Test of significant difference from zero for GCA and SCA effects was performed using t-test. Combining ability analysis of the purple maize waxy inbred lines is important in hybrid waxy maize (Shieh., et al. 2004). Estimation of combining ability to identify candidates for promising hybrid combinations to develop new hybrid variety (R.N. Mahto and D.K.Ganguli, 2003). Combining ability analysis of seven waxy maize inbred lines of variance and means for grain yield with three replications. Significant differences (P < 0.05) between genotypes were detected in the combined analysis with mean squares 207.3 and Ft is 656.75 as table 5a.

Mean squares from GCA analysis between genotypes (hybrids) for fresh yield is 94.363 and Ft was 598.676 , and SCA was 107.439 and Ft was 681.637 such significant differences (P < 0.05) between genotypes were detected in the combined analysis as table 5b.

For maize yield, we found that GCA was relative more important than SCA for unselected inbred lines, whereas SCA was more important than GCA for previously selected lines (Luciano Lourenço Nass., et al. 2000). This study was identified 7 inbred lines consisted of 6 lines have GCA value positive (D29, D161, D61, D86, D601 and D74), but only two lines (D601 and D74) have GCA value was not higher LSD.01 level and there were four lines have GCA value over LSD_0.5 at significant level (D29, D161, D61 and D86). Among them D61 have GCA value highest was (+5.444) and one line have GCA at negative value (-3,156) not further utilization for hybrid breeding.

The combining ability analysis of diallel data across replications showed highly significant effects (P<0.01). Specific combining ability analysis showed that the largest positive and negative SCA effects were observed with line D29 x line D161 (9.47), next was D161 x D61 (8.70), D161 x D601 (8.21) and D61 x D86 (5.94). Crosses between two inbred lines from difference origin showed SCA value higher as lLine D29 have origin from China, D161 from Viet Nam, line 601 have origin China. Han., et al. (1991), Vasal., et al. (1992), and Gama., et al. (1995) reported that, on average, crosses produced by crossing interpopulation lines have more positive SCA effects than those produced by crossing intrapopulation lines which tend to have more negative SCA effects.

The waxy maize inbred lines while GCA and having SCA are D29, D161 and D61, among them lines conformable female are D29 and D161, line D61 is conformable to male line in order to develop new waxy maize hybrid. Three elite lines were selected from this study can used further ion waxy maize breeding programme.

The utilization of molecular markers for identifying QTLs related to thinner pericarp
QTL for thinner pericarp traits of the 21 waxy maize hybrids detected by 4 specific primers according to Eunsoo Choe, (2010) and have high effect detected QTL control thinner pericarp in experiment evaluated 60 waxy maize inbred lines previous mention, marker pair umc2189-ZCT131 used for detection of QTL control thickness pericarp in UA (Upper Abgerminal) and LA (Lower Abgerminal) and CWN (Crown). Marker pair umc2118-bmc1325 used to detecte QTL control thickness pericarp in CWN, UA, LA, LG (Lower Germinal) and UG (Upper Germinal). Marker pair bmc1396-mmc0143 used to detecte QTL control thickness pericarp in UG, LG, UA, LA and CWN. Marker pair umc1550-phi096 used to detecte QTL control for LG.

Marker pair umc 2189-ztc131 used to detecte 17 per 21 hybrids contained QTL control thickness pericarp with size allele ranged from 100 to 200bp. Marker pair umc 2118- bmc 1325 used to detecte 20 hybrids that contained QTL. Marker pair bmc 1369 - mmc 0143 used to detecte 17 hybrids contanined QTL and marker pair mmc 1550 - dupssr28used to detecte 18 hybrids contained QTL control the thickness pericarp. Basically, there were 10 hybrids among 21 hybrids contained QTLs control thinner pericarp in five regions of kernel pericarp were THL1 (D29/D161), THL2 (D29/D71), THL3 (D29/D601), THL4 (D29/D61), THL5 (D29/D86), THL6 (D29/D74), THL8 (D161/D601), THL9 (D161/D61), THL16 (D601/D61), THL20 (D61/D74). Another hybrids contained QTL control thinner pericarp only in two to three regions of kernel pericarps.

In this study, the additive effect of QTL was negative for thickness pericarp therefore markers were choice to detect QTL control thickness pericarp were suitable and can utilized for MAS in hybrid waxy maize breeding with thinner pericarp trait. Marker assisted selection (MAS) may be useful for validating QTL effects and pyramiding favorable alleles in a fresh waxy corn breeding program. All five pericarp thickness traits were highly positively correlated, ranging from 0.78 to 0.93. Hybrids were varies significantly for different regions within a single kernel, and differs some among positions within a single ear. This indicated that some aspects of phenotypic breeding for pericarp thickness have difficulties due to experimental measuring errors. Therefore, early generation selection of QTL with additive effects for pericarp thickness traits may be promising for simultaneous indirect improvement of TP.

Evaluation of the growth, development, agronomical characteristics and fresh yield of 60 waxy maize inbred lines, we identified 35 inbred lines have thickness pericarps ranged from 35-60 μm and grain yield attained was higher 1.5 ton per hectare. Among them, 14 inbred lines origined from Viet Nam (D2, D14, D161, D21, D22, D33, D36, D42, D45, D60, D61, D68, D89, and D90), four inbred lines origined from Laos (D18, D26, D27, and D88), and 18 inbred lines origined from China (D15, D28, D29, D30, D31, D52, D601, D70, D71, D72, D73, D74, D75,D76, D77, D78, D80, and D86).

Using SSR markers showed that the correlation between phenotypic screening and molecular markers was ditermined in 24 out of 60 lines that have thinner pericarp traits. Combination of the thinner pericarp traits and yield was selected 21 lines within 24 lines considered promising lines, in there 9 lines origined from Viet Nam were D14, D161,D 21, D22, D42, D45, D60, D61 and D90, one line from Laos was D18 and 11 lines from China were D29, D30, D31, D52, D601, D70, D71, D72, D74, D82 and D85.

The waxy maize inbred lines have GCA and SCA are D29, D161 and D61, among them lines conformable female were D29 and D161, line D61 was conformable to male line in order to develop new waxy maize hybrid. Three elite lines were selected from this study can be used further ion waxy maize breeding programme. There were 10 hybrids among to 21 hybrids contained QTLs control thinner pericarp in five regions of kernel pericarp were THL1 (D29/D161), THL2 (D29/D71), THL3 (D29/D601), THL4 (D29/D61), THL5 (D29/D86), THL6 (D29/D74), THL8 (D161/D601), THL9 (D161/D61), THL16 (D601/D61), THL20 (D61/D74). Through evaluating lines and hybrids base on phenotype and SSR markers was evidence that the QTL information could be utilized through MAS to reduce pericarp thickness while maintaining favorable ear traits important to fresh waxy corn hybrid breeding.