Effect of high-molecular-weight glutenin subunit deletion on soft wheat quality properties and sugar-snap cookie quality estimated through near-isogenic lines

High-molecular-weight glutenin subunits（HMW-GSs） play a critical role in determining the viscoelastic properties of wheat dough. The HMW-GSs are encoded by Glu-A1, Glu-B1, and Glu-D1 loci on the long arms of chromosomes 1A, 1B, and 1D, respectively. In the present study, four near-isogenic lines with different HMW-GS deletions and compositions at the Glu-A1 and Glu-D1 loci in Yangmai 18 background were used for quality analysis. Deletion in Glu-D1 showed much weaker gluten quality and dough strength than null Glu-A1 genotype and wild genotype（WT）, based on the measurements of sodium dodecyl sulfate（SDS）-sedimentation, lactic acid solvent retention capacity（SRC）, gluten index, development time, stability time, and alveograph P and L values. The deletion of Glu-D1 did not significantly affect grain hardness, grain protein content, water SRC, sodium carbonate SRC, and sucrose SRC. Double null genotype in Glu-A1 and Glu-D1 and single null genotype in Glu-D1 showed significantly higher cookie diameter, crispness, and lower cookie height compared with single null genotype in Glu-A1 and WT. These indicate that the null Glu-D1 genotype is useful for improvement of biscuit quality, and use of this germplasm would be a viable strategy to develop new wheat varieties for biscuit processing.

Mapping a Novel Gene of Cold Tolerance at Booting Stage by Using Near-Isogenic Lines in japonica Rice

Genetic analysis showed that cold tolerance at booting stage of near-isogenic lines （NILs） of Kunmingxiaobaigu was controlled by a gene with large phenotypic variance. One hundred and sixty-four simple sequence repeats （SSR） distributed over 12 chromosomes were used to screen polymorphism between Towata （recurrent parent, RP） and near-isogenic line pool （NILP）, and two SSR markers at the long arm of chromosome 5 showed polymorphism in comparison with RP genome. Of the two markers, RM31 was found possibly linked with the cold tolerance gene at booting stage through one-way ANOVA. Twelve SSR markers around RM31 were then used to detect polymorphism between RP and NIL, and only RM7452 had polymorphism. The gene of cold tolerance at booting stage was further mapped on chromosome 5 between RM7452 and RM31 with genetic distances of 4.8 cM and 8.0 cM, respectively. This gene explained 10.50% of phenotypic variance and 5.10% of phenotypic variance of fully filled grains, and was tentatively designated as Ctb（t）.

Physiological Macro-lesions Enhanced Resistance to Blast (Magnaporthe grisea) in Rice Near-isogenic Lines

Roll-leaf-1 （rl-1） and spot-leaf-1 （spl-1） were two near-isogenic lines, which were obtained after 3 to 4 backcrosses with early season indica rice Zhefu 802 as recurrent parent. Henna macro-lesions, referred as physiological or morphological markers, began to appear on leaves at 4.5- to 6.0-leaf stage. The rice seedlings were inoculated at 3.5-, 5.0- and 7.0-leaf stages with high pathogenic races Zhong A1 and Zhong B1 of Magnaporthe grisea, respectively. The resistance of rl-1, spl-1 and Zhefu 802 against blast was significantly different. The seedlings of Zhefu 802 at 3.5- to 7.0-leaf stage were susceptible to races Zhong A1 and Zhong B1 of M. grisea, whereas those of rl-1 and spl-1 at 3.5-, 5.0- and 7.0-leaf stages were susceptible, moderately resistant and resistant, respectively. These results suggested that the enhanced resistance of rl-1 and spl-1 related to the appearance of their morphological marker lesions. The experiment provided a basis for studying lesion mimic and hypersensitive response in association with disease resistance.

Breeding of near-isogenic japonica rice lines with three major genes for resistance to bacterial blight

From 1986 to 1993, a set of near-isogenic japonicarice Iines with three major genes Xα-3, Xα - 4,and Xα-12 for resistance to bacterial blight(Xan-thomonas oryzae pv.oryzae)were developed anddesignated as CBB3, CBB4, and CBB12 respective-

Development of Chinese Near-isogenic Line of Rice and Their Differentiating Ability of Pathogenic Races of Blast Fungus

The rice( Oryza L. ) variety, Lijiangxintuanheigu(LTH), is a universally susceptible variety. A set of near-isogenic lines (NILs) of rice which has single resistance gene for each line was developed by use of LTH as recurrent parent and Kusabue,Tsuyuake, K1, PiNo. 4, K60 and BL1, which are from Kiyosawa's differential varieties(KDV), as donor parents. Differentiating ability of these NILs to Philippine isolates was compared with that of their donor parents, KDV and IRRI' s NILs. The results of comparative studies were as follows: (1) Differentiating ability of Chinese NILs to Philippine isolates was much stronger than that of their donor parents and that of KDV. (2) Chinese NILs had similar ability to differentiate races to IRRI's NILs or higher differentiating ability than them. But IRRI's NILs almost had no differentiating ability to isolates from japonica rice-growing regions. They can not be used to differentiate races of isolates from japonica ricegrowing regions. Whereas, Chinese NILs can be used to differentiate races of isolates from both japonica ricegrowing regions and indica rice-growing regions. It was concluded that the set of Chinese NILs can be used as international differentials.

The Construction of Heat-resistant Near Isogenic Line (NIL) of Bombyx mori and the Development of Molecular Marker Breeding Technique

[Objective] The study aimed to provide theoretical basis for development and application of molecular marker breeding technique to obtain Bombyx mori near-isogenic lines （NILs）. [Method] Thermotolerance gene was introduced into sensitive variety Ou17 by developing NILs and recurrent backcross,then through six generations of backcross,thermotolerance-assisted selection,and two generations of self-cross. [Result] Bombyx mori NILs carrying thermotolerance gene （new germplasm） were produced. Meanwhile,thermotolerance level of progenies of each backcross and molecular markers of NILs were determined,and then attempts were made to produce practical thermotolerance hybrids by using thermotolerance varieties whose thermotolerance gene is linked to SSR markers. [Conclusion] The study successfully construct thermotolerance NILs,monitor thermotolerance level and breeding results of progenies of each backcross,and determine molecular marker of NILs.

孕穗期NILs的耐冷相关性状的相关分析及聚类分析

用耐冷性极强的地方品种与十和田配制的NILs及亲本共84份材料,分别种在昆明和阿子营,通过相关和聚类分析,结果表明:(1)在昆明,结实率与花药长、倒二节长、实粒数、秕粒数之间相关系数较大,结实率、花药长、倒二节长、实粒数、秕粒数这几个性状与耐冷性的关系较密切。(2)在阿子营,结实率与花药长、株高、穗长、实粒数、秕粒数、总粒数之间相关达极显著水平,表明移栽至成熟期极端冷害下造成结实率降低,对整个植株生长发育均有抑制作用。(3)在昆明,84个材料18个性状聚类没有明显的类间区别,13个性状聚类可划分为2大类;在阿子营,78个材料8个性状聚类可划分为4类。(4)结实率作为耐冷性指标是较可靠的,在极端冷害下,可参考花药长等其他耐冷性指标。

粳稻孕穗期耐冷性NILs的形态评价和分子验证

2004年在海拔1 916 m昆明两种冷害(水温19.5±0.7℃,低田温冷泉水温17.8±1.1℃)、阿子营冷害(海拔2 150 m,水温18.2±0.22℃)条件下对5个亲本及其25个近等基因系进行耐冷性鉴定,用Statistica对17个农艺性状进行形态聚类和SSR分子标记聚类分析。结果表明:(1)已培育的穗期耐冷性NILs与轮回亲本十和田的形态极为相似,但与耐冷性相关的性状(穗颈长、实粒数、结实率、花药长和花药体积)有明显的差异;(2)从78个SSR标记筛选出了7个标记在十和田和NILs间存在多态性,从分子水平上验证了近等基因系与十和田耐冷性差异大。证明了这些NILs是水稻穗期耐冷基因精细定位和克隆的理想材料。

Qualitative Difference Between HMW-GS 5+10 and 2+12 NILs of Four Spring Wheat Cultivars with High-Quality Genetic Background

To investigate the effect of high molecular weight glutenin subunits (HMW-GS) 5+10 inhigh quality spring wheat, four near-isogenic lines (NILs) developed by 6 consecutivebackcrosses sharing the same genetic background and differing only at the Glu-D1 locusin each pair were employed in this study. The results showed that gluten indexes were allincreased by 3.1-27.7% (P<0.01) after the 5+10 subunits introduced into the fourcultivars. Their stability time was increased by 128, 104, 233 and 36%, where the maximumresistance raised by 130, 56.04, 95.10 and 16.33% in Kefeng6, Longmai20, Longfumai10and Xiaobingmai33, respectively.

Study on the Quality of NILs of Wheat cv. Longfumai 3 Possessing HMW-GS Null and 1 Subunits

To determine the genetic differences between high molecular weigh glutenin subunits （HMW-GS） null and 1. HMW-GS 1 was introduced into Longfumai 3 （N, 7＋8, 5＋10） by 5 consecutive backcrosses with biochemical marker assisted selection. The near isogenic lines （NILs） of HMW-GS null and 1 were obtained and planted in the experimental field of Crop Breeding Institute of Heilongjiang Academy of Agricultural Science in 2004 and 2005. The field experiments were designed using the method of two-line contrast arrangement with four replicates. The result of two years experiment showed that the statistic differences of quality parameters between Longfumai 3 with subunit 1 and with null were not significant in flour protein content, dry gluten content, and water absorption. However the gluten index,＇ Zeleny sedimentation, the ratio of sedimentation/dry gluten, the development time, stability, and the breakdown time in the NIL with 1 subunit were increased by 5.8% （P〈0.01）, 9.3% （P〈0.01）, 8.6% （P=0.01）, 127.3% （P〈0.01）, 79.2% （P〈0.01）, and 53.6% （P〈0.01）, and the ratio of wet gluten/dry gluten and the degree of softening were decreased by 1.7% （P = 0.05） and 16.5% （P = 0.13）, respectively. The impact of the HMW-GS 1 on the gluten strength was highly positive in NILs containing HMW-GS 5 ＋ 10, suggesting that HMW-GS 1 can be an indispensable subunit for breeding strong gluten wheat.

Characterization of QTL for unique agronomic traits of new-plant-type rice varieties using introgression lines of IR64

To enhance the yield potential of an elite indica rice cultivar,an introgression(BC_3-derived) line of IR64,YTH288,was developed using a new-plant-type cultivar,IR66215-44-2-3,as a donor parent.YTH288 has agronomically valuable characteristics such as large panicles,few unproductive tillers,and large leaves inherited from NPT.To identify the genetic basis of these traits,we used 167 F_2 plants derived from a cross between IR64 and YTH288 to conduct QTL analysis for five agronomic traits:days to heading(DTH),culm length(CL),flag leaf length(FLL),flag leaf width(FLW),and filled spikelet number per panicle(FSN).Six putative QTL were detected:four on chromosome4(for CL,FLL,FLW,and FSN) and two on chromosome 2(for DTH and FLL).All QTL with the IR66215-44-2-3 allele,except that for FLL on chromosome 2,had positive effects on each trait.To confirm the effects of these putative QTL,we developed NILs with the IR64 genetic background by marker-assisted selection.We observed significant differences in several agronomic traits between IR64 and NILs that carried these QTL on chromosomes2 and 4.Additionally,four IR64-NILs carrying chromosomal segments derived from different NPT varieties on the long arm of chromosome 4 exhibited similar pleiotropic To enhance the yield potential of an elite indica rice cultivar, an introgression(BC3-derived) line of IR64, YTH288, was developed using a new-plant-type cultivar,IR66215-44-2-3, as a donor parent. YTH288 has agronomically valuable characteristics such as large panicles, few unproductive tillers, and large leaves inherited from NPT.To identify the genetic basis of these traits, we used 167 F2 plants derived from a cross between IR64 and YTH288 to conduct QTL analysis for five agronomic traits: days to heading(DTH), culm length(CL), flag leaf length(FLL), flag leaf width(FLW), and filled spikelet number per panicle(FSN). Six putative QTL were detected: four on chromosome4(for CL, FLL, FLW, and FSN) and two on chromosome 2(for DTH and FLL). All QTL with the IR66215-44-2-3 allele, except that for FLL on chromosome 2, had positive effects on each trait. To confirm the effects of these putative QTL, we developed NILs with the IR64 genetic background by marker-assisted selection. We observed significant differences in several agronomic traits between IR64 and NILs that carried these QTL on chromosomes2 and 4. Additionally, four IR64-NILs carrying chromosomal segments derived from different NPT varieties on the long arm of chromosome 4 exhibited similar pleiotropiceffects for unique agronomic traits. These NILs can be used as research materials for studying each trait and as breeding materials for yield improvement of indica rice cultivars.

Fine mapping and characterization of a major QTL for grain length,QGl.cau-2D.1,that has pleiotropic effects in synthetic allohexaploid wheat

Grain size is one of the determinants of grain yield,and identifying the genetic loci that control grain size will be helpful for increasing grain yield.In our previous study,a quantitative trait locus(QTL)for grain length(GL),QGl.cau-2D.1,was identified from an F2 population developed from the cross between the natural(TAA10)and synthetic(XX329)allohexaploid wheat.In the present study,we mainly fine mapped and validated its genetic effects.To this end,multiple near-isogenic lines(NILs)were obtained through marker-assisted selection with TAA10 as the recurrent parent.The secondary populations derived from 25 heterozygous recombinants were used for fine mapping of QGl.cau-2D.1,and the allele from XX329 significantly increased GL,thousand-grain weight(TGW),total spikelet number per spike(TSN)and spike compactness(SC).Using NILs for XX329(2D+)and TAA10(2D−),we determined the genetic and pleiotropic effects of QGl.cau-2D.1.The target sequences were aligned with the wheat reference genome RefSeq v2.1 and spanned an~0.9 Mb genomic region.TraesCS2D03G0114900(ortholog of Os03g0594700)was predicted as the candidate gene based on whole-genome re-sequencing and expression analyses.In summary,the map-based cloning of QGl.cau-2D.1 will be useful for improving grain weight with enhanced GL and TSN.

Molecular Mapping of Sterility QTLs qSS-3, qSS-6a and qSS-7 as Single Mendelian Factors via NIL strategy

Hybrid sterility between Oryza glaberrima and O. sativa seriously hampers the introgression of favorable genes from each other. In order to further understand this issue, identification and isolation of hybrid sterility QTLs as single Mendelian factors are an effective strategy. A genetic map was constructed using a BC1 F1 population derived from a cross between an O. safiva japonica cultivar and an O. glaberrima accession. Four main-effect QTLs for pollen sterility were detected in the BC1F1. Five BC8F1 advanced backcross populations were developed via successive backcrosses based on phenotype and molecular selections. The BC8F1 populations showed bimodal distribution for pollen fertility and could be classified into semi-sterile and fertile types, fitting single Mendilian factor inheritance ratios. Three QTLs detected in the BC1F1 corresponding to qSS-3, qSS-6a and qSS-7 were mapped on chromosomes 6, 3 and 7, respectively, as single Mendilian factors.

Identification of KASP markers and putative genes for pre-harvest sprouting resistance in common wheat(Triticum aestivum L.)

Common wheat(Triticum aestivum L.)is the most important crop in the world and a typical allopolyploid with a large and complex genome.Pre-harvest sprouting(PHS)leads to a significant reduction in grain quality worldwide.PHS is a complex trait with related QTL located on different chromosomes.However,the study of markers and genes related to PHS resistance is limited especially for whitegrained wheat.Four pairs of near isogenic lines(NILs)from a white-grained wheat cross of Chara
DM5637B*8 targeting a major QTL for PHS resistance(Qphs.ccsu-3A.1)on wheat chromosme 3AL were genotyped using the 90K SNP Illumina iSelect array.Ten SNPs were identified,with a 75%-100%consistency between genotype and phenotype in the resistant or susceptible isolines.The 10 SNPs were converted to cost-effective kompetitive allele-specific PCR(KASP)markers.Screening of 48 wheat cultivars with different phenotypes of PHS identified four KASP markers with 81.3%-85.4%conformity between genotype and phenotype.Further investigation revealed that the four SNPs(BS00022245_51,Kukri_c49927_151,BS00022884_51 and BS00110550_51)corresponding to the four validated KASP markers are residing in three independent genes(TraesCS3A03G1072800,TraesCS3A03G1072400,TraesCS3A03G1071800)close to each other with a distance of 4.28-4.48 Mb to the targeted QTL.These three annotated genes have potential functions related to PHS resistance.Our study revealed that combined use of NILs and the 90K SNP chip is a powerful approach for developing KASP markers and mining functional genes in wheat.The KASP markers for PHS resistance on chromosome 3AL are useful for high-throughput evaluation and marker-assisted selection,and the three identified genes could lead to a better understanding of the genetic pathways controlling PHS.

Screening and Cloning of RAPD Marker of Fluoride Tolerance Gene in Silkworm,Bombyx mori

In this study,silkworm strain T6,tolerant to fluoride,and silkworm strain 733xin,highly sensitive to fluoride,were used to construct the near-isogenic lines.300 random primers were used in RAPD amplification to DNAs of these lines.A molecular marker named S207 was found linked to the fluoride tolerance gene.Examination to F 2 segregated individuals of the above lines verified that this molecular marker was reliable.Subsequently,the molecular marker was cloned into a T vector (pUCm-T) for sequencing.Comparing with sequences available in the GenBank showed that this molecular marker was novel.We plan to convert it into a SCAR marker to facilitate establishment of a molecular marker assisted breeding system.

龙辐麦3号小麦品种HMW-GS Null和1近等基因系间品质差异的研究

【目的】确定Glu-A1位点高分子量麦谷蛋白亚基(HMW-GS)Null和1间的遗传差异。【方法】利用生化标记和选择性回交的方法将1亚基转移到了黑龙江省小麦品种龙辐麦3号中,获得了龙辐麦3号的N亚基和1亚基的HMW-GS近等基因系。2004和2005年这2个近等基因系被种植在黑龙江省农科院育种所的实验地里,田间设计采用双列对比排列,4次重复。【结果】两年的品质分析结果表明,1亚基龙辐麦3号与N亚基龙辐麦3号两年平均数相比,其面粉蛋白质含量、干面筋和吸水率在统计学上无显著差异;面筋指数、沉降值、沉降值/干面筋、形成时间、稳定时间和断裂时间分别提高5.8%、9.3%、8.6%、127.3%、79.2%、53.6%,而湿面筋/干面筋和软化度分别低1.7%和16.5%。【结论】在5+10亚基的遗传背景下,1亚基对面筋强度仍有较大的影响,因此在选育强筋小麦时,1亚基也是必需被考虑的亚基之一。

5+10亚基对超强面筋小麦品质的影响

为了了解5+10亚基与2+12亚基在超强筋小麦遗传背景条件下遗传效应的差异,利用生化标记和选择性回交(6次)的方法将5+10亚基转移到了超强面筋麦小冰麦33中。品质分析结果表明:含5+10亚基的小冰麦33在蛋白质含量和干面筋含量上与2+12亚基小冰麦33是相同的。在两者的籽粒蛋白质含量为18%(干基)时,5+10亚基的小冰麦33比2+12亚基的小冰麦33在面筋指数、形成时间、稳定时间、断裂时间、评价值、最大抗延阻力和拉伸面积上分别高3.1%、76.9%、36.7%、27.9%、10.0%、16.3%、7.7%;在湿面筋、粉质吸水率和延伸性上5+10亚基的小冰麦33略低于2+12亚基的小冰麦33。

小麦抗白粉病的遗传改良及多系品系的配制

为了培育抗白粉病的综合性状优良的小麦品种,采用滚动回交与遗传标记相结合的方法,排除了供体亲本中的不良性状,把抗白粉病基因Pm4a、Pm2、Pm6、Pm21以及Compton的慢白粉病性等转入综合性状较好的轮回亲本背景,与背景材料的丰产、早熟、优质、抗病等数量性状相结合,育成了一系列优良的近等基因系,其中扬麦10号、11号、12号等已经通过品种审定,在生产上大面积推广。同时构建了抗白粉病多系品系。并提出了把一系列重要农艺性状象转育Pm基因一样转育成综合丰产性好的近等基因系,然后在它们之间进行逐一回交,以构建聚合育种体系的构想。

龙麦20小麦品种中7+8~*亚基和17+18亚基近等基因系间的品质差异

【目的】确定高分子麦谷蛋白亚基17+18和7+8*间的遗传差异。【方法】利用生化标记和选择性回交(5次)的方法将拥有超量表达的Bx7亚基的加拿大超强筋小麦品种Glenlea的7+8*亚基转移到了小麦品种龙麦20亚基组成为1,17+18,5+10的近等基因系(NILs)中,获得了龙麦20的17+18亚基和7+8*亚基的近等基因系。2005年这对近等基因系被种植在黑龙江农业科学院育种研究所的试验地里,试验设计采用随机取组,6次重复。【结果】品质分析结果表明,7+8*亚基类型的龙麦20与17+18亚基龙麦20类型相比,面粉蛋白质含量提高5%(P=0.012)、干面筋含量增加4%(P=0.018)、湿面筋/干面筋降低2%(P=0.043)、沉降值提高10%(P=0.013)、形成时间提高13%(P=0.258)、稳定时间提高256%(P=0.029)、断裂时间提高86%(P=0.020)、软化度降低35%(P=0.007)。【结论】7+8*亚基对面筋强度有较大的正向影响。

水稻灌浆期耐热性近等基因系的选育研究

在广泛搜集、耐热性筛选与鉴定的基础上,从大量的品种资源中选择对灌浆期高温胁迫耐性很强的早籼纯系品种"BG33-2"作供体亲本,灌浆期耐热性敏感的早籼纯系品种"赣早籼38号"为轮回亲本,经过9年11个世代的杂交、连续单株鉴定选择与回交、花药培养、连续自交,育成了一套赣早籼38号近等基因系。33个特征特性的鉴定及性状测定值的统计分析结果表明,本研究育成的G38HT、G38MT、G38HS其性状均已纯合稳定,灌浆期耐热性分别为高抗、中抗、敏感,这3个纯系是灌浆期耐热性有显著差异的赣早籼38号近等基因系。这套近等基因系可为进一步深入系统地探明水稻灌浆期耐热性的生理机制和遗传机理,提供理想的实验材料。