Diversity, Structure, and Marker-Trait Association Analysis of the Maize Recombinant Inbred Line Population

Association mapping has emerged as a new tool to elucidate complex quantitative trait loci in maize, but there are few reports about systematic association analysis for the specific SSR markers with agronomic traits of interest in China. We investigated the morphological and genetic diversity and population structure for 76 maize recombinant inbred lines, and then association analysis were further performed between 48 simple sequence repeat loci and 17 morphological traits, consisting of nine ear-related traits and eight other traits. The 48 SSR markers were screened out and further classified into two groups including a group of loci in regions harboring reported quantitative trait loci that affect ear shape and a group of markers distributing on the whole genome randomly. The result indicated that the population of recombinant inbred lines was structured, showing five subpopulations. Our association results revealed that there were 82, 59, and 40 significant associations detected by K-test, logistic regression, and both analysis, respectively. When the 17 traits were considered separately, the significant associations between Q-SSRs and E-traits were raised to 27.8%, whereas the other groups of combinations ranged between 2.3 and 6.3%. As the proportion of significant associations is higher among the Q-SSR subset of markers and the subset of traits related to ear shape than those for all of the other combinations, we conclude that this approach is valid for establishing true positive marker-trait relationships. Our results also demonstrated that association mapping could complement and enhance previous QTL information for marker-assisted selection.

Transfer and Detection of barstar Gene to Maize Inbred Line 18-599 (White) by Particle Bombardment

In China, the purity of maize hybrid strain is discomforting to the development of seed industrialization. Finding a new method for reproduction of maize hybrid strain is necessary. In this study, using particle bombardment, barstar gene was transferred into maize inbred line 18-599 （White）, which is an antiviral and high quality maize inbred line. By molecular detection of the anther of transgenic maize, two plants transferred with barstar gene were gained in this study, which are two restorer lines. The two plants showed normal male spike, and lively microspores. But the capacity of the two restorer lines should be studied in the future. The aim of this study is to find a new method of reproduction of maize hybrid strain using engineering restorer lines and engineering sterility lines by gene engineering technology.

Low-Temperature Response to Major Agronomic Traits by Using Recombinant Inbred Line(RIL) Populations Derived from Towada × Kunmingxiaobaigu

Development of the recombinant inbred line populations （RILs） is important basis to detect QTLs for cold tolerance at booting stage in rice. A set of 230 RILs derived from the cross of Towada and Kunmingxiaobaigu were used for evaluation of low-temperature response on major agronomic traits of plant height （PH）, panicle length （PL）, panicle exsertion （PE）, spikelet fertility （SF）, specific spikelet fertility （SSF）, and spikelets per panicle （SPP） under natural low-temperature growing environments in Yunnan Province, China. The results showed PH, PE, and SPP were mainly attributed by genotypes. PL was mainly influenced interactively by the genotypes × environments. SF and SSF were mainly controlled by the environments. Under the five different growth environments, F values of the six agronomic traits mentioned above ranged from 4.019 to 97.284. Significant difference was revealed between the lines. Under every environment, it indicated significantly positive correlation between SF and SSF, with correlation coefficients ranged from 0.826 to 0.885. It indicated significantly positive correlation between PH, PL, and PE. Under five different growing environments, variation coefficients of the six characters ordered in SSF （66.3%） 〉 PE （57.4%） 〉 SP （37.2%） 〉 SPP （16.2%） 〉 PH （9.6%） 〉 PL （6.4%）. SSF, PE and SF were most sensitive to low temperature stress at booting stage, while SPP, PH and PL being least. The RILs of Towada/ Kunmingxiaobaigu can be used as a genetic population to investigate cold tolerance at booting stage. SSF, PE and SF are most sensitive to cold tolerance at booting stage in rice. So far the the variation of PH, PL, and SPP related to cold tolerance are not clear under natural low-temperature environment. More tested environments and years are required to identify and evaluate cold tolerance at booting stage in rice.

Evaluation of drought tolerance in ZmVPP1-overexpressing transgenic inbred maize lines and their hybrids

The vacuolar proton-pumping pyrophosphatase gene(VPP)is often used to enhance plant drought tolerance via genetic engineering.In this study,the drought tolerance of four transgenic inbred maize lines overexpressing ZmVPP1(PH4CV-T,PH6WC-T,Chang7-2-T,and Zheng58-T)and their transgenic hybrids was evaluated at various stages.Under normal and drought conditions,the height and fresh weight were greater for the four transgenic inbred maize lines than for the wild-type(WT)controls at the germination and seedling stages.Additionally,the transgenic plants exhibited enhanced photosynthetic efficiency at the seedling stage.In irrigated and non-irrigated fields,the four transgenic lines grew normally,but with increased ear weight and yield compared with the WT plants.Moreover,the ear weight and yield of the transgenic hybrids resulting from the PH4CV-T×PH6WC-W and Chang7-2-T×Zheng58-W crosses increased in the non-irrigated field.Our results demonstrated that the growth and drought tolerance of four transgenic inbred maize lines with improved photosynthesis were enhanced by the overexpression of ZmVPP1.Moreover,the Chang7-2 and PH4CV transgenic lines may be useful for future genetic improvements of maize hybrids to increase drought tolerance.

Adaptability and recovery capability of two maize inbred-line foundation genotypes, following treatment with progressive water-deficit stress and stress recovery

Two maize inbred lines, the foundation genotype Y478 and its derived line Z58, are widely used to breed novel maize cultivars in China, but little is known about which traits confer Z58 with superior drought tolerance and yield. In the present study, responses in growth traits, photosynthetic parameters, chlorophyll fluorescence and leaf micromorphological characteristics were evaluated in Y478 and Z58 subjected to water-deficit stress induced by PEG 6000. The derived line Z58 showed greater drought tolerance than Y478, which was associated with higher leaf relative water content (RWC), root efficiency, and strong growth recovery. Z58 showed a higher stomatal density and stomatal area under the non-stressed condition;in these traits, both genotypes showed a similar decreasing trend with increased severity of water-deficit stress. In addition, the stomatal size of Y478 declined significantly. These micromorphological differences between the two lines were consistent with changes in physiological parameters, which may contribute to the enhanced capability for growth recovery in Z58. A non-linear response of Fv/Fm to leaf RWC was observed, and Fv/Fm decreased rapidly with a further gradual decline of leaf RWC. The relationship between other chlorophyll fluorescence parameters (photochemical quenching and electron transport rate) and RWC is also discussed.

Evaluation of maize inbred lines for resistance to pre-harvest aflatoxin and fumonisin contamination in the field

Two important mycotoxins, aflatoxin and fumonisin, are among the most potent naturally occurring carcinogens, contaminating maize(Zea mays) and affecting crop yield and quality.Resistance of maize to pre-harvest mycotoxin contamination, specifically aflatoxin produced by Aspergillus flavus and fumonisin produced by Fusarium verticillioides, is a goal in breeding programs that screen for these important traits with the aim of developing resistant commercial hybrids. We conducted two years of field evaluations on 87 inbred lines originating primarily in China and Mexico and not previously screened for resistance.The objectives of our study were to identify resistant germplasm for breeding purposes and to examine possible relationships between resistances to the two mycotoxins. Aflatoxin and fumonisin were present in samples harvested from all lines in both years.Concentrations of total aflatoxin ranged from 52.00 ± 20.00 to 1524.00 ± 396.00 μg kg^(-1),while those of fumonisin ranged from 0.60 ± 0.06 to 124.00 ± 19.50 mg kg^(-1). The inbred lines TUN15, TUN61, TUN37, CY2, and TUN49 showed the lowest aflatoxin accumulation and CN1, GT601, TUN09, TUN61, and MP717 the lowest fumonisin accumulation. TUN61 showed the lowest accumulation of both mycotoxins. This study confirmed previous observations that high levels of aflatoxin can coexist with fumonisin, with 55 maize lines showing a positive correlation coefficient between the concentrations of aflatoxin and fumonisin and 32 lines showing a negative correlation coefficient. These selected lines,particularly TUN61, may provide sources of resistance to mycotoxin contamination in breeding programs. However, the mechanism of resistance in this germplasm remains to be identified. Future research should also address factors that influence the fungus–plant interaction, such as herbivory and environmental stress.

Identification of QTLs for Blast, Bacterial Blight, and Planthopper Resistance Using SNP-Based Linkage Maps from Two Recombinant Inbred Rice Lines

Rice is the most significant global food security. Several biotic factors limit rice production, breeding biotic-resistant rice has, therefore, become an increasingly important goal. Two elite rice lines, IR71033-121-15 (IR71033) and IR57514-PMI-5-B-1-2 (IR57514), provide potential genes for biotic stress resistance traits. In this study, genotyping by sequencing (GBS) for single nucleotide polymorphism (SNP)-based linkage map construction was used to detect quantitative trait loci (QTLs) for blast (BL), bacterial blight (BB), whitebacked planthopper (WBPH), and brown planthopper (BPH) resistance. IR71033 was derived from Oryza minuta and carried BL, BB, WBPH, and BPH resistance QTLs. IR57514 is a well-adapted rainfed lowland line that carries BL and BB resistance QTLs. Two sets of recombinant inbred line (RIL) populations derived from crosses of KDML105 × IR71033 and KDML105 × IR57514 were used to dissect the genetic basis of disease and insect pest resistance. The RIL populations were evaluated for BL, BB, WBPH, and BPH resistance from 2016 to 2018 at four rice research centers in Thailand. From these, we identified a large number of SNPs through GBS and constructed high-resolution linkage maps. By combining phenotypic evaluation with the GBS data, a total of 24 QTLs on four chromosomes were detected that confered pest resistance and explained 7.3% - 61.4% of the phenotypic variance. These findings should facilitate identifying novel resistance genes and applying marker-assisted selection for resistance to the four major rice pests investigated here. These strategies will improve the resilience and reliability of rice varieties adapted to the low-yielding environment of rainfed lowland areas worldwide.

ESTABLISHMENT OF A MAMMARY CANCER CELL LINE Ca 761-86 AND ITS BIOLOGICAL CHARACTERISTICS IN INBRED 615 MICE

A cell line designated as Ca 761-86 has been established from the solid mouse mammary cancer (Ca 761) by suspension culture. It has been passaged for more than 212 generations. Moderate round cells were predominant and most of them were mononuclear. Some characteristics of malignant cells and A-type viral-like particles were observed by electron microscopy. The results of cytochemical studies (DNA, RNA, SDH, 5' AMPase, ACP etc.) were comparable to the ultramicroscopic results. It multiplied approximately 27.4 fold on day 5 with mitotic index reaching 1.8% on day 3. This cell line was a hyperdiploid with karyotype of 45 or 45, -2X, tril2, tri17, +M1-5. Cell agglutination was observed when treated with ConA (≥7 fig, ml). Spontaneous agglutination might also take place without adding any ConA. After 5×106 cells of Ca 761-86 suspension were transplanted into the normal inbred 615 mice by different ways (subcutan eous, intrafoot-pad or intraperitoneal), the transplan lability rate reached 100%. Spontaneous remission was never observed and its metastatic ability reserved. PPLO were not detected. Ca 761-86 may be of value for practical purposes.

Identifying Genomic Regions Conferring Morphological and Yield-related Traits in Soybean Based On A Four-way Recombinant Inbred Line Population

Improvement of seed yield of soybean(Glycine max(L.)Merr.)is generally achieved by combining morphological and yield-related traits,such as plant height(PH),node number on main stem(NN),pod number per plant(NP),seed number per plant(NS),100-seed weight(HSW)and seed weight per plant(SWPP).Identifying quantitative trait loci(QTLs)for morphological and yield-related traits is therefore important for breeding.In this study,a four-way recombinant inbred line population comprising 160 lines derived from the cross(Kenfeng14×Kenfeng15)×(Heinong48×Kenfeng19)was planted in five different environments and morphological and yield-related trait data were used to identify QTLs by the inclusive composite interval mapping method.Totally 38 QTLs for PH,40 QTLs for NN,26 QTLs for NP,10 QTLs for NS,26 QTLs for HSW and 49 QTLs for SWPP were detected in 125 genomic regions.Single QTLs explained 2.17%-14.60%,2.00%-10.04%,2.37%-9.77%,2.62%-8.61%,0.47%-6.51%and 0.14%-12.39%of the phenotypic variation for PH,NN,NP,NS,HSW and SWPP,respectively.Among these 125 genomic regions,120 were newly associated with morphological and yield-related traits.The results would facilitate the molecular breeding of morphological and yield-related traits in soybean.

Genetic analysis and QTL mapping of growth period traits and plant height traits in soybean recombinant inbred lines from Dongnong 47 × PI 317334-B

High and stable yield is the main goal of soybean genetic improvement.In this study,association analysis was used to detect the quantitative trait loci(QTL)for the plant height,and soybean growth period using 182 SSR markers in the RIL population of 136 F_(4:8) lines,which developed from a cross between photoperiod-insensitive cultivar‘Dongnong 47’and photoperiod-sensitive variety PI317334–B.The results showed that 33 QTLs related to soybean growth period and plant height traits were detected by compound interval mapping,and were located on 12 linkage groups including N,C1,C2,J,D1a,B2,E,G,A2,O,L,I,with the contribution rate of 7.85–33.84%.These QTL loci and linkage markers related to soybean photoperiod sensitivity,would be helpful to identify key genes that control soybean photoperiod sensitivity,and provide an important basis for the breeding of new photoperiod-insensitive soybean varieties based on molecular design breeding.

Cluster Analysis of Chinese Pumpkin Inbred Lines

A cluster analysis was carried out based on Euclidean genetic distances through UPGMA method in Chinese pumpkin inbred lines.7 important agronomic traits of 46 Chinese pumpkin inbred lines were investigated.The result indicated that 46 pumpkin inbred lines were clustered into 4 groups and the inter-groups distances was larger than that in intra-group.The genetic distances of parents were related to F1 performance and the results of cluster would increase effectiveness in the Chinese pumpkin crossing breeding.

Analysis on Combining Ability and Genetic Parameters of Main Agronomic Characters of 22 Maize Inbred Lines

The general combining ability（GCA）, special combining ability（SCA） and genetic parameter of ten characters of 22 maize inbred lines including plant height and ear height were analyzed using 10×12 through incomplete diallel cross（NC Ⅱ）.The results showed that：（1） Among the 22 maize inbred lines, the yield GCAs of11 HN 097, 11 HN 099, 11 HN 105 and 11 HN 110 were high, which were elite inbred lines to collocate hybridized combinations with strong heterosis. The yield of11 HN110 × 11 HN097, 11 HN110 × 11 HN105, 11 HN112 × 11 HN 097 and 11 HN 106 × 11 HN 104 were in the first four place. The yielding abilities, adaptabilities and yielding stabilities of the four combinations can be further identified by experiment. The heredities of the ten characters were mainly controlled by additive gene effect whereas the influence of non-addictive gene effect was small. The narrow heritabilities of plant height, ear height, ear rows, ear length, kernels per row,100-grain weight and seed-producing percentage were more than 50%. The variances were mainly caused by heredity and early-generation selection should be conducted. The narrow heritabilities of ear diameter, bare tip length and yield was low, which should not be selected in early-generation.

Combining Ability and Correlation of Ear Characteristics of Twenty Maize Inbred Lines

The combining ability and correlation of eight ear characteristics in 99 maize hybrids generated by crossing nine female parents with 11 male parents were analyzed by incomplete diallel cross （NC II ） design. The results showed that the line F6 had the highest general combining ability （GCA） for yield, followed by F7, M3, M4 and M8. All of the five lines have great potential in maize breeding. The cross combination M3xF10 had the highest specific combining ability （SCA） for yield, showing strong heterosis. Heritability analysis of ear characteristics showed that GCA variance was higher than SCA variance in ear diameter, number of rows per ear and seed rate, and they were mainly controlled by the additive gene effect, indicating that that the selections for these traits are effective at early generations. The other three traits had lower SCA, for which the selections should be carried out at late generations. The correlation analysis revealed that ear length, number of grains per row, ear diameter, number of rows per ear, 100-seed weight and seed rate had extremely significant positive correlations with grain yield per plant. Among them, number of grains per row had the most significant effect on yield per plant. Barren tip length had a significant negative correlation with grain yield per plant. Therefore, we concluded that the combinations with more grains per row and shorter barren tip should be selected to achieve high yield of maize.

Combining Ability Analysis on Yield Traits of Recombinant Inbred Lines in Hybrid Rice

A population of 140 recombinant inbred lines at F8 generation were obtained after seven successive generations of self-pollination using single seed descent（SSD） method from the F2 hybrids of three-line restorers Luhui 8258 with high combining ability and Yanghui 34. Then, the 140 inbred lines obtained above and their parents Luhui 8258 and Yanghui 34 were crossed with three different types of cyto-plasmic male sterile（CMS） lines（Gang 46 A, Ⅱ-32 A and Lu 98A） according to NCⅡ design. The resulting 426 combinations were planted at Deyang and Suining bases to test the combining ability and inheritance of five yield traits： yield per plant, panicle number per plant, filled grain number per panicle, seed setting rate and 1 000-grain weight. The results showed that the variances of both general and specific combining abilities of the five traits all reached a significant or extremely significant level at the two sites. The broad and narrow heritability of the yield traits（except 1 000-grain weight whose broad and narrow heritability were both over70%） were all below 50% at the two experimental bases, suggesting that the four traits were easily subjected to environment influence. Very significant positive correlation of general combining ability was found between yield per plant and other traits except 1 000-grain weight. The general combining ability variance showed a normal distribution among the recombinant inbred lines at two sites, right in line with inheritance of quantitative traits. So, the genes controlling rice general combining ability can be targeted by QTL mapping.

Combining Ability Analysis of Panicle Traits in Six CIMMYT Maize Inbred Lines

In this study, six CIMMYT maize inbred lines and five representative do- mestic maize inbred lines were used as parental lines. By using incomplete diallel cross design, 30 hybrid combinations were developed to analyze the general com- bining ability （GCA）, specific combining ability （SCA） and total combining ability （TCA） of seven panicle traits in six CIMMYT maize inbred lines. The results showed that CIMBL98 and GEMS13 were excellent inbred lines with good compre- hensive performance; CIMBL98 × 340 and GEMS13×Chang 7-2 were superior combinations.

Mapping of QTLs for Sheath Blight Resistance Using Recombinant Inbred Lines of Rice(Oryza sativa L.)

A recombinant inbred line （RIL） population composed of 157 lines derived from an inter-subspecific hybrid of Daguandao/IR28 by the single seed descent method was used as materials, and the quantitative trait loci （QTLs） coffering the resistance to sheath blight in the 157 RILs and the parents were detected using the toothpick inoculation method. The disease indexes of rice sheath blight in the two parents and 157 RILs were scored and the QTLs responsible for rice sheath blight resistance were detected accordingly by QTL Cartographer software. The results showed that a total of 4 QTLs （qsbl, qsb2, qsb5-1, qsb5-2） conferring sheath blight resistance were detected on chromosomes 1, 2 and 5, and their variance explained ranged from 10.41% to 36.92%. The additive effect of qsb5-1 was negative, indicat- ing that the QTLs derived from donor parent IR 28 could enhance the resistance to sheath blight. However, the additive effects of qsbl, qsb2 and qsb5-2 were positive, indicating that the QTLs derived from donor parent Daguandao weakened the resis- tance to sheath blight.

Selection of Maize Inbred Lines with High Regeneration and Susceptibility to Agrobacterium tumifacien

Ten-maize inbred lines of maize （Zea mays L.） with high-induction rate and proliferation ability of embryonic calli were selected from 70-maize inbred lines by immature embryo culturing. Some of the embryonic calli were transferred onto regeneration medium to examine the ability of regeneration, some were transformed via Agrobacterium tumifaciens C58 carrying intron-β-glucuronidase （gus） gene, and GV3301 carrying the green fluorescent protein （gfp） gene to study the susceptibility of different genotypes in maize to A. tumifaciens. All embryonic calli initiated from 10-maize inbred lines were able to regenerate into plantlets, and the regeneration frequencies of inbred lines 6010, 6038, 6015, 6051, and 6060 were 61.11%, 31.94%, 45%, 33.33%, and 56.94%, respectively, which were higher than that of other lines. Analysis of variance indicated that the susceptibility of the various genotypes in maize to A. tumifacien C58 showed a significant difference among each other, and the inbred lines 6010, 6015, 6051, 6050, 6058, 6060, 6069, 6077 were susceptible to A. tumifacien C58, of which frequency of gus expression were over 70%. Expression of GFP was observed in six-inbred lines （6050, 6015, 6051, 6058, 6069, 6077）. The inbred lines 6051, 6010, 6015, 6060, and 6050 had the high regeneration and the susceptibility to A. tumifaciens C58; and the inbred lines 6051, 6015, and 6060 had the high regeneration and the susceptibility to Agrobacterium tumifaciens GV3301.

Genetic Relation Analysis on Ramie[Boehmeria nivea(L.)Gaud.]Inbred Lines by SRAP Markers

The objective of this article is to reveal the variations of ramie inbred lines in DNA level and discuss their molecular background to provide a theoretical basis for ramie cross breeding. In the present study, the genetic relationships among 33 inbred line accessions and two wild types that originated from China and Brazil were estimated using sequence-related amplified polymorphism （SRAP） markers. The results showed that 33 out of 81 primer combinations turned out to be polymorphic and 332 polymorphism bands were obtained. On the basis of the appearance of the markers, the genetic relationships were analyzed using unweighted pair-group method of arithmetic average cluster analysis （UPGMA）, and the genetic Jaccard similarity coefficients were calculated. The inbred-lines originating from China and Brazil formed a cluster suggesting a possibility that the Brazilian cultivars could have developed from cultivars introduced from China. Within ramie inbred-lines, the groupings also indicated that the greatest genetic relationship among cultivars was correlated to the region of origin of cultivars. The results provided the evidence that SRAP was an efficient approach, suitable for taxonomic analysis of ramie inbred lines, To the authors＇ knowledge, this is the first application of SRAP marker on the systematics of ramie inbred lines.

Identification of the miniature pig inbred line by skin allograft

Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identification of swine inbred lines by skin grafting has been reported. At present, Wuzhishan miniature pig （WZSP） inbred line has acquired the F24 individuals in China. In order to verify whether WZSP inbred line had D^en cultivated successfully, allogeneic skin grafts and related research were performed on F20 individuals of WZSP inbreeding population, compared with a control group of autologous transplantation. We observed the transplant recipients＇ wounds, detected peripheral blood-related indicators interleukin-2, 4 and 10, CD4~ and CD8~ lymphocytes, and conducted hematoxylin-eosin （HE） and Masson＇s staining of skin to judge whether the immune rejection reactions occurred within 28 days after transplantation. Chr. 7 genomic heterozygosity of 48 WZSP individuals from F20 to F22 was analyzed by high-density single nucleotide polymorphism （SNP） chips （60 000 SNPs）. The result showed that there were no significant differences in graft skin, the plasma interleukin-2, 4, 10, CD4~ and CD8~, HE and Masson＇s staining results between the allograft and autograft groups, and no immune rejection occurred on the allograft group. We found that 11 genes in Chr. 7 of major histocompatibility complex （MHC） I and MHC II were homozygous which confirmed that immune antibody of the allograft and autograft groups were highly identical and also provided a theoretical basis to no immune rejection occurred on the allograft in the inbred WZSP. The result proved that the WZSP inbred line had been cultivated successfully for the first time in the world. The test methods also provide a scientific basis for the identification of swine and mammal inbred lines.

Genetic analysis and QTL mapping of stalk cell wall components and digestibility in maize recombinant inbred lines from B73 × By804

The cell wall composition and structure of the maize stalk directly affects its digestibility and in turn its feed value.Previous studies of stem quality have focused mostly on common maize germplasm,and few studies have focused on high-oil cultivars with high grain and straw quality.Investigation of the genetic basis of cell wall composition and digestibility of maize stalk using high-oil maize is desirable for improving maize forage quality.In the present study,a high-oil inbred line(By804)was crossed as male parent with the maize inbred line B73 to construct a population of 188 recombinant inbred lines(RILs).The phenotypes of six cell-wall-related traits were recorded,and QTL analysis was performed with a genetic map constructed with SNP markers.All traits were significantly correlated with one another and showed high broad-sense heritability.Of 20 QTLs mapped,the QTL associated with each trait explained 10.0%–41.1%of phenotypic variation.Approximately half of the QTL each explained over 10%of the phenotypic variation.These results provide a theoretical basis for improving maize forage quality by marker-assisted selection.