From Markers to Genome Based Breeding in Horticultural Crops: An Overview

Molecular markers,genome sequencing and genome editing are considered as efficient tools to accomplish demands of plant breeders for crop improvement programs.Morphological and biochemical markers have not been extensively used as these are greatly influenced by environmental factors.Different molecular markers and sequencing techniques are routinely used in evaluation of genetic diversity and evolutionary relationship,accurate classification or taxonomy,characterization of germplasm,identification of hybrids and phylogenetic studies.Desired and undesired traits controlled by genes can be identified through different molecular markers technology all over the globe.These molecular markers are well established and have successfully been used for genetic analysis of different plants during last two decades.Recently,advanced techniques of molecular markers have been developed,which provide advance genotypic platform and tends to merge valuable properties of many basic systems.New class of markers also includes little modifications in basic methods to enhance the sensitivity and resolution.Biotechnologists,plant breeders and strong investment are strongly linked for crop improvement purposes.Current review provides detailed description of different markers,genome sequencing and genome editing that have been utilized for different genetic analyses of horticultural crops.Genome editing technologies based on CRISPR/Cas are now successfully applied in horticultural crops.Moreover,current review encourages the use of molecular marker technology for DNA fingerprinting,bar coding,sequencing,re-sequencing QTL mapping,genome association mapping and genome editing.It is need of time that diverse germplasm should be identified with different molecular techniques and further utilized in breeding purposes to achieve higher yielding and resistant cultivars against biotic and abiotic stresses.

Assembly and marker analysis of mitochondrial genomes provide insights into origin,evolution and spread of Brassica juncea(L.)Czern.et Coss.

The release of mitochondrial genome sequences provides the basis for characterizing interspecific and intraspecific variation in Brassica mitochondrial genomes.However,few B.juncea(mustard)mitochondrial genomes have been published.We assembled the mitochondrial genomes of three B.juncea subspecies and compared them with previously published genomes.The genomes were phylogenetically classified into A,B,C,and Bna clades.Two variant sites,a transversion(C→A)at nt 79,573 and a 31-bp copy-number variation between nts 65,564 and 65,596,were identified.Based on these variant sites,mitotype-specific sequence markers were developed to characterize the variation among worldwide 558 B.juncea accessions.Three mitochondrial genome types(mitotypes MT1–MT3)were identified.In terms of geographical distribution,MT1 and MT2 accessions were distributed mainly to the north and MT3 to the south of 34°N.Root mustards carried only MT1,leaf and stem mustards carried mainly MT3,and seed mustards carried all three mitotypes,implying that the mitotypes underwent selection during B.juncea domestication.A new form of oil mustard evolved by hybridization between two gene pools in southwest China.

Genetic Map of Cotton with Molecular Markers

Cotton (Gossypium spp.) is the most important natural fiber in the world, and its seeds are also used as a food source. Breeding cotton for traits of interest, such as production and processing of fibers, will ensure that this natural product is as competitive as renewable synthetic fibers derived from petroleum. Thus, the mapping of the cotton genome for traits of interest may be the basis for its subsequent use in breeding programs. This work consists of a literature review, with the aim of bringing together works from different research groups working with the mapping of the cotton genome with molecular markers.

Development of genome-wide insertion/deletion markers in rice based on graphic pipeline platform

DNA markers play important roles in plant breed- ing and genetics. The Insertion/Deletion （InDel） marker is one kind of co-dominant DNA markers widely used due to its low cost and high precision. However, the canonical way of searching for InDel markers is time-consuming and labor- intensive. We developed an end-to-end computational solution （InDel Markers Development Platform, IMDP） to identify genome-wide InDel markers under a graphic pipeline environment. IMDP constitutes assembled genome sequen- ces alignment pipeline （AGA-pipe） and next-generation re- sequencing data mapping pipeline （NGS-pipe）. With AGA-pipe we are able to identify 12,944 markers between the genome of rice cultivars Nipponbare and 93-11. Using NGS-pipe, we reported 34,794 InDels from re-sequencing data of rice cultivars Wu-Yun-Geng7 and Guang-Lu-Ai4. Combining AGA- pipe and NGS-pipe, we developed 2o5,659 InDels in eight japonica and nine indica cultivars and 2,681 InDels showed a subgroup-specific pattern. Polymerase chain reaction （PCR） analysis of subgroup-specific markers indicated that the precision reached 90% （86 of 95）. Finally, to make them available to the public, we have integrated the InDels/markers information into a website （Rice InDel Marker Database, RIMD, http：I/2o2.12o.45.71/）. The application of IMDP in rice will facilitate efficiency for development of genome-wide InDel markers, in addition it can be used in other species with reference genome sequences and NGS data.

Genetic Diversity Within a Jackfruit (Artocarpus heterophyllus Lam.) Germplasm Collection in China Using AFLP Markers

Jackfruit is cross-pollinated and mostly seed propagated, a wide range of variation exists in fruit quality. With the development of efficient vegetative propagation methods, excellent genotypes selected from these seed propagated seedlings will gradually replace other genotypes in jackfruit producing areas. In this study, genetic diversity of 50 jackfruit accessions from three provinces in China was analyzed based on amplified fragment length polymorphic （AFLP） markers. A total of 320 unambiguous bands were produced by eight primer combinations, and 65 （20.3%） of them were polymorphic. Genetic similarity coefficients ranged from 0 to 0.9841, with an average of 0.5000, indicating a moderate genetic diversity in this collection. The dendrogram derived by unweighted pair group method with arithmetic mean algorithm （UPGMA） analysis revealed five groups, and no correlation between genetic relationship and geographical origin were found. Accessions of soft and firm flesh type were not clustered into distinct groups, neither could yearly bearing once, or twice fruit accessions. This study has provided useful information for collection and preservation of jackfruit germplasm worldwide.

Cloning,characterization of two female-specific AFLP markers and development of PCR-based sex identification method for the half-smooth tongue sole Cynoglossus semilaevis

Two female-specific AFLP(amplified fragment length polymorphism)markers(named CseF464 and CseF136)were isolated by using one selective primer combination(E-AGC/M-CTG)from the genomic DNA of 20 females and 20 males of the half-smooth tongue sole Cynoglossus semilaevis.Both the markers were re-amplified,recovered from the agarose gels,cloned and sequenced.Bioinformatics analysis indicated that the length of the two markers were 468 bp and 134 bp,respectively,and the sequences showed no similarity to each other,as well as to the known sequences deposited in the GenBank database using BLASTn.Two pairs of SCAR(sequence characterized amplified regions)primers were designed based on the sequences of the two female-specific markers.Furthermore,PCR-based genetic sex identification method was developed in Cynoglossus semilaevis.A specific fragment was amplified in all females but not in any males by using these SCAR primers on the initial 20 female and 20 male individuals of Cynoglossus semilaevis.The feasibility of the two SCAR primer pairs was confirmed in additional 100 individuals(50 females and 50 males).This allowed for reliable,rapid molecular identification of genetic sex of the species,genetic mapping on the sex chromosomes and better understanding of the sex determination and sex differentiation in the half-smooth tongue sole.

Identification of AFLP Markers Linked to Lr19 Resistance to Wheat Leaf Rust

AFLP analyses were carried out on Thatcher, 23 near-isogenic lines and F2 generation of TcLr19 × Thatcher, to develop molecular markers for gene Lr19 resistance to wheat leaf rust. Seven markers linked to Lr19 resistance trait were obtained, which were P-AGT/M-GAG289 bp （3.3 cM）, P-ACA/M-GGT102 bp （4.1 cM）, P-ACA/M-GGT106 bp （4.1 cM）, P-AAC/M-CAG123 bp （4.9 cM）, P-AAC/M-GGT203bp （5.0 cM）, P-ACA/M-GGT290bp （5.7 cM）, and P-ATC/M-GAG293bp （9.6 cM）. All of these specific fragments were isolated from the polyacrylamide gels, reamplified, cloned, and sequenced. The research may facilitate genetic mapping, physical mapping, and the eventual cloning of Lr19.

Identification of Heat Tolerance Linked Molecular Markers of Chinese Cabbage(Brassica campestris L.ssp.pekinensis)

Genetically stable population of recombination inbred line (RIL) was derived from a cross between a heat tolerant line 177 and a heat sensitive line 276 of Chinese cabbage (Brassica campestris L. ssp. pekinensis) by single seed descent. The RILs were analyzed using isozyme, RAPD and AFLP techniques in order to find molecular markers that are linked to heat tolerance quantitative trait loci (QTL). The results of variance analysis of single factor indicated that there were 9 molecular markers closely linked with heat tolerance QTL, including 5 AFLP markers, 3 RAPD markers and 1 PGM isozyme marker. Total genetic contribution of these makers to heat tolerance was 46.7%. Five of the nine markers distributed in one linkage group, the remaining 4 markers were located in separate groups. Thus the 9 heat tolerance linked markers distributed in 5 independent locations in the genome of Chinese cabbage.

Molecular Diversity of Kenyan Lablab Bean (<i>Lablab purpureus</i>(L.) Sweet) Accessions Using Amplified Fragment Length Polymorphism Markers

Lablab purpureus (L.) Sweet is a multipurpose legume that combines use as human food and animal feed in addition to serving as a cover crop for soil conservation. In this work, molecular diversity in Lablab purpureus was assessed using amplified fragment length polymorphism markers on fifty Kenyan lablab accessions obtained from farmers’ fields and the Kenya National gene bank. One hundred and eighty polymorphic bands were revealed using fifteen selective primer pairs. The overall mean expected heterozygosity (He) for the five populations was 0.189. Estimates of components of molecular variance revealed that most of the genetic variation resided within populations (99%) and only 1% variance was among the populations, while Principal Coordinate Analysis showed an overlap between accessions from different geographic origins. The UPGMA cluster analysis generated from the distance matrix of the 50 assayed accessions, revealed low diversity among most of the accessions. The low diversity observed may be due to the narrow genetic base for breeding stocks, and extensive exchange of germplasm among smallholder farmers across the country. Results obtained from this study are discussed in light of the need to enhance the genetic management and improvement of this multipurpose crop species.

Research Progress of Molecular Markers in Genetic Diversity of Rapeseed

In recent years,with the continuous improvement and development of molecular technology in the application process,different types of DNA molecular markers have made rapid progress in the study of genetic diversity of rapeseed. The study of genetic diversity is conducive to the correct formulation of the strategy of collecting and in situ preservation of genetic resources of rapeseed,and it is the genetic basis for the improvement of rapeseed varieties. This article mainly starts with the three DNA molecular markers( SSR,RAPD,AFLP) widely used in the study of genetic diversity of rapeseed. By introducing the application principles and characteristics of SSR,RFPD and AFLP molecular markers,research progress of these three marker technologies in genetic diversity of rapeseed is briefly described.

Identification of Molecular Markers Linked to the Wilt Resistance Gene FuJ7(t) in Flax

A cross between wilt resistant flax variety Jinya7 and susceptible variety Jinya1 wasmade for mapping wilt resistance gene(s). The inoculation test of F1 and F2 progeny provedthat the resistance of Jinya7 to wilt is controlled by two dominant genes. With 48 EcoRⅠ/MseⅠ primer combinations, amplified fragment length polymorphisms (AFLP) analysis wasperformed on two parents and their F2 resistance and susceptibility bulks. A total ofabout 3300 distinguishable bands were amplified, of which three bands had stabledifferences. The genetic linkage analysis of the three polymorphic DNA fragments withthe resistance gene(s) was made in the F2 segregating population derived from the crossbetween Jinya7 and Jinya1. The DNA fragment AG/CAG was found closely linked to one of thewilt-resistant genes, which with a genetic distance of 5.2cm, was tentatively named FuJ7(t).The cloned fragment AG/CAG was sequenced and then converted successfully to a sequencecharacterized amplified region (SCAR) marker, which can be used more conveniently in theidentification and marker-assisted selection for the wilt resistance gene FuJ7(t) toflax wilt.

Molecular Footprint of Kenya’s Gene Bank Repositories Based on the <i>cp</i>-Genome Signatures

While the mutational processes that subsume biological diversity can be revealed in great detail through phylogenetic inferencing using plastid markers, few studies document their use. Accurate phylogenic inference can provide a framework for addressing a host of important evolutionary questions including a context to reconstruct molecular evolution of an organism. Despite the obvious utility of plastid markers in illuminating biological enquiry, many important questions still abound. The use of cp-DNA gene sequence data for phylogenetic inference can have an enormous impact on plant phylogenetics and systematics. The repertoire of genetic diversity of Kenya’s Gene Bank repositories can be explored based on cp-genome signatures. This is because cp-DNA-based mutational changes are an important additional tool to the previous evidence available on plant evolution yet to be explored in biodiversity studies in Kenya. Taken together, these evolutionary changes can inspire development of realistic algorithms for phylogenetic inferencing based on molecular data. Phylogenetic reconstructions are at the very core of molecular evolution. Comparative sequence analyses of plastid markers can have utility beyond the study of phylogeny. The pattern of nucleotide substitution observed over evolutionary time can reflect functional constraints imposed due to natural selection. In line with this, it is possible to detect subtle anatomical variations associated with small fitness effects that can account for genetic diversity at varietal level. The lack of sequence information in Kenyan cowpea has limited the robust advancement of molecular markers use in dissecting diversity based on the putative plastid markers?[1]. The present study sought to generate and upscale novel technologies such as genomics, DNA barcoding and bio-informatics in understanding molecular diversity of cowpea accessions from the Gene Bank of Kenya and ecotypes. A total of 298 sequences of cowpea germplasm conserved as in situ and ex situ in Kenya but sourced from phylogeographically diverse settings were examined and their genetic profiles were characterized and evaluated using molecular tools. The Gene Bank materials were purposefully sampled to develop subsets representative of the diversity in the genepool’s collection. We present an extensive study on characterizing the genetic diversity of cp-DNA gene sequence data for the cowpea accessions from the Nation Gene Bank of Kenya. The comparative sequence analyses and phylogenetic clustering of seven plastid markers widely used in the DNA barcoding of land plants provide insights on the molecular evolution of this vascular plant. The detailed and in-depth genome characterization herein greatly enriches the genetic profile of this important crop, which can help in reconstructing realistic models of mutational process during plant evolutionary history. This study addressed this gap by employing a DNA barcode library for cowpea to determine the loci that yield the best species resolution. As well, this study examined the efficacy of custom DNA barcode loci for identification success, and compared phylogenetic diversity measures between sites and among variants.

Evaluation of Genetic Diversity in Wild Diploid Wheat Triticum boeoticum from Iran Using AFLP Markers

Although Iran is a main center of distribution of wild wheats, but little information is available regarding genetic variation in wild wheat relatives from this country (Tabatabaei and Massoumi, 2001). The habitats of

Study Genetic Variation Using DNA Molecular Markers and Identification Physiological Races of Wheat Stripe （yellow） Rust Puccinia striiformis f.sp tritici during 2010-2014 in Some Regions of Syria

Yellow Rust （stripe） rust （Puccinia striiformis West. f. sp. tritici） is one of the most epidemic diseases infect wheat in cold and wet regions. In 1988, this disease caused a loss of seasonal production amounted 70% on wheat variety Mexipak in Syria, and recurrent infection in 2010, caused by a virulent race called Yr27, caused a considerable loss in the production of bread wheat cultivars （Cham 8, Cham 6 particularly） amounted 90%. Recently, 15 races of yellow rust had been addressed in Syria for seasons 2010-2014; 159E256, 166E254, 166E256, 255 E112, 0 E0, 64 E 6, 230 El50, 0 E 18, 198 El30, 166 El50, 102 El60, 128 E0, 126 El50, 214E150, and 6E16. The race 6E16 was the most frequent during the two seasons, while the race 255El12 was the most virulent, followed by the race 230E222 and the race 0E0 was the weakest one. This study revealed the presence of fourteen newly observed races in Syria. Molecular Variance Analysis of Molecular Variance （AMOVA） of 55 yellow rust Puccinia striiformis f.sp tritici isolates examined by Amplify Fragment Length Polymorphism （AFLP） revealed high genetic variation within population, and the dimensional scale analysis （MSD） and tree diagram showed that the Syrian yellow rust isolates were clustered in three groups： the first group contained isolates derived from durum wheat, the second one contained bread wheat isolates, but the third was made of isolates derived from both durum and bread wheat species.

Genetic Diversity Analysis of Jatropha Species from Costa Rica Using AFLP Markers

The genetic diversity from species of the genus Jatropha collected from Costa Rica was analyzed by AFLP (amplified fragments length polymorphism). The study consisted of 114 accessions from 15 populations of 4 different species: J. curcas, J. costaricensis, J. gossypifolia and J. stevensii. These were collected from different locations in Costa Rica. Three different primers were used, resulting in 428 loci, and they were classified in three categories: unique and double bands (UBD), rare bands (RB) and shared bands (SB). The UBD were excluded for a total of 339 polymorphic loci used for the UPGMA dendrogram and principal component analysis (PCA). The species that obtained the highest average of polymorphic loci was J. curcas, which obtained the highest percentage of polymorphic loci (80.24%), followed by J. gossypifolia (79.35%), J. costaricensis (78.76%), and finally J. stevensii (40.71%). The average for the polymorphic loci was of 69.76%. Thus, the phylogeny of the Jatropha species in Costa Rica was elucidated, showing J. curcas more related to J. stevensii and this one with J. costaricensis, and J. gossypifolia as the most distant member of the genus. For the best of our knowledge this is the first report of a genetic analysis of J. costaricensis and J. stevensii. The obtained molecular evidence showed high levels of polymorphisms in the present study compared with reports from Africa, India, and China. The molecular diversity estimated in our analysis, together with agronomical or morphological data, can be very useful for plant breeding programs, given the importance of Jatropha species in oil production. Interestingly, Central American Jatropha material can be used to increase the genetic base of J. curcas populations localized in Asia and Africa, where reduced genetic diversity has been reported.

枣树品种品系的AFLP分析

为建立完善枣种质资源分类鉴定技术体系,采用了基因组DNA-AFLP分子标记技术,从32对引物中筛选出了E-AAC/M-CCT、E-AAC/M-CAT、E-ACT/M-CCG、E-AAG/M-CCC和E-ACC/M-CAT等5个多态性好、分辨率高的引物组合,共扩增213条带,其中多态性带172条,多态性百分率76.16%。通过计算品种间单匹配系数,不加权算术平均值法绘制聚类图,可将28个供试品种和品系全部区分开;通过观察指纹图谱,也可明显看出新品系与其原种的差异带的多少,可快速准确地区分和确定新变异品系;另外,还可消除枣种质资源同名异物或同物异名现象。从而表明,AFLP技术是鉴定枣品种的有效方法。探讨了基因组DNA-AFLP分析技术的各关键步骤,并展望AFLP技术在枣品种鉴定上的应用前景。

利用AFLP技术筛选与银杏性别相关的分子标记

利用 amplified fragment length polymorphism(AFLP)技术 ,应用 4 8个引物组合 ,检测了雌雄银杏基因组DNA的多态性 ,筛选与银杏性别相关的分子标记 ,其中 3个引物组合各提供了 1个与雌性相关的分子标记 .经Southern点杂交证实有两个标记为银杏雌性基因组所特有 .这些分子标记的获得 ,为寻找性别特异的探针或PCR引物 。

贵州小型香猪基因组DNA的AFLP检测研究

报道了AFLP标记在研究贵州小型香猪遗传多态性方面的应用和该品系猪个体基因组DNA的AFLP扩增结果 ,分析了贵州小型香猪的群体遗传结构。实验应用 10条AFLP引物 ,用PstⅠ酶切 ,对 17头猪基因组DNA进行AFLP反应 ,共获得 116个AFLP标记 ,单引物获得的标记数在 2～ 2 2间 ,贵州小型香猪群体相似系数AFLP研究结果为 0 .86 6 (0 .76 0～ 0 .96 7) ,该研究为评价贵州小型香猪的遗传稳定性提供了相关的参数 。

家蚕AFLP连锁框架图谱的构建

利用改进的AFLP分子标记方法 ,对家蚕Bombyxmori回交一代BC1群体进行连锁图谱的构建。经 17组AFLP引物的选择性扩增 ,共得到 4 30个多态位点 ,卡方检验后有 2 5 3个为有效位点。利用Mapmaker Exp (Version 3 0b)软件作连锁分析 ,其中 16 3个标记分属 2 8个连锁群 ,连锁群标记数变化范围是 2～ 2 8个 ,平均每个连锁群标记数为 5 8个 ,该图覆盖的基因组长度为 2 998 9cM(图距单位 ) ,连锁群长度变化范围为 4 5～ 6 5 2 8cM ,连锁群的平均长度为 10 7 1cM ,平均图距为4 5～ 36 7cM。

运用近等基因系(NIL)、AFLP、RFLP和SCAR标记对玉米S组育性恢复基因(Rf_3)的研究

以１对近等基因系（ＮＩＬ）及其回交群体（ＢＣ１）为材料，采用ＢＳＡ法，利用ＡＦＬＰ技术，筛选与Ｒｆ３基因连锁的分子标记。在筛选的１２８个ＡＦＬＰ引物组合中，有２个能在ＮＩＬ及其可育池、不育池间扩增出多态性条带ＲＲ６和ＲＲ７。１００个ＢＣ１个体验证结果表明，ＡＦＬＰ标记ＲＲ６扩增产物中仅出现２个重组体，重组率２％，由此估测ＲＲ６距Ｒｆ６基因约２．０ｃＭ。并成功地将此标记转化为 ＳＣＡＲ标记，进行了ＮＩＬ和 ＢＣ１个体的特异性扩增。在来自综 ３ × Ｐ１３８的Ｆ２：３的群体上经ＲＦＬＰ分析后，将ＲＲ６定位于第二染色体的长臂上。不仅为辅助育种奠定了基础，而且为克隆Ｒｆ３基因提供了有益的信息。